Patent Grant
10328139
This application contains a Sequence Listing electronically submitted via EFS-Web to the U.S. Patent and Trademark Office as an ASCII text file entitled “265-00890101-2nd-SubstSequence Listing ST25” having a size of 50 kilobytes and created on Aug. 7, 2018. The information contained in the Sequence Listing is incorporated by reference herein.
Provided herein are genetically modified Y. pestis. In one embodiment, a genetically modified Y. pestis includes three alterations compared to a control Y. pestis. The first alteration includes decreased mRNA, decreased protein, or a combination thereof, encoded by a lpp coding region. The second alteration includes decreased mRNA, decreased protein, or a combination thereof, encoded by a msbB coding region. The third alteration is selected from an alteration of an intergenic region and decreased mRNA, decreased protein, or a combination thereof, encoded by a coding region. The alteration of an intergenic region includes the intergenic region between the coding regions ypo1119 and ypo1120. The decreased mRNA, protein, or a combination thereof, is encoded by a coding region selected from pla, ypo1717, ypmt1.80c, rbsA (ypo2500), ypo0498, vasK (ypo3603), ypo3164, hxuB (ypo3248), ypo1616, ypo1119, ypo1120, and ail.
In one embodiment, the genetically modified Y. pestis includes decreased mRNA, decreased protein, or a combination thereof, encoded by the coding regions lpp, msbB, and ail. In one embodiment, the genetically modified Y. pestis includes decreased mRNA, decreased protein, or a combination thereof, encoded by the coding regions lpp, msbB, and rbsA (ypo2500). In one embodiment, the genetically modified Y. pestis includes decreased mRNA, decreased protein, or a combination thereof, encoded by the coding regions lpp, msbB, and vasK (ypo3603). In one embodiment, the genetically modified Y. pestis includes decreased mRNA, decreased protein, or a combination thereof, encoded by the coding regions lpp and msbB, and an alteration of the intergenic region between the coding regions ypo1119 and ypo1120.
In one embodiment, the decreased mRNA or decreased protein is a decrease of at least 0.1% compared to the mRNA or the protein in the control Y. pestis. In one embodiment, the mRNA or the protein encoded by the one or more of the altered coding regions is undetectable in the genetically modified Y. pestis.
In one embodiment, an alteration includes a mutation in a coding region selected from lpp, msbB, pla, ypo1717, ypmt1.80c, rbsA (ypo2500), ypo0498, vasK (ypo3603), ypo3164, hxuB (ypo3248), ypo1616, ypo1119, ypo1120, and ail. In one embodiment, the mutation includes a deletion of at least one nucleotide. In one embodiment, the mutation includes a deletion of a portion of the coding region, or a deletion of the entire coding region.
Also provided is a composition that includes a genetically modified Y. pestis described herein and a pharmaceutically acceptable carrier. In one embodiment, composition is formulated for intramuscular administration. In one embodiment, composition is formulated for intranasal administration.
Further provided are methods for using the genetically modified Y. pestis described herein. In one embodiment, a method includes administering to a subject an effective amount of a composition that includes a genetically modified Y. pestis described herein, wherein the subject has an immune response to the genetically modified Y. pestis. The immune response can include a humoral immune response, a cell-mediated immune response, or a combination thereof. In one embodiment, the immune response is protective against bubonic plague. In one embodiment, the immune response is protective against septicemic plague. In one embodiment, the immune response is protective against pneumonic plague. In one embodiment, the immune response is protective against bubonic plague and pneumonic plague.
In one embodiment, the subject has or is at risk of having plague. The plague can be bubonic, septicemic, pneumonic, or a combination thereof. The subject can be, for instance, a human or a laboratory animal such as a mouse, a rat, or a non-human primate. In one embodiment, the method also includes administration of an antibiotic.
As used herein, “genetically modified” refers to a Yersinia pestis that has been altered through human intervention. In one embodiment, the human intervention may be the introduction of an exogenous polynucleotide into a Y. pestis. An example of an exogenous polynucleotide includes a polynucleotide that is inserted in the genome, such as a transposon (for instance by transposition) or a plasmid (for instance by homologous recombination). The genome of a Y. pestis includes chromosomal DNA and the DNA of any plasmid present in a Y. pestis, such as pCD1, pPCP1, and pMT1. “Genetically modified” also refers to a Y. pestis that has been genetically manipulated such that one or more endogenous nucleotides have been altered. For example, a microbe is a genetically modified microbe by virtue of introduction into a suitable microbe of an alteration of endogenous nucleotides. For instance, an endogenous coding region could include a mutation. Such a mutation may result in a protein having a different amino acid sequence than was encoded by the endogenous polynucleotide. Such a protein may have reduced biological activity, including no detectable biological activity. An example of a mutation resulting in no detectable biological activity is a deletion. Another example of a genetically modified Y. pestis is one having an altered regulatory sequence, such as a promoter, to result in increased or decreased expression of an operably linked endogenous coding region.
As used herein, “sequence similarity” refers to the amount of identity between a nucleotide sequence described herein and a second candidate nucleotide sequence. Whether a polynucleotide has sequence similarity with a polynucleotide described herein can be determined by aligning the residues of the two polynucleotides (for example, a candidate polynucleotide and any appropriate reference polynucleotide described herein, e.g., the lpp coding region of CO92) to optimize the number of identical nucleotides along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical nucleotides, although the nucleotides in each sequence must nonetheless remain in their proper order. A reference polynucleotide may be a polynucleotide described herein. A candidate polynucleotide is the polynucleotide being compared to the reference polynucleotide. A candidate polynucleotide may be isolated, for example, from a plant, or can be produced using recombinant techniques, or chemically or enzymatically synthesized. A candidate polynucleotide may be present in the genome of a plant and predicted to encode a protein useful herein.
A pair-wise comparison analysis of nucleotide sequences can be carried out using the Blastn program of the BLAST search algorithm, available through the World Wide Web, for instance at the internet site maintained by the National Center for Biotechnology Information, National Institutes of Health. Preferably, the default values for all Blastn search parameters are used. Alternatively, sequence similarity may be determined, for example, using sequence techniques such as GCG FastA (Genetics Computer Group, Madison, Wis.), MacVector 4.5 (Kodak/IBI software package) or other suitable sequencing programs or methods known in the art.
Thus, as used herein, a candidate polynucleotide useful herein includes those with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide sequence identity to a reference nucleotide sequence.
As used herein, “structural similarity” refers to the amount of identity or similarity between an amino acid sequence described herein and a second amino acid sequence. Whether a protein is structurally similar to a protein described here can be determined by aligning the residues of the two proteins (for example, a candidate protein and any appropriate reference protein described herein) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. In one embodiment a reference protein is a protein described herein. A candidate protein is the protein being compared to the reference protein. A candidate protein can be isolated, for example, from a plant, or can be produced using recombinant techniques, or chemically or enzymatically synthesized.
Unless modified as otherwise described herein, a pair-wise comparison analysis of amino acid sequences can be carried out using the Blastp program of the Blastp suite-2sequences search algorithm, as described by Tatusova et al., (FEMS Microbiol Lett, 174, 247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website. The default values for all blastp suite-2sequences search parameters may be used, including general parameters: expect threshold=10, word size=3, short queries=on; scoring parameters: matrix=BLOSUM62, gap costs=existence: 11 extension: 1, compositional adjustments=conditional compositional score matrix adjustment. Alternatively, proteins may be compared using other commercially available algorithms, such as the BESTFIT algorithm in the GCG package (version 10.2, Madison Wis.).
In the comparison of two amino acid sequences, structural similarity may be referred to by percent “identity” or may be referred to by percent “similarity.” “Identity” refers to the presence of identical amino acids. “Similarity” refers to the presence of not only identical amino acids but also the presence of conservative substitutions.
Thus, as used herein, reference to an amino acid sequence disclosed herein can include a protein with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to the reference amino acid sequence.
Alternatively, as used herein, reference to an amino acid sequence disclosed herein can include a protein with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the reference amino acid sequence.
The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.
The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
It is understood that wherever embodiments are described herein with the language “include,” “includes,” or “including,” and the like, otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided.
Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.
Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
There is no FDA-approved vaccine available against Yersinia pestis infections. This microbe leads to different forms of plague, namely bubonic, septicemic, and pneumonic. The organism is a Tier-1 select agent and is classified by the World Health Organization as a reemerging human pathogen. Since both humoral and cell-mediated immunity are helpful in providing optimal protection to the host from plague, developing a live-attenuated vaccine(s) is a promising approach. However, developing a live-attenuated vaccine is not straightforward. Such a vaccine candidate should have minimal reactogenicity while providing protection against all forms of plague.
Identification of new virulence factors in Y. pestis and optionally understanding their molecular mechanisms during an infection process are helpful in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, 5,088 mutants of Y. pestis CO92 were created and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 LD50 of wild-type (WT) CO92. From this screen, 118 clones showing impairment in disseminating to spleen were obtained based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD50 when tested in a mouse model of bubonic plague, with 10/20 aforementioned mutants providing 40% or higher survival rates at an infectious dose of 40 LD50. Upon sequencing, six of the attenuated mutants carried interruptions in genes encoding hypothetical proteins or proteins with putative functions. In-frame deletion mutation of two of the genes identified from the screen, namely rbsA that codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11-12 LD50 in a mouse model of pneumonic plague.
Earlier, it was found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced virulence of the Δlpp ΔmsbB double mutant in mouse models of bubonic and pneumonic plague (Sha et al., 2013, Infect Immun 81:815-828). As described herein, deletion of ail, rbsA or vasK genes from the Δlpp ΔmsbB double mutant resulted in an unexpected synergistic increase of attenuation. The Δlpp ΔmsbB Δail triple mutant was unable to kill mice at a LD50 dose equivalent to 6,800 LD50s of the wild type CO92 in a mouse model of pneumonic plague. The Δlpp ΔmsbB Δrbs and Δlpp ΔmsbB ΔvasK triple mutants provided 90-100% survivability to mice in a pneumonic plague model at 20-50 LD50s. The Δlpp ΔmsbB Δail triple mutant-infected mice at 3600-6800 LD50s were 40% to 70% protected upon subsequent challenge with 20-28 LD50 of WT CO92. The alteration of three genes also advantageously reduces the likelihood of reversion. These data suggest that the triple mutants described herein are useful as a live attenuated plague vaccine.
Genetically Modified Y. pestis
A genetically modified Yersinia pestis is provided herein. A genetically modified Y. pestis may be any strain of Y. pestis. In one embodiment, a genetically modified Y. pestis is biovar Y.p., orientalis, such as strain CO92 (Parkhill et al., 2001, Nature, 413(6855):523-527). In one embodiment, a genetically modified Y. pestis is using biovar Y. p., medievalis, such as strain KIM (Deng et al., J Bacteriol., 184(16):4601-4611). In one embodiment, a genetically modified Y. pestis is produced using a strain that is virulent before a genetic modification described herein is made to the strain. As used herein, a Y. pestis is considered virulent if it causes disease in a mouse model of bubonic plague at an LD50 of 50 colony forming units (CFUs) or less, or pneumonic plague at an LD50 of 500 CFUs or less. In one embodiment, a genetically modified Y. pestis includes one, two, or three plasmids selected from pCD1, pPCP1 (also referred to as pPla or pPst) and pMT1 (also referred to as pFra). In one embodiment, a genetically modified Y. pestis includes all three plasmids. One or more of the plasmids may include an alteration as described herein.
A genetically modified Y. pestis is attenuated. An attenuated Y. pestis is able to replicate in an animal and induce an immune response, but has a reduced ability to cause the clinical signs and/or symptoms of disease in an animal. Whether a genetically modified Y. pestis is attenuated can be determined by testing using a mouse model system recognized in the art as relevant in the evaluation of putative vaccines for protection of humans from infection by Y. pestis. The murine model can be used to evaluate protection from bubonic, pneumonic, and/or septicemic plague caused by Y. pestis. In one embodiment, whether a genetically modified Y. pestis is attenuated can be determined as described in Example 1. Briefly, a genetically modified Y. pestis is administered to mice by either the intramuscular (I.M.) or intranasal (I.N.) route. The dosage of the test microbe administered is twenty 50% lethal doses, where the lethal dose is the LD50 of the Y. pestis without the genetic modifications. If there is at least 90% survivability of the mice 30 days after administering the triple mutant, then the genetically modified Y. pestis is attenuated. In one embodiment, the genetically modified Y. pestis is attenuated is there is 100% survivability of the mice after 30 days.
In one embodiment, a genetically modified Y. pestis causes an immune response that protects the recipient from subsequent infection by a Y. pestis. The ability of a genetically modified Y. pestis to protect a recipient from challenge can be determined using the mouse model. In one embodiment, whether a genetically modified Y. pestis protects a recipient can be determined as described in Example 1. A population of mice are immunized I.M. or I.N. with the genetically modified Y. pestis at fifty 50% lethal doses, where the lethal dose is the LD50 of the Y. pestis without the genetic modifications. Twenty one days later the mice are immunized again, and 21 days after the second immunization mice are challenged with 12 50% lethal doses. If at least 90% of the recipients survive, the genetically modified Y. pestis protects the recipient.
In one embodiment, a genetically modified Y. pestis includes at least three alterations compared to a control Y. pestis. The alteration can refer to a coding region or to an intergenic region. As used herein, a “control” Y. pestis is a Y. pestis before it is genetically modified to include the alteration. A control Y. pestis may be a wild-type Y. pestis or a Y. pestis that includes a genetic modification. As used herein, an “alteration” refers to a change a Y. pestis that attenuates virulence.
With respect to an alteration of a coding region (e.g., lpp, msbB, ail, and other coding regions described herein), the alteration can result in a reduced amount in the cell of the mRNA encoded by the coding region, a reduced amount in the cell of the protein encoded by the coding region, or a combination thereof, compared to a control Y. pestis. The decrease in the amount of an mRNA or a protein encoded by the coding region may be a decrease of at least 0.1%, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the amount of the mRNA or the amount of the protein in a the same Y. pestis that does not include the alteration. In one embodiment, the mRNA or protein encoded by an altered coding region is undetectable in the genetically modified Y. pestis. In one embodiment, the biological activity of a protein encoded by an altered coding region is reduced in the genetically modified Y. pestis, and in one embodiment the biological activity is undetectable.
With respect to an alteration of an intergenic region in the genome of a Y. pestis, the intergenic region is between coding regions ypo1119 and ypo1120. The intergenic region of Y. pestis CO92 is shown in
One of the three alterations results in decreased mRNA, decreased protein, or a combination thereof, encoded by the coding region lpp. An example of an lpp coding region of a Y. pestis is nucleotides 2691297-2691533 of the genomic sequence of CO92 at Genbank accession number AL590842.1 or NC 003143. The Lpp protein encoded by that lpp coding region is the amino acid sequence at Genbank accession number YP 002347360. Other lpp coding regions include those having sequence similarity to the lpp coding region of CO92. Other lpp coding regions also include those encoding a protein having structural similarity to the Lpp protein at Genbank accession number CAL21022.1 (SEQ ID NO:49).
The second of the three alterations results in decreased mRNA, decreased protein, or a combination thereof, encoded by the coding region msbB (ypo2063). An example of a msbB coding region of a Y. pestis is nucleotides 2341684-2342646 of the genomic sequence of CO92 at Genbank accession number AL590842.1 or NC 003143. The MsbB protein encoded by that msbB coding region is the amino acid sequence at Genbank accession number CAL20698.1. Other msbB coding regions include those having sequence similarity to the msbB coding region of CO92. Other msbB coding regions also include those encoding a protein having structural similarity to the MsbB protein at Genbank accession number CAL20698.1 (SEQ ID NO:50).
In one embodiment, the third of the three alterations results in decreased mRNA, decreased protein, or a combination thereof, encoded by coding region selected from pla, ypo1717, ypmt1.80c, rbsA (ypo2500), ypo0498, vasK (ypo3603), ypo3164, hxuB (ypo3248), ypo1616, ypo1119, ypo1120, and ail. In one embodiment, two or more of these coding regions is altered in a genetically modified Y. pestis.
An example of a pla coding region of a Y. pestis is nucleotides 6665-7603 of the pPCP1 plasmid sequence at Genbank accession number AL109969.1. The Pla protein encoded by that pla coding region is the amino acid sequence at Genbank accession number CAB53170.1 (SEQ ID NO:51). Other pla coding regions include those having sequence similarity to the pla coding region of CO92. Other pla coding regions also include those encoding a protein having structural similarity to the Pla protein at Genbank accession number CAB53170.1.
An example of a ypo1717 coding region of a Y. pestis is nucleotides 1961429-1961977 of the genomic sequence of CO92 at Genbank accession number AL590842.1 or NC_003143. The protein encoded by that ypo1717 coding region is the amino acid sequence at Genbank accession number CAL20360.1 (SEQ ID NO:52). Other ypo1717 coding regions include those having sequence similarity to the ypo1717 coding region of CO92. Other ypo1717 coding regions also include those encoding a protein having structural similarity to the protein at Genbank accession number CAL20360.1.
There are multiple copies of ypmt1.80c coding regions in Y. pestis CO92. Examples of ypmt1.80c coding regions include nucleotides 17906-19114, 1777851-1779059, 1807069-1808277, 1811445-1812653, 1842497-1843705, and 1972184-1973392 of the chromosome sequence of CO92 at Genbank accession number AL590842.1 or NC 003143 and at nucleotides 79777-80985 of the pMT1 plasmid sequence at Genbank accession number AL117211.1. The proteins encoded by that ypmt1.80c coding regions are the amino acid sequences at Genbank accession numbers CAL18701.1 (SEQ ID NO:53), CAL20207.1, CAL20228.1, CAL20234.1, CAL20266.1, and CAL20370.1 for the chromosomal coding regions, respectively, and at Genbank accession number CAB55262.1 for the plasmid coding region. Other ypmt1.80c coding regions include those having sequence similarity to a ypmt1.80c coding region of CO92. Other ypmt1.80c coding regions also include those encoding a protein having structural similarity to the protein at Genbank accession number CAL18701.1, CAL20207.1, CAL20228.1, CAL20234.1, CAL20266.1, CAL20370.1, or CAB55262.1.
An example of a rbsA (ypo2500) coding region of a Y. pestis is nucleotides 2809281-2810771 of the genomic sequence of CO92 at Genbank accession number AL590842.1 or NC_003143. The RbsA protein encoded by that rbsA coding region is the amino acid sequence at Genbank accession number CAL21128.1 (SEQ ID NO:54). Other rbsA coding regions include those having sequence similarity to the rbsA coding region of CO92. Other rbsA coding regions also include those encoding a protein having structural similarity to the RbsA protein at Genbank accession number CAL21128.1.
An example of a ypo0498 coding region of a Y. pestis is nucleotides 531628-532371 of the genomic sequence of CO92 at Genbank accession number AL590842.1 or NC_003143. The protein encoded by that ypo0498 coding region is the amino acid sequence at Genbank accession number CAL19178.1 (SEQ ID NO:55). Other ypo0498 coding regions include those having sequence similarity to the ypo0498 coding region of CO92. Other ypo0498 coding regions also include those encoding a protein having structural similarity to the protein at Genbank accession number CAL19178.1.
An example of a vasK (ypo3603) coding region of a Y. pestis is nucleotides 4012390-4015923 of the genomic sequence of CO92 at Genbank accession number AL590842.1 or NC_003143. The VasK protein encoded by that vasK coding region is the amino acid sequence at Genbank accession number CAL22191.1 (SEQ ID NO:56). Other vasK coding regions include those having sequence similarity to the vasK coding region of CO92. Other vasK coding regions also include those encoding a protein having structural similarity to the VasK protein at Genbank accession number CAL22191.1.
An example of a ypo3164 coding region of a Y. pestis is nucleotides 3527637-3528593 of the genomic sequence of CO92 at Genbank accession number AL590842.1 or NC_003143. The protein encoded by that ypo3164 coding region is the amino acid sequence at Genbank accession number CAL21759.1 (SEQ ID NO:57). Other ypo3164 coding regions include those having sequence similarity to the ypo3164 coding region of CO92. Other ypo3164 coding regions also include those encoding a protein having structural similarity to the protein at Genbank accession number CAL21759.1.
An example of a hxuB (ypo3248)coding region of a Y. pestis is nucleotides 3620645-3622435 of the genomic sequence of CO92 at Genbank accession number AL590842.1 or NC_003143. The protein encoded by that hxuB (ypo3248) coding region is the amino acid sequence at Genbank accession number CAL21842.1 (SEQ ID NO:58). Other hxuB (ypo3248) coding regions include those having sequence similarity to the hxuB (ypo3248) coding region of CO92. Other hxuB (ypo3248) coding regions also include those encoding a protein having structural similarity to the protein at Genbank accession number CAL21842.1.
An example of a ypo1616 coding region of a Y. pestis is nucleotides 1838652-1839194 of the genomic sequence of CO92 at Genbank accession number AL590842.1 or NC_003143. The protein encoded by that ypo1616 coding region is the amino acid sequence at Genbank accession number CAL20261.1 (SEQ ID NO:59). Other ypo1616 coding regions include those having sequence similarity to the ypo1616 coding region of CO92. Other ypo1616 coding regions also include those encoding a protein having structural similarity to the protein at Genbank accession number CAL20261.1.
An example of a ypo1119 coding region of a Y. pestis is nucleotides 1266799-1267131 of the genomic sequence of CO92 at Genbank accession number AL590842.1 or NC_003143. The protein encoded by that ypo1119 coding region is the amino acid sequence at Genbank accession number CAL19785.1 (SEQ ID NO:60). Other ypo1119 coding regions include those having sequence similarity to the ypo1119 coding region of CO92. Other ypo1119 coding regions also include those encoding a protein having structural similarity to the protein at Genbank accession number CAL19785.1.
An example of a ypo1120 coding region of a Y. pestis is nucleotides 1267263-1267664 of the genomic sequence of CO92 at Genbank accession number AL590842.1 or NC_003143. The protein encoded by that ypo1120 coding region is the amino acid sequence at Genbank accession number CAL19786.1 (SEQ ID NO:61). Other ypo1120 coding regions include those having sequence similarity to the ypo1120 coding region of CO92. Other ypo1120 coding regions also include those encoding a protein having structural similarity to the protein at Genbank accession number CAL19786.1.
An example of a ail (ypo1860) coding region of a Y. pestis is nucleotides 2109269-2109820 of the genomic sequence of CO92 at Genbank accession number AL590842.1 or NC_003143. The Ail protein encoded by that ail coding region is the amino acid sequence at Genbank accession number CAL20500.1 (SEQ ID NO:62). Other ail coding regions include those having sequence similarity to the ail coding region of CO92. Other ail coding regions also include those encoding a protein having structural similarity to the Ail protein at Genbank accession number CAL20500.1.
A genetically modified Y. pestis may include additional alterations. In one embodiment, a genetically modified Y. pestis includes an alteration in more than one ypmt1.80c coding region. In one embodiment, an additional alteration includes a mutation that attenuates the microbe. Other attenuating mutations of Y. pestis are known in the art.
Methods of Making
A genetically modified Y. pestis described herein can be made using any routine method known in the art for reducing expression of a coding region, or altering an intergenic region, in a microbe like Y. pestis. For instance, a coding region or an intergenic region may be deleted in whole or in part, a regulatory region of a coding region may be modified, or a protein that modulates expression of a coding region may be modified.
A genetically modified microbe can be produced using classical genetic methods, recombinant methods, or a combination thereof. Classic genetic methods include the use of transduction and conjugation to introduce mutations into a Y. pestis. Recombinant methods include, for instance, transformation of a Y. pestis with an artificial polynucleotide, such as a plasmid. For instance, DNA integration cassettes (also referred to as DNA mutagenic cassettes) can be used to replace a genomic coding region in a Y. pestis by homologous recombination. Such cassettes typically include the mutation to be inserted, homologous nucleotide sequences to target the mutation to the coding region, and optionally a marker sequence. The actual nucleotide sequence of a specific coding region to be altered in a Y. pestis may vary slightly from a publicly available sequence; however, the actual nucleotide sequence of the specific coding region can be easily determined using routine methods.
Examples of mutations that can be used in the production of a genetically modified Y. pestis, including a genetically modified Y. pestis having an alteration of a coding region or an alteration of an intergenic region, include a deletion, an insertion, and a point mutation, such as transition and/or transversion point mutations. A deletion may include deletion of part or an entire nucleotide sequence of a coding region or an intergenic region, or deletion of a regulatory region of a coding region. The genetically modified Y. pestis may encode a fragment of a protein encoded by the coding region, or not encode the protein. A mutation useful to produce a genetically modified Y. pestis described herein is stable and reverts at a low frequency. In one embodiment, a mutation useful to produce a genetically modified Y. pestis described herein is non-reverting.
Compositions
Provided herein are compositions that includes a genetically modified Y. pestis. Such a composition may include a pharmaceutically acceptable carrier. As used herein “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration and not deleterious to a recipient thereof. The skilled person will recognize that a pharmaceutically acceptable carrier is chosen that will not kill the genetically modified Y. pestis or inhibit its ability to effectively grow and cause an immune response in a subject receiving the attenuated microbe. A composition described herein may be referred to as a vaccine. The term “vaccine” as used herein refers to a composition that, upon administration to an animal, will increase the likelihood the recipient is protected against Y. pestis. Without intending to be limiting, administration of the composition to an animal typically produces a humoral and cellular immunological response that results in immunity.
A composition may be formulated in pharmaceutical preparations in a variety of forms adapted to the chosen route of administration, including routes suitable for stimulating an immune response. Thus, a composition described herein can be administered via known routes including, for example, parenteral (e.g., intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular), enteral (e.g., oral), and topical (e.g., epicutaneous, intranasal, transmucosal) administration. In one embodiment, the route of adminstration is intramuscular. In one embodiment, the route of administration is intranasal. Appropriate dosage forms for enteral administration of the genetically modified Y. pestis may include tablets, capsules or liquids. Appropriate dosage forms for parenteral administration may include intramuscular administration. Appropriate dosage forms for topical administration may include nasal sprays, metered dose inhalers, dry-powder inhalers or by nebulization. It is foreseen that administration of a composition to a mucosal surface, such as by administration to the nasal or respiratory mucosa (e.g., via a spray or aerosol), may stimulate mucosal immunity, such as production of secretory IgA antibodies, throughout the subject's body.
A formulation may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing the active compound (e.g., a genetically modified Y. pestis) into association with a carrier that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations. In general, a composition can be formulated to be compatible with its intended route of administration. More specifically, the compositions described herein may be administered to any tissue of a subject, including, but not limited to, muscle (such as skeletal muscle), skin, lung tissue, intestinal tissue, and the like. A composition described herein may be administered to any internal cavity of an animal, including, but not limited to, lungs, mouth, nasal cavity, stomach, peritoneal cavity, intestine, veins, and the like.
A composition including a pharmaceutically acceptable carrier can also include an adjuvant. An “adjuvant” refers to an agent that can act in a nonspecific manner to enhance an immune response to a genetically modified Y. pestis, thus potentially reducing the quantity of microbe necessary in any given immunizing composition, and/or the frequency of injection necessary in order to generate an adequate immune response to the genetically modified microbe. Adjuvants may include, for example, IL-1, IL-2, emulsifiers, muramyl dipeptides, dimethyl dioctadecyl ammonium bromide (DDA), avridine, aluminum hydroxide, oils, saponins, alpha-tocopherol, polysaccharides, emulsified paraffins, ISA-70, RIBI and other substances known in the art.
In another embodiment, a composition described herein including a pharmaceutically acceptable carrier can include a biological response modifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-alpha, IFN-gamma, and other cytokines that effect immune cells.
A composition described herein may be administered in an amount sufficient to treat or prevent infection by Y. pestis. For instance, the amount of genetically modified microbe present in a composition can vary. For example, the dosage of genetically modified microbes can be present at a concentration of, for instance, at least 102 bacteria/ml, at least 103 bacteria/ml, at least 104 bacteria/ml, at least 105 bacteria/ml, at least 106 bacteria/ml, at least 107 bacteria/ml, at least 108 bacteria/ml, or at least 109 bacteria/ml. In one embodiment, the genetically modified microbe is present in the composition in an amount that the total volume of the composition administered is 0.5 ml to 5.0 ml, typically 1.0 to 2.0 ml. The amount administered will vary depending on various factors including, but not limited to, the specific genetically modified Y. pestis chosen, the weight, physical condition and age of the subject, and the route of administration. Thus, the unit dosage form can vary widely, and depends upon factors such as the species, age, weight and physical condition of the subject, as well as the method of administration. Such factors can be determined by one of skill in the art.
Methods of Use
Provided herein are methods for using the genetically modified Y. pestis. The method can include administering to a subject effective amount of a composition that includes a genetically modified Y. pestis described herein. As used herein, an “effective amount” of a composition that includes a genetically modified Y. pestis is the amount able to elicit the desired response in the recipient. The subject can be, for instance, a human, or a laboratory animal such as a murine (e.g., a mouse or a rat), a guinea pig, or a non-human primate. In one embodiment, a human subject is a person likely to be exposed to Y. pestis, such as a member of the military. In one embodiment, a subject is immunocompromised. The methods may further include additional administrations (e.g., one or more booster administrations) of the composition to the subject to enhance or stimulate a secondary immune response. A booster can be administered at a time after the first administration, for instance, 1 to 8 weeks, preferably 2 to 4 weeks, after the first administration of the composition. Subsequent boosters can be administered one, two, three, four, or more times annually.
In one embodiment, the method may be directed to making the animal cause a protective immune response to the genetically modified Y. pestis. As used herein, “protective” means that the immune response contributes to the lessening of any symptoms associated with infection of a subject with Y. pestis. For example, a genetically modified Y. pestis may induce an immune response that helps to reduce symptoms associated with Y. pestis infection or reduce the morbidity and mortality associated with infection with the microbe.
An immune response to a genetically modified Y. pestis described herein may include a humoral immune response, a cell-mediated immune response, or a combination thereof. In one embodiment, an immune response includes both a humoral immune response and a cell-mediated immune response. In one embodiment, the presence of a humoral immune response may be determined by measuring the increase of serum IgG to one or more antigens produced by the genetically modified Y. pestis. Examples of suitable antigens that can be detected include the capsular antigen F1 and the low-calcium response V antigen of the type 3 secretion system. In one embodiment, the presence of a cellular immune response can be determined by measuring T-cell proliferation, where T-cell proliferation to Y. pestis antigens is increased in a subject after administration of a composition described herein. Cytokine production by T-cells in response to Y. pestis antigens can also be measured. Increased cytokine/chemokine production, e.g., IFN-γ TNF-α, IL-6, IL-1β, and/or IL-10 can result when T-cells are stimulated with Y. pestis antigens after immunization with a genetically modified Y. pestis described herein.
In one embodiment, the method may be directed to treating an animal that has, or is at risk of having, an infection by Y. pestis. As used herein, the term “infection” refers to the presence of a Y. pestis in an subject's body, which may or may not be clinically apparent. An infection by Y. pestis may result in plague, such as bubonic plague, septicemic plague, pneumonic plague, or a combination thereof.
Treating an infection can be prophylactic or, alternatively, therapeutic—in this context, treatment after a subject manifests one or more indication of infection by Y. pestis. Generally, treatment that is prophylactic—in this context, initiated before a subject is infected by a Y. pestis or while an infection remains subclinical—is referred to herein as treatment of a subject that is “at risk” of infection. As used herein, the term “at risk” refers to a subject that may or may not actually possess the described risk—in this context, a subject that may or may not be infected by Y. pestis. Thus, typically, a subject “at risk” of infection by Y. pestis is a subject present in an area where individuals have been identified as infected by the Y. pestis and/or is likely to be exposed to the Y. pestis even if the subject has not yet manifested any detectable indication of infection by the Y. pestis and regardless of whether the subject may harbor a subclinical amount of the microbe. For example, a subject at risk includes a subject likely to be exposed to a vector of Y. pestis, such as a flea. Accordingly, administration of a composition can be performed before, during, or after the subject has first contact with Y. pestis. Treatment initiated after the animal's first contact with the microbe may result in decreasing the risk of death, increasing likelihood of survival, decreasing the severity of symptoms and/or clinical signs of infection by the microbe, completely removing the microbe, and/or decreasing the likelihood of experiencing a clinically evident infection compared either to the animal before administration of the composition or to an animal to which the composition is not administered. The method includes administering an effective amount of the composition as described herein to an animal having, or at risk of having, an infection caused by Y. pestis, and determining whether the infection has decreased. Methods for determining whether an infection is caused by Y. pestis are routine and known in the art, as are methods for determining whether the infection has decreased.
In another aspect, provided herein are methods for treating one or more symptoms or clinical signs of certain conditions in an animal that may be caused by infection by Y. pestis. The method includes administering an effective amount of a composition as described herein to an animal having or at risk of having a condition, or exhibiting symptoms and/or clinical signs of a condition, and determining whether at least one symptom and/or clinical sign of the condition is changed, preferably, reduced.
Treatment of symptoms and/or clinical signs associated with these conditions can be prophylactic or, alternatively, therapeutic—in this context, treatment initiated after the subject exhibits one or more symptoms or clinical signs associated with a condition caused by infection by Y. pestis. As used herein, the term “symptom” refers to subjective evidence of disease or condition experienced by the patient and caused by infection by a microbe. As used herein, the term “clinical sign” or, simply, “sign” refers to objective evidence of disease or condition caused by infection by Y. pestis. Symptoms and/or clinical signs associated with conditions referred to herein and the evaluations of such symptoms are routine and known in the art. Treatment that is prophylactic—in this context, treatment that is initiated before a subject manifests symptoms or signs of a condition caused by Y. pestis—is referred to herein as treatment of a subject that is “at risk” of developing the condition. Thus, typically, an animal “at risk” of developing a condition is an animal present in an area where animals having the condition have been diagnosed and/or is likely to be exposed to a Y. pestis causing the condition even if the animal has not yet manifested symptoms or signs of any condition caused by the microbe. For example, an animal at risk includes an animal likely to be exposed to a vector of Y. pestis, such as a flea. Accordingly, administration of a composition can be performed before, during, or after the occurrence of the conditions described herein. Treatment initiated after the development of a condition may result in decreasing the severity of the symptoms of one of the conditions, completely removing the symptoms, or increasing the likelihood of survival.
Kits
Also provided herein are kits for immunizing a subject. The kit includes a composition described herein in a suitable packaging material in an amount sufficient for at least one administration. Optionally, other reagents such as a buffer solution (either prepared or present in its constituent components, where one or more of the components may be premixed or all of the components may be separate), and the like, are also included. Instructions for use of the packaged composition are also typically included.
As used herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. The packaging material has a label which indicates that the composition can be used for immunizing a subject to protect against infection by Y. pestis. In addition, the packaging material contains instructions indicating how the materials within the kit are employed. As used herein, the term “package” refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits a composition described herein. Thus, for example, a package can be a glass vial used to contain appropriate quantities of an attenuated Y. pestis, in an aqueous solution or dried form (e.g., lyophilized).
Previously, it was shown that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection. This Example is also available as Tiner et al. 2015, Infect. Immun., 83:1318-1338.
Introduction
Pathogenic yersiniae lead to two types of diseases: yersiniosis (typified by gastroenteritis caused by Yersinia enterocolitica and Y. pseudotuberculosis) (Galindo et al., 2011, J Pathog 2011:182051) and plague (evoked by Y. pestis) (Perry et al., 1997, Clin Microbiol Rev 10:35-66; Prentice et al., 2007, Lancet 369:1196-1207). Y. pestis has evolved from Y. pseudotuberculosis within the last 20,000 years by acquiring additional plasmids and pathogenicity islands as well as by deactivating some genes (Achtman et al., 2004, Proc Natl Acad Sci USA 101:17837-17842; Achtman et al., 1999, Proc Natl Acad Sci USA 96:14043-14048; Skurnik et al., 2000, Mol Microbiol 37:316-330). This evolutionary adaptation allowed the plague bacterium to maintain a dual life-style in fleas and rodents/mammals and conferred the ability to survive in the blood instead of the intestine (Prentice et al., 2007, Lancet 369:1196-1207). Plague manifests itself in three forms: bubonic (acquired from an infected rodent through a flea bite), pneumonic (acquired either directly by aerosol transmission from an infected host's lungs through respiratory droplets or secondarily from bubonic plague), and septicemic (severe bacteremia either directly due to a flea bite or subsequent to bubonic or pneumonic plague) (Perry et al., 1997, Clin Microbiol Rev 10:35-66). The latter two forms of plague are almost always fatal without treatment or if the administration of antibiotics is delayed (Quenee et al., 2011, Vaccine 29:6572-6583; Centers for Disease Control and Prevention, 17 Nov. 2008, Protecting the American public by ensuring safe and secure possession, use, and transfer of select agents and toxins that pose a threat to public health. CDC Select Agent Program, Centers for Disease Control and Prevention, Atlanta, Ga.). Historically, Y. pestis has been credited for causing three pandemics and >200 million deaths worldwide (Perry et al., 1997, Clin Microbiol Rev 10:35-66). Currently classified as a reemerging pathogen by the World Health Organization, numbers of Y. pestis outbreaks are increasing with current climate changes and shifting of the rodent carrier range (World Health Organization Media Center. 6 Aug. 2009. Plague: questions and answers about plague. World Health Organization, Geneva, Switzerland). Y. pestis is classified as a Tier-1 select agent by the Centers for Disease Control and Prevention (CDC) due to the ease of weaponizing the organism and its associated high mortality rate in humans (Centers for Disease Control and Prevention. 17 Nov. 2008. Protecting the American public by ensuring safe and secure possession, use, and transfer of select agents and toxins that pose a threat to public health. CDC Select Agent Program, Centers for Disease Control and Prevention, Atlanta, Ga.; Inglesby et al., 2000, JAMA 283:2281-2290; Pearson et al., 1998, Biological weapons proliferation: reasons for concern, courses of action. Henry L. Stimson Center, Washington, D.C.).
Braun lipoprotein (Lpp) and lipopolysaccharide (LPS) are the most abundant components of the outer membrane of Gram-negative bacteria in the Enterobacteriaceae family, to which Y. pestis belongs (Glauser et al., 1991, Lancet 338:732-736; Braun et al., 1974, Annu Rev Biochem 43:89-121). Both Lpp and LPS trigger toxic and biological responses in the hosts through the interaction of their lipid domains with Toll-like receptor 2 (TLR-2) and TLR-4, respectively, and by evoking the production of inflammatory cytokines such as tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and interferon gamma (IFN-γ) (Neilsen et al., 2001, J Immunol 167:5231-5239; Aliprantis et al., 1999, Science 285:736-739). Also, the complement and coagulation cascades are activated by both Lpp and LPS, and the production of other damaging inflammatory mediators contributes to the severity of infection (Neilsen et al., 2001, J Immunol 167:5231-5239; Pernerstorfer et al., 1999, Arterioscler Thromb Vasc Biol 19:2517-2523; Jacob et al., 2007, Lab Invest 87:1186-1194; Bashir et al., 2011, Int J Biochem Res Rev 1:1-13).
While Lpp links the peptidoglycan layer to the outer membrane of Y. pestis (Hantke et al., 1973, Eur J Biochem 34:284-296), MsbB is an acyltransferase located in the inner membrane of the bacterial cell wall and catalyzes the addition of lauric acid (C12) to the lipid A moiety of LPS, thus increasing its biological potency (Rebeil et al., 2006, J Bacteriol 188:1381-1388; Clementz et al., 1996, J Biol Chem 271:12095-12102; Clementz et al., 1997, J Biol Chem 272:10353-10360; Somerville et al., 1996, J Clin Invest 97:359-365). Y. pestis synthesizes a rough LPS devoid of the O antigen and exists in different acylated forms depending upon bacterial growth temperatures (Rebeil et al., 2006, J Bacteriol 188:1381-1388; Rebeil et al., 2004, Mol Microbiol 52:1363-1373; Anisimov et al., 2007, J Med Microbiol 56:443-453; Knirel et al., 2005, Biochemistry 44:1731-1743; Pérez-Gutiérrez et al., 2010, Infect Immun 78:2768-2781; Oyston et al., 2003, J Med Microbiol 52:289-294; Montminy et al., 2006, Nat Immunol 7:1066-1073; Kawahara et al., 2002, Infect Immun 70:4092-4098). For example, lipid A of Y. pestis LPS shifts from a hexa-acylated form at 21° C. to 27° C. (flea temperature) to a tetra-acylated form at 37° C. (human temperature), due in part to the inactivity of MsbB at 37° C., which prevents the activation of TLR-4 (Rebeil et al., 2006, J Bacteriol 188:1381-1388; Clementz et al., 1996, J Biol Chem 271:12095-12102; Clementz et al., 1997, J Biol Chem 272:10353-10360; Somerville et al., 1996, J Clin Invest 97:359-365).
Y. pestis must be able to survive in the blood to establish an infection and to increase its chances of transmission, and consequently, the organism must have evolved ways to evade and disarm the host immune system. Ail (attachment-invasion locus), also referred to as OmpX, is a major contributor to serum resistance and complement evasion in Y. pestis (Bartra et al., 2008, Infect Immun 76:612-622; Hinnebusch et al., 2011, Infect Immun 79:4984-4989; Kolodziejek et al., 2010, Infect Immun 78:5233-5243; Kolodziejek et al., 2007, Microbiology 153:2941-2951) and accounts for 20 to 30% of the total outer membrane proteins in yersiniae at 37° C. (Myers-Morales et al., 2007, Appl Environ Microbiol 73:5750-5759; Pieper et al., 2009, Microbiology 155:498-512; Pieper et al., 2009, Proteome Sci 7:5). Ail proteins of Y. enterocolitica and Y. pestis are ˜69% homologous (Kolodziejek et al., 2007, Microbiology 153:2941-2951) and bind, as well as regulate, several mediators of the complement system, e.g., complement protein 4-binding protein (Ho et al., 2012, J Immunol 188:4450-4459; Kirjavainen et al., 2008, PLoS Pathog 4:e1000140; Ho et al., 2014, Eur J Immunol 44:742-751) and complement factor H (FH) (Ho et al., 2012, J Immunol 189:3593-3599; Biedzka-Sarek et al., 2008, Infect Immun 76:4100-4109; Biedzka-Sarek et al., 2008, Infect Immun 76:5016-5027). In addition to serum resistance, Ail of Y. pestis facilitates the adhesion/invasion of bacteria in host cells (Kolodziejek et al., 2010, Infect Immun 78:5233-5243; Kolodziejek et al., 2007, Microbiology 153:2941-2951; Felek et al., 2009, Infect Immun 77:825-836; Tsang et al., 2010, Infect Immun 78:3358-3368; Tsang et al., 2012, J Biol Chem 287:16759-16767), inhibits inflammatory responses (Hinnebusch et al., 2011, Infect Immun 79:4984-4989; Felek et al., 2009, Infect Immun 77:825-836), and assists in the translocation of damaging Yersinia outer membrane proteins (Yops) to host cells (Felek et al., 2009, Infect Immun 77:825-836; Tsang et al., 2010, Infect Immun 78:3358-3368; Yamashita et al., 2011, Structure 19:1672-1682; Felek et al., 2010, Infect Immun 78:4134-4150).
In our previous study, we investigated the effects of the deletion of the lpp and msbB genes on the pathogenesis of a highly virulent Y. pestis CO92 strain (Sha et al., 2013, Infect Immun 81:815-828). Both Δlpp single and Δlpp ΔmsbB double mutants exhibited significant attenuation (70 to 100%) compared to the wild-type (WT) bacterium in pneumonic and bubonic plague mouse models at a dose of 3 50% lethal doses (LD50s) (Sha et al., 2013, Infect Immun 81:815-828). Importantly, only animals initially challenged with the double mutant in a pneumonic plague model were significantly protected (55%) upon subsequent pneumonic infection with 10 LD50s of WT CO92 (Sha et al., 2013, Infect Immun 81:815-828). The attenuated phenotype of the Δlpp ΔmsbB double mutant in mouse models correlated with its reduced survivability in murine RAW 264.7 macrophages (Sha et al., 2013, Infect Immun 81:815-828). Furthermore, the Δlpp ΔmsbB double mutant evoked reduced levels of inflammatory cytokines compared to those induced by the WT bacterium in a pneumonic plague mouse model, which coincided with overall decreased dissemination of the mutant to the peripheral organs of mice (Sha et al., 2013, Infect Immun 81:815-828). However, it is important to mention that while the Δlpp ΔmsbB double mutant was much more impaired in its ability to disseminate than the Δlpp single mutant, substantial numbers of the double mutant bacteria were still detected at the initial infection site (lungs) in some mice at 3 days postinfection (p.i.) (Sha et al., 2013, Infect Immun 81:815-828). Similarly, the Δlpp ΔmsbB double mutant persisted in the spleen of mice by day 6 p.i. when animals were challenged by the subcutaneous route (Sha et al., 2013, Infect Immun 81:815-828), suggesting the need to delete an additional virulence factor-encoding gene(s) from this Δlpp ΔmsbB double mutant to increase attenuation.
It has been reported that the virulence potential of Ail is modulated by the LPS core saccharide length and that Ail's biological activity could be masked by LPS (Kolodziejek et al., 2010, Infect Immun 78:5233-5243). However, since the LPS of Y. pestis lacks 0 antigen, Ail is believed to contribute significantly to the pathogenesis of Y. pestis infections (Skurnik et al., 2000, Mol Microbiol 37:316-330). Indeed, a recent study by Kolodziejek et al. showed that Ail of Y. pestis CO92 contributed to virulence in a rat model of pneumonic plague (Kolodziejek et al., 2010, Infect Immun 78:5233-5243). Thus, we aimed to determine whether a deletion of the ail gene from WT CO92 or its Δlpp single and Δlpp ΔmsbB double mutants would further attenuate the bacterium. Our data showed that the Δlpp ΔmsbB Δail triple mutant of Y. pestis CO92 was severely attenuated while retaining immunogenicity.
Materials and Methods
Bacterial strains and plasmids. All bacterial strains and plasmids used in this study are listed in Table 1. Y. pestis was grown in heart infusion broth (HIB) (Difco, Voigt Global Distribution Inc., Lawrence, Kans.) at 28° C. with constant agitation (180 rpm). On the solid surface, Y. pestis was grown on either HIB agar or 5% sheep blood agar (SBA) plates (Teknova, Hollister, Calif.). Luria-Bertani (LB) medium was used for growing recombinant Escherichia coli at 37° C. with agitation. Strains containing plasmid pBR322 or its tetracycline-sensitive (Tcs) variant were grown in media with the addition of 100 μg/ml ampicillin. All of our studies were performed in a Tier-1 select-agent facility within the Galveston National Laboratory (GNL), University of Texas Medical Branch (UTMB). Restriction endonucleases and T4 DNA ligase were obtained from Promega (Madison, Wis.). Advantage cDNA PCR kits were purchased from Clontech (Palo Alto, Calif.), and all digested plasmid DNA or DNA fragments from agarose gels were purified by using QIAquick kits (Qiagen Inc., Valencia, Calif.). All tissue culture cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, Va.).
Y. pestis CO92
E. coli K-12 DH5α
Deletion of the all gene. The up- and downstream DNA sequences flanking the all gene were PCR amplified by using primer pairs Aup5-Aup3 and Adn5-Adn3 (Table 2), respectively, with genomic DNA of Y. pestis CO92 as the template. Additionally, primer pair Km5-Km3 (Table 2), specific for plasmid pKD13, was used to amplify the kanamycin resistance (Kmr) gene cassette with flippase (FLP) recombinase recognition sites (Choi et al., 2005, BMC Microbiol 5:30; Datsenko et al., 2000, Proc Natl Acad Sci USA 97:6640-6645). The upstream DNA fragment flanking the ail gene, the Kmr gene cassette, and the downstream DNA fragment flanking the ail gene were ligated in that order by using appropriate restriction enzyme sites and cloned into the pDMS197 suicide vector (Edwards et al., 1998, Gene 207:149-157). The resulting recombinant plasmid, pDMS197-Δail (Table 1), was then transformed into the WT strain, the Δlpp single mutant, and the Δlpp ΔmsbB double mutant of Y. pestis CO92 via electroporation (Genepulser Xcell; Bio-Rad, Hercules, Calif.). Transformants were plated onto LB agar plates containing 5% sucrose and 100 μg/ml kanamycin, and Kmr colonies were screened by using PCR to ensure genomic replacement of the ail gene with the antibiotic-resistant cassette. The correct clones were retransformed with plasmid pFlp2, which contains the FLP recombinase, to remove the Kmr gene cassette. Plasmid pFlp2 was eventually cured by growing colonies on 5% sucrose (Sha et al., 2013, Infect Immun 81:815-828), leading to the generation of the single (Δail), double (Δlpp Δail), and triple (Δlpp ΔmsbB Δail) mutants. Subsequent PCR analysis with primer pairs (Up5-Dn3 and Ail5-Ail3) and genomic sequencing with primer SqAil (Table 2) further confirmed the in-frame deletion of the ail gene from all three mutant strains.
Y. pestis CO92
Y. pestis CO92
pestis CO92 including its promoter for
Complementation of the Δail mutant strains of Y. pestis CO92. By using primers Apbr5-Apbr3 (Table 2), the coding region of the ail gene with its promoter was PCR amplified with genomic DNA of WT CO92 as the template. The amplified DNA fragment was cloned into the pBR322 vector, creating recombinant plasmid pBR322-ail (Table 1). Through electroporation, plasmid pBR322-ail was transformed into the Δail single and Δlpp ΔmsbB Δail triple mutant strains, resulting in the creation of the complemented Δail:pBR322-ail and Δlpp ΔmsbB Δail:pBR322-ail Y. pestis strains (Table 1). These complemented strains were sensitive to tetracycline due to the replacement of a large portion of the tetracycline resistance (Tcr) cassette from plasmid pBR322 with the ail gene. The Tcs variant of the pBR322 vector (Galindo et al., 2010, Comp Funct Genomics 2010:342168) without the ail gene was also electroporated into WT CO92 and the Δail single, Δlpp ΔmsbB double, and Δlpp ΔmsbB Δail triple mutants for generating empty vector controls (e.g., WT:pBR322, Δail:pBR322, Δlpp ΔmsbB:pBR322, and Δlpp ΔmsbB Δail:pBR322, respectively) (Table 1).
Absence of Ail and unchanged levels of Lpp in the membranes of Y. pestis CO92 Δail mutants. The generated mutant strains were grown overnight in HIB medium at 28° C. with shaking at 180 rpm, and the resulting bacterial cell pellets (representing similar CFU) were dissolved by boiling in SDS-PAGE sample buffer. An aliquot of the samples was then analyzed by immunoblotting using polyclonal antibodies to Ail and monoclonal antibodies to Lpp that were available in the laboratory (Erova et al., 2013, Clin Vaccine Immunol 20:227-238; Sha et al., 2008, Infect Immun 76:1390-1409). As a loading control for the Western blots, the presence of DnaK in the bacterial pellets of the mutants and WT CO92 was assessed by using anti-DnaK monoclonal antibodies (Enzo, Farmingdale, N.Y.).
Growth kinetics and membrane alteration of the Y. pestis CO92 triple mutant.WT CO92 and its Δlpp ΔmsbB Δail triple mutant were grown in 100 ml of HIB medium contained in 500-ml HEPA filter Top polycarbonate Erlenmeyer culture flasks (Triforest Labware, Irvine, Calif.) at 28° C. with constant shaking (180 rpm). Samples from each flask were taken at 1- to 2-h intervals until the cultures reached their plateau phases. CFU were determined by plating (Sha et al., 2011, J Clin Microbiol 49:1708-1715). For visualization of membrane alterations, bacterial strains were grown to exponential phase at 28° C. (optical density at 600 nm [OD600] of 0.6). The cells were washed, pelleted, fixed, and subjected to transmission electron microscopy (van Lier et al., 2014, Infect Immun 82:2485-2503).
Sensitivity of the Y. pestis CO92 mutants to gentamicin. The MICs of gentamicin against WT Y. pestis CO92:pBR322 and the Δail:pBR322, Δail::pBR322-ail, Δlpp ΔmsbB:pBR322, Δlpp ΔmsbB Δail:pBR322, and Δlpp ΔmsbB Δail:pBR322-ail mutants were determined by an E-test (bioMérieux Inc., Durham, N.C.) (Sha et al., 2013, Infect Immun 81:815-828). Briefly, the bacterial cultures were spread evenly onto 5% SBA and LB agar plates, and predefined gentamicin (range, 0.016 to 256 μg/ml) E-test strips were placed onto the plates. The plates were incubated for 48 h at 28° C., and the MICs were recorded.
Evaluation of essential Y. pestis virulence factors in various mutants of Y. pestis CO92. The intactness and functionality of the type 3 secretion system (T3SS), crucial for plague pathogenesis and immunity, were then evaluated. Through the T3 SS, the plague bacterium secretes Yops such as YopE, YopH, and LcrV (low-calcium-response V antigen) in response to a low-calcium signal. Consequently, WT CO92 and Δail single, Δlpp ΔmsbB double, and Δlpp ΔmsbB Δail triple mutant cultures grown overnight were diluted 1:20 and grown in either HIB or calcium-depleted modified M9 medium (42 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 18.6 mM NH4Cl, 0.001 mg/ml FeSO4, 0.0001% thiamine, 1 mM MgSO4, 0.4% dextrose, and 1% Casamino Acids) at 28° C. with shaking (180 rpm) for 3 h and then at 37° C. for 2 h.
When the bacteria were grown in HIB, 5 mM EGTA (Sigma-Aldrich, St. Louis, Mo.) was added to trigger the low-calcium response 5 min before harvesting of the cultures. Aliquots of the cultures grown in either medium (representing similar CFU) were removed, and 1 ml of the supernatants was precipitated with 55 ρl of 100% trichloroacetic acid (TCA) on ice for 2 h. The TCA precipitates were dissolved in SDS-PAGE buffer and analyzed by immunoblotting with antibodies to YopE, LcrV (Santa Cruz Biotechnology, Santa Cruz, Calif.), and YopH (Agrisera, Stockholm, Sweden) (Sha et al., 2013, Infect Immun 81:815-828; Sha et al., 2008, Infect Immun 76:1390-1409; van Lier et al., 2014, Infect Immun 82:2485-2503). The anti-DnaK monoclonal antibody (Enzo) was employed to probe bacterial pellets to ensure that the bacterial supernatants were obtained from similar numbers of bacteria across the tested strains.
To evaluate the translocation of T3SS effectors by the Y. pestis mutants, a digitonin extraction assay was used (Sha et al., 2008, Infect Immun 76:1390-1409). Briefly, Y. pestis cultures grown overnight in HIB were diluted in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS). The diluted bacteria were cultivated at 28° C. for 30 min and then at 37° C. for 60 min. HeLa cells (in a 12-well plate) were then infected with the above-described Y. pestis cultures at a multiplicity of infection (MOI) of 30. After 4 h of infection at 37° C., the cells were washed twice with phosphate-buffered saline (PBS) and lysed with 200 μl of digitonin (1% in PBS). The cells were dislodged from the surface of the plate, collected, and centrifuged at 13,000 rpm for 5 min to obtain the supernatant and the pellet fractions. The YopE polyclonal antibodies were then used to detect YopE in both the fractions, while anti-actin (Santa Cruz Biotechnology) and anti-DnaK monoclonal antibodies were employed for the supernatant and pellet fractions, respectively, to monitor equivalent sample loading during Western blot analyses.
For examining capsular antigen (F1) production by WT CO92 and its triple mutant, bacteria grown at 37° C. were subjected to a commercially available plague detection kit, the Yersinia pestis (F1) Tetracore RedLine Alert kit (Tetracore, Rockville, Md.), as we previously described (Sha et al., 2011, J Clin Microbiol 49:1708-1715). A Δcaf1-negative mutant of CO92 devoid of F1 antigen was used as a control (Sha et al., 2011, J Clin Microbiol 49:1708-1715). We further analyzed F1 production by WT CO92 and its triple mutant by flow cytometry (Sha et al., 2011, J Clin Microbiol 49:1708-1715). Briefly, the above-mentioned Y. pestis cultures (106 CFU/sample) were fixed with 4% paraformaldehyde. After being washed with PBS, the bacteria were incubated with a primary antibody to the F1 antigen (Santa Cruz Biotechnology), followed by a secondary antibody (goat anti-mouse IgG1) conjugated with Alexa Fluor 488. The bacterial cells were then washed and resuspended in 500 μl of fluorescence-activated cell sorter (FACS) buffer (1% FBS in PBS) before analysis. Samples were read on an LSRII Fortessa instrument (UTMB Core Facility) and analyzed with FlowJo software. We also examined F1 production by WT CO92, the Δcaf1-negative mutant, and the Δlpp ΔmsbB Δail triple mutant by immunofluorescence (IF) staining using anti-F1 antibodies and microscopy (Sha et al., 2011, J Clin Microbiol 49:1708-1715).
The Pla (plasminogen activator) surface protease of Y. pestis is a multifunctional protein that contributes to bacterial adherence to host cells, intracellular survival, complement resistance, and bacterial dissemination by virtue of possessing fibrinolytic and coagulase activities (Felek et al., 2010, Infect Immun 78:4134-4150; van Lier et al., 2014, Infect Immun 82:2485-2503; Sebbane et al., 2006, Proc Natl Acad Sci USA 103:11766-11771; Sodeinde et al., 1992, Science 258:1004-1007; Agar et al., 2009, Microbiology 155:3247-3259; Cowan et al., 2000, Infect Immun 68:4523-4530; Korhonen et al., 2013, Front Cell Infect Microbiol 3:35; Caulfield et al., 2014, Cell Host Microbe 15:424-434). To examine whether the deletion of three membrane protein-encoding genes (lpp, msbB, and ail) from WT CO92 altered Pla levels, we performed Western blot analysis. The various Y. pestis strains grown overnight were diluted 1:20 in fresh HIB and grown at 28° C. with shaking (180 rpm) for 3 h and then at 37° C. for 2 h. The bacterial cell pellets were harvested and dissolved in SDS-PAGE sample buffer. An aliquot of the samples was then analyzed by immunoblotting using polyclonal antibodies to Pla that were available in the laboratory (Erova et al., 2013, Clin Vaccine Immunol 20:227-238). The anti-DnaK monoclonal antibody was also employed to monitor equivalent sample loading during Western blot analyses.
To ensure that Pla was properly displayed on the bacterial surface and enzymatically active, we measured its protease activity by using a fluorimetric assay with the Pla substrate (Agarkov et al., 2008, Bioorg Med Chem Lett 18:427-431). Briefly, all of the tested strains were plated onto HIB agar plates at 28° C. for 36 h. The strains were then replated onto fresh HIB agar plates and incubated at either 28° C. or 37° C. (representing flea and human body temperatures, respectively) for 20 to 22 h. Bacteria from each plate were suspended in PBS and adjusted to optical densities (OD600) of 0.1 (5×107 CFU/ml) and 0.5 (2.5×108 CFU/ml) by using a Bio-Rad SmartSpec 300 instrument. For each strain, 50 μl of the suspension was added to the wells of a black microtiter plate (Costar Corning Inc., Corning, N.Y.) in triplicate. The hexapeptide substrate 4-{[4′-(dimethylamino)phenyl]azo}benzoic acid (DABCYL)-Arg-Arg-Ile-Asn-Arg-Glu{5-[(2′-aminoethyl)amino]naphthalene sulphonic acid (EDANS)}-NH2, at a final concentration of 2.5 μg/well, was added to the bacterial cells. The kinetics of substrate cleavage by Pla displayed on the bacterial surface was measured every 15 min for 2 h by a fluorimetric assay (extinction/emission wavelength of 360 nm/460 nm) at 37° C. by using a BioTek Synergy HT spectrophotometer (BioTek Instruments Inc., Winooski, Vt.).
Animal studies with the Y. pestis CO92 mutant strains. Six- to eight-week-old female Swiss Webster mice (17 to 20 g) were purchased from Taconic Laboratories (Germantown, N.Y.). All of the animal studies were performed in an animal biosafety level 3 (ABSL-3) facility under an approved UTMB Institutional Animal Care and Use Committee protocol. Mice were challenged intranasally (i.n.) with 1.3×104 CFU (representing 26 50% lethal doses [LD50s] of the WT bacterium, with 1 LD50 corresponding to 500 CFU [van Lier et al., 2014, Infect Immun 82:2485-2503]) of WT CO92 or the Δail single, Δlpp ΔmsbB or Δlpp Δail double, or Δlpp ΔmsbB Δail triple mutant strain. Also, the animals were challenged by the i.n. route with the Δlpp ΔmsbB Δail triple mutant at higher doses of 4.0×104, 1.7×105, 5.9×105, 1.8×106, and 3.4×106 CFU (corresponding to 80, 340, 1,180, 3,600, and 6,800 LD50s of WT CO92). Mice were assessed for morbidity and/or mortality as well as clinical symptoms over the duration of each experiment (24 to 30 days p.i.).
Survivors after initial infection with the mutants were subsequently challenged with 1×104 to 1.4×104 CFU (20 to 28 LD50s) of the bioluminescent WT Y. pestis CO92 luc2 strain (Sha et al., 2013, Microb Pathog 55:39-50), which contains the luciferase (luc) gene and its substrate, allowing in vivo imaging of mice in terms of bacterial dissemination in real time. Naive mice of the same age and infected with the WT CO92 luc2 strain were used as controls. On day 3 and/or day 7 p.i., the animals were imaged by using an Ivis 200 bioluminescence and fluorescence whole-body imaging workstation (Caliper Corp., Alameda, Calif.) in the ABSL-3 facility.
Bacterial dissemination and histopathological studies of the triple mutant of Y. pestis CO92. Mice infected with 2.5×106 CFU (representing 5,000 LD50s of WT CO92) of the Δlpp ΔmsbB Δail triple mutant or WT CO92 by the i.n. route were euthanized by using a mixture of ketamine and xylazine, followed by cervical dislocation on days 2, 3, and 6 p.i. For each time point, five mice per group were used, and the lungs, liver, and spleen were removed immediately following animal sacrifice. Blood was collected from these animals by cardiac puncture. The tissues were homogenized in 1 ml of PBS, and serial dilutions of the homogenates were spread onto SBA plates to assess dissemination of the bacteria to peripheral organs (Sha et al., 2013, Infect Immun 81:815-828).
Portions of each organ (lung, liver, and spleen) from 5 mice at each time point were also removed and immersion fixed in 10% neutral buffered formalin (Sha et al., 2008, Infect Immun 76:1390-1409; Agar et al., 2009, Microbiology 155:3247-3259). The tissues were processed and sectioned at 5 μm, and the samples were mounted onto slides and stained with hematoxylin and eosin (H&E). Tissue lesions were scored on the basis of a severity scale, which correlated with estimates of lesion distribution and the extent of tissue involvement (minimal, 2 to 10%; mild, >10 to 20%; moderate, >20 to 50%; severe, >50%), as previously described (Sha et al., 2008, Infect Immun 76:1390-1409; Agar et al., 2009, Microbiology 155:3247-3259). The histopathological evaluation of the tissue sections was performed in a blind fashion.
Cytokine and chemokine levels and antibody responses in mice infected with the triple mutant of Y. pestis CO92. Concurrently, blood was collected from infected (with WT CO92 versus the Δlpp ΔmsbB Δail triple mutant) animals on days 2, 3, and 6 p.i. (for cytokine analysis). Blood was also collected from all animals prior to infection and on day 14 p.i. to determine antibody responses. Serum samples were filtered by using Costar 0.1-μm centrifuge tube filters (Corning Inc.). The levels of cytokines/chemokines, namely, IL-12, IFN-γ, IL-4, IL-5, IL-6, and TNF-α, in sterile serum samples were analyzed by using a mouse 6-plex Bioplex assay (eBioscience, San Diego, Calif.).
Total levels of IgG and antibody isotypes in the sera of animals infected with the Δlpp ΔmsbB Δail triple mutant were determined at 14 days p.i. by an enzyme-linked immunosorbent assay (ELISA). Briefly, ELISA plates were coated with either the F1-V fusion protein (1 ng/ml; BEI Resources, Manassas, Va.) or whole bacterial cells of WT CO92 overnight at 4° C. For the whole-bacterial-cell ELISA, microtiter plates were first coated with poly-L-lysine (10 μg/ml), as we previously described (Tao et al., 2013, PLoS Pathog 9:e1003495).
The sera were serially diluted (either 1:5 or 1:10), and horseradish peroxidase (HRP)-conjugated secondary antibodies were used to determine total IgG titers and IgG isotype responses by employing goat anti-mouse IgG-HRP, IgG1-HRP, IgG2a-HRP, and IgG2b-HRP (SouthernBiotech, Birmingham, Ala.). The substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (ThermoScientific, Waltham, Mass.) was used for color development, and the plates were read at 450 nm by using a spectrophotometer (Tao et al., 2013, PLoS Pathog 9:e1003495).
Serum resistance of various mutants of Y. pestis CO92. Normal human and mouse sera were purchased from Sigma-Aldrich, and nonhuman primate (NHP) sera were collected from naive animals that were housed at the GNL. Prior to use, an aliquot of each serum sample was also heated at 56° C. for 30 min to inactivate complement and served as a control. WT CO92:pBR322, Δail:pBR322, Δlpp ΔmsbB:pBR322, Δlpp ΔmsbB Δail:pBR322, Δail:pBR322-ail, and Δlpp ΔmsbB Δail:pBR322-ail strains were grown overnight, harvested, and then diluted in PBS to an OD600 of 0.8 (˜4×108 CFU/ml). A 50-μl volume of the diluted bacteria (˜2×107 CFU) was mixed with 200 μl of either normal (unheated) or heated sera. The samples were incubated at 37° C. for 2 h with shaking at 500 rpm. The number of surviving bacteria (CFU) in each sample was determined by serial dilutions and plating onto SBA plates (Sha et al., 2013, Infect Immun 81:815-828; van Lier et al., 2014, Infect Immun 82:2485-2503). Percent bacterial survival was calculated by dividing the average number of CFU in samples incubated in normal serum by the average number of CFU in samples incubated in heat-inactivated serum and multiplying by 100.
Adherence, invasion, and intracellular survival of various Y. pestis CO92 mutants in HeLa and A549 epithelial cells. Twelve-well tissue culture plates were seeded with either HeLa (from human cervix) or A549 (human alveolar) epithelial cells at a concentration of 4×105 cells in 1 ml of DMEM, 10% FBS (HeLa) or F-12K (Kaighn's) medium, and 10% FBS (A549) (Cowan et al., 2000, Infect Immun 68:4523-4530; Liu et al., 2006, Infect Immun 74:5636-5644). The cells were incubated at 37° C. in 5% CO2 until a confluent monolayer was established.
Bacterial strains grown overnight were used to infect host cells at an MOI of 100. The plates were centrifuged at 1,200 rpm for 10 min to ensure bacterial contact with the host cells. After 2 h of incubation, one set of the triplicate wells was not washed, and the total numbers of bacteria used for infection that were present in the culture medium and those adhering to and/or invading the host cells were recovered by scraping and vortexing the host cells. Another set of triplicate wells was gently washed twice with 1 ml of Dulbecco phosphate-buffered saline (DPBS), and the adherent and invading bacteria in the host cells were then enumerated after lysing epithelial cells with 1 ml of ice-cold water.
The last set of the host cells was similarly washed twice with DPBS, and a gentamicin (50 μg/ml) protection assay was used to discriminate between invading and extracellular bacteria (Cowan et al., 2000, Infect Immun 68:4523-4530). After 1 h of incubation in gentamicin-containing medium to kill extracellular bacteria, the host cells were washed twice with 1 ml of DPBS, and intracellular bacteria were then enumerated after the addition of 1 ml of ice-cold water to each well (Cowan et al., 2000, Infect Immun 68:4523-4530). The percentages of invasion and adhesion were then determined.
The intracellular survival of various CO92 mutants in HeLa and A549 cells was assessed in a manner similar to that described above for the invasion assay. The 0-h samples corresponded to a time point immediately after gentamicin treatment. The intracellular bacteria in HeLa and A549 cells were then enumerated by serial dilution and plating after 12 h of incubation in medium containing 10 μg/ml gentamicin (Sha et al., 2008, Infect Immun 76:1390-1409).
Survival of WT Y. pestis CO92 and its mutant strains in murine alveolar macrophages and human monocyte-derived macrophages and production of cytokines. Murine MH-S alveolar macrophages were infected with WT CO92 and its mutant strains at an MOI of 10. After 30 min of infection, the host cells were treated for 45 min with 20 μg/ml gentamicin to kill extracellular bacteria. The surviving bacteria inside the macrophages were then enumerated immediately after gentamicin treatment (0-h time point) and subsequently at 2 and 4 h of incubation in medium containing 10 μg/ml gentamicin. The number of bacteria present inside the macrophages was determined by serial dilution and plating (Sha et al., 2008, Infect Immun 76:1390-1409).
Human buffy coats were obtained from three different healthy individuals in 10-ml Vacutainer tubes without additive (Becton Dickinson Labware, Franklin Lakes, N.J.) from the UTMB blood bank. The EDTA-treated blood was handled under endotoxin-free conditions and diluted 1:1 with PBS, and peripheral blood mononuclear cells (PBMCs) were purified by centrifugation over a Ficoll-sodium diatrizoate solution (Ficoll-Paque Plus; GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Monocytes were then purified from PBMCs by positive selection using human CD14 microbeads and a magnetic column separation system from Miltenyi Biotec (Auburn, Calif.). Monocyte-derived macrophages were subsequently differentiated from purified CD14+ monocytes.
Briefly, monocytes were cultured in RPMI 1640 medium supplemented with 10% FBS, 1-glutamine, HEPES, sodium pyruvate, penicillin-streptomycin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (100 ng/ml) (Leukine [sargramostim]; Genzyme Corp., Cambridge, Mass.). Monocytes were seeded into 24-well tissue culture plates at 106 cells/ml, and adherent monocyte-derived macrophages were obtained at 6 days of culture.
These human monocyte-derived macrophages (HMDMs) were infected with WT CO92 and its mutant strains at an MOI of 1. The infected macrophages were incubated at 37° C. with 5% CO2 for 45 min, followed by 1 h of treatment with 10 μg/ml gentamicin. The surviving bacteria inside the macrophages were enumerated immediately after gentamicin treatment (0-h time point) and subsequently at 2 h and 4 h (Sha et al., 2008, Infect Immun 76:1390-1409). The concentration of gentamicin used in the gentamicin protection assay was optimized for each host cell type used in this study.
Supernatants from infected macrophages during the intracellular survival assay were collected at each of the time points tested and filtered. A Bio-Rad mouse 6-plex assay kit (IL-1β, IFN-γ, IL-10, IL-17, IL-6, and TNF-α) or a Bio-Rad human 8-plex assay kit (GM-CSF, IFN-γ, IL-2, IL-4, IL-6, IL-8, IL-10, and TNF-α) was used to measure cytokine and chemokine levels.
Statistical analysis. For the majority of the experiments, one-way analysis of variance (ANOVA) was used with the Bonferroni correction, except for the intracellular survival experiments, in which Tukey's post hoc test was employed for data analysis. We used Kaplan-Meier survival estimates for animal studies, and P values of <0.05 were considered significant for all of the statistical tests used.
Results
In vitro characterization of Δail mutants of Y. pestis CO92. The in-frame deletion of the ail gene from WT CO92 and the Δlpp single and Δlpp ΔmsbB double mutants of Y. pestis CO92 was confirmed by PCR analysis using specific primers (Table 2) as well as by DNA sequencing of the flanking regions of the ail gene on the chromosome. The above-mentioned genetic manipulations resulted in the creation of authentic Δail single, Δlpp Δail double, and Δlpp ΔmsbB Δail triple mutants of Y. pestis CO92. As shown in
Since both Ail and Lpp are outer membrane proteins and the MsbB acyltransferase modifies LPS, WT CO92 and its Δlpp ΔmsbB Δail triple mutant were subjected to transmission electron microscopy to evaluate if there were any membrane alterations. Except for finding that the Δlpp ΔmsbB Δail triple mutant had somewhat decreased periplasmic space compared to that of WT CO92 (
The Δlpp ΔmsbB Δail triple mutant of Y. pestis CO92 produces essential Y. pestis virulence and immunogenic factors. The T3 SS is an essential virulence mechanism in Y. pestis. Through the T3 SS, an immunoreactive antigen (LcrV) as well as other Yops such as YopE and YopH (which destroy actin monofilaments and interfere with phagocyte signal transduction machinery, respectively) are secreted. Therefore, T3 SS-dependent protein secretion in response to a low-calcium signal was measured for the generated ail mutants. This in vitro assay mimics the environment during eukaryotic host cell contact with the bacterial T3SS needles. In calcium-depleted M9 medium, the Δlpp ΔmsbB Δail triple mutant showed significantly increased levels of YopH and YopE in the culture supernatants compared to those of WT CO92 and its Δlpp ΔmsbB double mutant (
To demonstrate that the higher level of the specific effector YopH observed in the culture supernatant of the Δlpp ΔmsbB Δail triple mutant (
To simulate in vivo conditions and to measure the translocation of Yops, HeLa cells were infected with various mutant strains of Y. pestis. A digitonin extraction assay was used to evaluate the translocation of YopE into the host cells. While WT CO92 and Δlpp ΔmsbB mutant bacteria had similar levels of YopE translocation, the ail deletion mutants (both Δail and Δlpp ΔmsbB Δail) had significantly decreased translocation of YopE into the cytosol of the host cells (
Pla, another important virulence factor of Y. pestis, is a multifunctional protein (Felek et al., 2010, Infect Immun 78:4134-4150; van Lier et al., 2014, Infect Immun 82:2485-2503; Sebbane et al., 2006, Proc Natl Acad Sci USA 103:11766-11771; Sodeinde et al., 1992, Science 258:1004-1007; Agar et al., 2009, Microbiology 155:3247-3259; Cowan et al., 2000, Infect Immun 68:4523-4530). To evaluate whether the levels of Pla remained unaltered in the Δail mutants, Western blot analysis was performed. Essentially, similar levels of the Pla protein were noted for the various mutant strains tested (Δail single, Δlpp ΔmsbB double, Δlpp ΔmsbB Δail triple, as well as ail-complemented mutant strains) compared to that of WT CO92 at 37° C., a temperature that increases Pla production and activity (Pieper et al., 2009, Microbiology 155:498-512; Chromy et al. 2005 J Bacteriol 187:8172-8180; Suomalainen et al., 2010, Infect Immun 78:2644-2652) (
Not only is the capsular antigen (F1) a major immunoreactive protein of Y. pestis, it also exhibits antiphagocytic properties (Du et al., 2002, Infect Immun 70:1453-1460). Thus, F1 production was verified in WT CO92 versus its triple mutant by using a commercially available plague immunochromatographic dipstick (impregnated with F1 antibodies) test, which allows rapid, in vitro qualitative identification of Y. pestis. Both WT CO92 and its Δlpp ΔmsbB Δail triple mutant produced purple bands in the reaction and control lanes of similar intensities, whereas the Δcaf1 mutant (negative control) was positive only in the control lane (
Evaluation of Y. pestis CO92 Δail mutants in a pneumonic plague mouse model. To gauge the virulence potential of the Δail mutant strains, mice (n=10/group) were infected by the i.n. route with similar doses (1.3×104 CFU, representing 26 LD50s of the WT bacterium) of the Δail single, Δlpp Δail or Δlpp ΔmsbB double, or Δlpp ΔmsbB Δail triple mutant strain as well as WT CO92. While animals inoculated with WT CO92 died by day 4 p.i., all of the mice infected with the Δail single mutant died by day 10 p.i., showing an increased mean time to death (
To evaluate the specific immunity to Y. pestis that developed, sera from all of the surviving mice infected with various mutants of CO92 (
The extent of attenuation of the virulence potential of the Δlpp ΔmsbB Δail triple mutant was then ascertained by infecting mice by the i.n. route with increasing doses ranging from 4.0×104 to 5.9×105 CFU, representing 80 to 1,180 LD50s of the WT bacterium (
To further assess the specific immunity induced in mice after initial infection with the Δlpp ΔmsbB Δail triple mutant strain (
The surviving mice were imaged on day 3 p.i. by using an in vivo imaging system, and the first animal on the left in each imaging panel was uninfected and served as a control (
In our subsequent experiment, initial infection doses of the Δlpp ΔmsbB Δail triple mutant given to the mice i.n. were increased to 1.8×106 and 3.4×106 CFU, which corresponded to 3,600 and 6,800 LD50s of WT CO92, respectively. As shown in
As expected, protection levels increased to 70% when the animals were initially infected with the Δlpp ΔmsbB Δail triple mutant at a dose of 3.4×106 CFU (6,800 LD50s of WT CO92) and then challenged i.n. on day 24 with a higher dose (28 LD50s or 1.4×104 CFU) of the WT CO92 luc2 strain (
Bacterial dissemination and histopathological lesions in mice challenged with the Δlpp ΔmsbB Δail triple mutant of Y. pestis CO92 by the intranasal route. Mice were challenged with 2.5×106 CFU of either WT CO92 (5,000 LD50s) or its Δlpp ΔmsbB Δail triple mutant. Animals were sacrificed on days 2, 3, and 6 p.i., and their lungs, liver, spleen, and blood were harvested and subjected to bacterial load determination. Mice challenged with WT bacteria had a high bacterial load in each of these organs on both day 2 (ranging from 1.1×107 to 1.7×109 CFU/organ) and day 3 (1.3×109 to 2.8×109 CFU/organ). No data were collected on day 6 since all of the mice succumbed to infection within 80 h. Animals challenged with the Δlpp ΔmsbB Δail triple mutant had minimal to no bacterial load in the organs examined, except for the lungs on day 2 (ranging from 2.8×102 to 1.9×103 CFU/organ) (
Organs from the infected mice were also removed for histopathological analysis. At between 48 and 60 h p.i., two of the five WT bacterium-infected mice succumbed to infection, and their organs were not harvested. The remaining three mice succumbed to infection at between 60 and 72 h p.i., and their lungs had mild to moderate neutrophilic inflammation (
All of the livers of WT-infected animals had bacteria (
The Δlpp ΔmsbB Δail triple mutant of Y. pestis CO92 evokes reduced levels of inflammatory cytokines in a pneumonic plague mouse model. Samples of the lung homogenates and sera collected from the above-mentioned infected animals were assessed for cytokine production by using an eBioscience 6-plex Bioplex assay. There was a statistically significant difference in the presence of TNF-α, IFN-γ, and IL-6 in both the lungs and the sera between the WT- and the Δlpp ΔmsbB Δail triple mutant-infected animals on both days 2 and 3 p.i. (
Δail mutants of Y. pestis CO92 have host-dependent serum sensitivities. Ail has been reported to function in providing serum resistance to Y. pestis (Bartra et al., 2008, Infect Immun 76:612-622; Hinnebusch et al., 2011, Infect Immun 79:4984-4989; Kolodziejek et al., 2010, Infect Immun 78:5233-5243; Kolodziejek et al., 2007, Microbiology 153:2941-2951). Consequently, WT CO92 and its Δail single, Δlpp ΔmsbB double, and Δlpp ΔmsbB Δail triple mutants were tested for their ability to be killed by the complement cascade. As shown in
To confirm that Ail is responsible for this phenotype, the complemented strains (Δail:pBR322-ail and Δlpp ΔmsbB Δail:pBR322-ail) were exposed to normal (unheated) NHP and human sera. Both of these strains exhibited ≥100% survival (
Decreased adherence and invasion of Y. pestis CO92 Δail mutants in epithelial cells. Since Ail functions in the adherence and subsequent invasion of bacteria in host cells, these virulence phenotypes of WT CO92 and its various mutants were first examined in HeLa cells at an MOI of 100 (
Since Y. pestis infects the lungs during pneumonic plague, bacterial adherence to and invasion of A549 human alveolar epithelial cells were then examined to mimic a natural infection scenario. As with the HeLa cell infection model, both the Δail single mutant and the Δlpp ΔmsbB Δail triple mutant had similar significantly decreased rates of adherence (34% and 10%, respectively) (
Host-dependent survivability of Y. pestis CO92 Δail mutants in murine and human macrophages and epithelial cells. To determine the role of Ail in intracellular survival within macrophages, MH-S murine alveolar macrophages were infected with WT CO92 or the Δail single, Δlpp ΔmsbB double, or Δlpp ΔmsbB Δail triple mutant at an MOI of 10. The macrophages showed an increased uptake of the Δlpp ΔmsbB Δail triple mutant (29%) compared to WT CO92 and the Δail single mutant strain (12% and 13%, respectively) (data not shown). At 2 h p.i., 46% of the Δlpp ΔmsbB double mutant and 39% of the Δlpp ΔmsbB Δail triple mutant cells survived in MH-S cells, compared to 77% of WT CO92 cells (
To further investigate the intracellular survivability of mutant bacteria, human monocyte-derived macrophages (HMDMs) were used. The Δlpp ΔmsbB Δail triple mutant strain was most impaired in survival intracellularly (18%) in HMDMs compared to the Δlpp ΔmsbB double mutant (25%) and WT CO92 (38%) at 2 h p.i. (
We then infected HeLa epithelial cells with various CO92 mutants in two independent experiments (
Finally, we infected A549 human lung epithelial cells with WT CO92 and its Δail single, Δlpp ΔmsbB double, or Δlpp ΔmsbB Δail triple mutant strain. Both the Δlpp ΔmsbB double mutant and Δlpp ΔmsbB Δail triple mutant strains had decreased survival rates (49% and 11%, respectively) at 12 h p.i. (
Host-dependent inflammatory cytokine secretion by Y. pestis CO92 Δail mutants in infected macrophages. Supernatants from the above-mentioned infected macrophages were collected and assessed for cytokine production by using either a Bio-Rad mouse 6-plex assay kit for MH-S cells or a Bio-Rad human 8-plex assay kit for HMDMs. The Δlpp ΔmsbB double mutant-infected MH-S macrophages maintained levels of TNF-α and IL-6 secretion comparable to those of WT-infected cells at 2 h p.i., both of which were significantly decreased compared to those in WT-infected MH-S macrophages at 4 h p.i. (
The Δlpp ΔmsbB Δail triple mutant-infected HMDMs had statistically significant increases in levels of TNF-α and IL-6 at 2 h p.i. compared to those in both the WT CO92- and Δlpp ΔmsbB double mutant-infected macrophages (
Discussion
We made an in-frame deletion of the all gene from an already existing Δlpp ΔmsbB double mutant of WT strain CO92. The Δlpp ΔmsbB double mutant was attenuated in evoking both bubonic and pneumonic plague in mouse and rat models (Sha et al., 2013, Infect Immun 81:815-828). This double mutant retained immunogenicity to partially protect rodents against pneumonic plague upon subsequent infection with lethal doses (8 to 10 LD50s) of WT CO92 (Sha et al., 2013, Infect Immun 81:815-828). Our goal was to discern whether the deletion of the ail gene from the Δlpp ΔmsbB double mutant of WT CO92 would further attenuate the bacterium in vivo while retaining immunogenicity, to serve as a possible background strain from which additional genes could be deleted for future live-attenuated vaccine development against plague. Indeed, the triple mutant was so highly attenuated that it did not kill any mice, even at a dose as high as 3.4×106 CFU (corresponding to 6,800 LD50s) of WT CO92 (
In previous studies, the Δlpp ΔmsbB double mutant of Y. pestis CO92 and the Δail mutant of Y. pestis KIM5 were reported to have a decreased ability to disseminate to peripheral organs of mice compared to their respective parental strains; however, both of these mutant strains persisted for 3 to 7 days p.i. in mouse organs (Felek et al., 2009, Infect Immun 77:825-836; Sha et al., 2013, Infect Immun 81:815-828). On the contrary, the Δlpp ΔmsbB Δail triple mutant was more rapidly cleared from animals (by days 2 to 3 p.i.) (
The Δlpp ΔmsbB Δail triple mutant produced levels of F1, Pla, and LcrV essentially similar to those produced by WT CO92 (
Brown Norway rats infected with the Δail mutant of Y. pestis CO92 were recently found not only to survive pneumonic infection (Hinnebusch et al., 2011, Infect Immun 79:4984-4989), but also to have an influx of neutrophils in the draining lymph nodes when challenged by the intradermal route, leading to the development of large purulent abscesses (Hinnebusch et al., 2011, Infect Immun 79:4984-4989). In addition, Ail seemed necessary for specifically targeting neutrophils for T3SS translocation of effectors in the lungs when animals were infected intranasally with either the WT strain or the Δail mutant of Y. pseudotuberculosis (Paczosa et al., 2014, Cell Microbiol 16:247-268). In our study, the lungs of the triple mutant-infected animals had minimal to mild neutrophilic inflammation on day 2 p.i. The life span of neutrophils in mice is estimated to be up to 12.5 h (Pillay et al., 2010, Blood 116:625-627), and therefore, the influx of neutrophils and other inflammatory cells in mice at the infection site by the Δlpp ΔmsbB flail triple mutant at earlier time points clearly represents a possibility that will be investigated in the future.
In addition to neutrophils, Y. pestis preferentially infects host macrophages, probably via the recognition of specific surface-associated CCR5 molecules, and survives within these phagocytic cells during early stages of infection (Elvin et al., 2004, Nature 430:417). The intracellular survival and growth of Y. pestis in macrophages seem to play a role in the pathogenesis of the plague bacterium, as the organism acquires the ability to evade subsequent phagocytosis (e.g., by synthesizing capsule) and is protected from contact with other immune components (Pujol et al., 2003, Infect Immun 71:5892-5899). Thus, the impaired survival of the Δlpp ΔmsbB flail triple mutant in macrophages (both murine and human) (
Although Y. pestis is a facultative intracellular pathogen (Perry et al., 1997, Clin Microbiol Rev 10:35-66), its ability to invade epithelial cells has been reported only recently (Kolodziejek et al., 2010, Infect Immun 78:5233-5243; Felek et al., 2009, Infect Immun 77:825-836; Cowan et al., 2000, Infect Immun 68:4523-4530; Tsang et al., 2013, PLoS One 8:e83621). Ail is a major mediator responsible for the adherence and invasion of Y. pestis KIM strains or when the ail gene is overexpressed in nonadherent and noninvasive E. coli strains in human epithelial cells of cervical origin (HeLa and Hep-2) (Kolodziejek et al., 2010, Infect Immun 78:5233-5243; Kolodziejek et al., 2007, Microbiology 153:2941-2951; Felek et al., 2009, Infect Immun 77:825-836; Tsang et al., 2010, Infect Immun 78:3358-3368). Likewise, mutated versions of the ail gene from Y. pestis, when expressed and produced in E. coli, exhibited phenotypes of decreased adherence and invasion in HeLa cells, compared to E. coli strains expressing the nonmutated ail gene (Tsang et al., 2013, PLoS One 8:e83621). We provided the first evidence that Ail of Y. pestis has a role in adherence to and invasion of the human alveolar A549 epithelial cell line (
In addition to Ail, Pla and pH 6 antigen (Psa) also mediate the binding of Y. pestis KIM5 to Hep-2 cells (Felek et al., 2010, Infect Immun 78:4134-4150). However, Ail is the most critical adhesion molecule, followed by Pla and then Psa (Tsang et al., 2013, PLoS One 8:e83621). We have shown that the levels of Pla and F1 as well as the enzymatic activity of Pla were not affected in the Δlpp ΔmsbB Δail triple mutant (
Our previous studies have shown that both Lpp and Pla, but not MsbB, contributed to WT CO92 survival in murine RAW 264.7 macrophages (Sha et al., 2013, Infect Immun 81:815-828; Sha et al., 2008, Infect Immun 76:1390-1409; van Lier et al., 2014, Infect Immun 82:2485-2503; Agar et al., 2009, Microbiology 155:3247-3259). In various cell types that we studied (e.g., murine alveolar macrophages and HeLa and A549 epithelial cells), the single ail gene deletion did not affect the intracellular survival of Y. pestis (
We consistently detected an increased amount of YopH in culture supernatants of ail deletion mutants compared to that in WT CO92 supernatants (
Yops have been reported to be quickly degraded by proteases in the extracellular milieu, which may explain the marginally higher levels of YopE detected in the supernatant of the Δail single mutant than in WT CO92 (
A recent study showed that the presence of outer membrane vesicles (OMVs) containing membrane and periplasmic components was increased in the Δlpp mutant of Y. pestis under specific conditions (Eddy et al., 2014, PLoS One 9:e107002). While our electron microscopy studies revealed no OMVs in the Δlpp ΔmsbB Δail triple mutant, an increase in the release of OMVs would not explain the increased levels of some Yops in the supernatant of the Δlpp ΔmsbB Δail triple mutant (
YopE and YopH act as deterrents to bacterial phagocytosis, while YopP/YopJ inhibits inflammatory cytokine responses when injected into host cells (Boland et al., 1998, Infect Immun 66:1878-1884). Although the translocation of all the Yops by the Δlpp ΔmsbB Δail triple mutant in host cells was not assessed in this study, we observed an increased uptake of the triple mutant by MH-S murine macrophages compared to that of WT CO92 (data not shown). Importantly, this increased uptake of the triple mutant correlated with an early burst of inflammatory cytokine secretion in the triple mutant-infected murine and human macrophages compared to that in macrophages infected with WT CO92 and the Δlpp ΔmsbB double mutant (
The mutant strains of CO92 deficient in Ail production were highly sensitive to both human and NHP sera but remained resistant to mouse serum. Thus, serum sensitivity did not seem to play a role in the attenuation of these mutants in a mouse model of pneumonic plague. Differences in serum resistance of bacteria are most likely due to differences in the immune systems of the host. One obvious difference among various hosts is within the amino acid sequences of the complement proteins. For example, FH consists of 20 short consensus repeat (SCR) domains (Oyston et al., 2003, J Med Microbiol 52:289-294), and Ail is predicted to bind FH near SCR7 (Glauser et al., 1991, Lancet 338:732-736; Braun et al., 1974, Annu Rev Biochem 43:89-121; Neilsen et al., 2001, J Immunol 167:5231-5239). Mouse SCR6-8 has 55% identity and 70% homology with human SCR6-8 (Montminy et al., 2006, Nat Immunol 7:1066-1073), indicating that differences in this region of FH may influence interactions with Ail. Furthermore, other outer membrane proteins of Y. pestis may bind and recognize the mouse SCR-binding region, allowing Δail mutants to remain resistant to murine serum.
Despite the fact that the Δlpp ΔmsbB Δail triple mutant was highly attenuated and rapidly cleared from mouse tissues, animals infected with this triple mutant were still able to mount balanced TH1 and TH2 responses (
Interestingly, in our previous study, Ail was also identified by mass spectrometric analysis along with several other outer membrane antigens to which antibodies were generated when rats were exposed to WT CO92 by the intranasal route to mimic pneumonic plague and then rescued by an antibiotic, levofloxacin, given 24 h postinfection for 6 days (Erova et al., 2013, Clin Vaccine Immunol 20:227-238). Importantly, immunization of rats with the recombinant Ail protein provided partial protection to animals from a lethal challenge dose of WT CO92 in a pneumonic plague model, indicating that Ail also has some immunogenic potential (Erova et al., 2013, Clin Vaccine Immunol 20:227-238). Therefore, further manipulation of the ail gene to reduce its virulence potential while retaining immunogenicity in the Δlpp ΔmsbB background strain would provide a promising strategy in our future study.
In summary, we were able to determine the mechanistic basis of attenuation of the Δlpp ΔmsbB Δail triple mutant of Y. pestis CO92. The Δlpp ΔmsbB Δail triple mutant of Y. pestis CO92 was severely attenuated, with minimal damage to the host, while retaining immunogenicity. The possibility that this mutant may provide a platform for deleting additional genes to develop a viable live-attenuated plague vaccine for immunocompetent military and health care workers is encouraging and will be pursued in future studies. Overall, our goal is to develop several highly attenuated Y. pestis mutant strains (Sha et al., 2013, Infect Immun 81:815-828; Sha et al., 2008, Infect Immun 76:1390-1409; van Lier et al., 2014, Infect Immun 82:2485-2503; Agar et al., 2009, Microbiology 155:3247-3259) as candidate vaccines, which possibly may also be effective in an immunocompromised population.
As described in Example 1, the Δlpp ΔmsbB Acid triple mutant of Yersinia pestis CO92 deleted for genes encoding Braun lipoprotein (Lpp), an acyltransferase (MsbB), and the Attachment Invasion Locus (Ail), respectively, was avirulent in a mouse model of pneumonic plague. In this study, we further evaluated the immunogenic potential of the Δlpp ΔmsbB Acid triple mutant and its derivative by different routes of vaccination. Mice were immunized via the subcutaneous (s.c.) or the intramuscular (i.m.) route with two doses (2×106 CFU/dose) of the above-mentioned triple mutant with 100% survivability of the animals. Upon subsequent pneumonic challenge with 70-92 LD50 of WT CO92, all of the mice survived when immunization occurred by the i.m. route. Since Ail has both virulence and immunogenic potential, a mutated version of Ail devoid of its virulence properties was created, and the genetically modified ail replaced the native ail gene on the chromosome of the Δlpp ΔmsbB double mutant, creating a Δlpp ΔmsbB::ailL2 vaccine strain. This newly generated mutant was similarly attenuated as the Δlpp ΔmsbB Δail triple mutant when administered by the i.m. route, and provided 100% protection to animals against subsequent pneumonic challenge. Not only did both of the above-mentioned mutants cleared rapidly from the initial i.m. site of injection in animals with no histopathological lesions, the immunized mice did not exhibit any disease symptoms during immunization and after subsequent exposure to WT CO92. These two mutants triggered balanced Th1- and Th2-based antibody responses and cell-mediated immunity. A substantial increase in IL-17 from T-cells of vaccinated mice, a cytokine of the Th17 cells, further augmented their vaccine potential. Thus, both Δlpp ΔmsbB Δail triple and Δlpp ΔmsbB::ailL2 mutants represent excellent vaccine candidates for plague, with the latter mutant still retaining Ail immunogenicity but much diminished virulence potential. This Example is also available as Tiner et al., 2015, Clinical Vaccine Immunol., 22:1255-1268.
Introduction
Yersinia pestis is the causative agent of plague (Perry et al., 1997, Clin Microbiol Rev 10:35-66), and there has been a rise in the number of plague cases globally in recent years due possibly to climate changes and shifting of the rodent carrier range (World Health Organization Media Center. 6 Aug. 2009, posting date. Plague: questions and answers about plague., World Health Organization, Geneva, Switzerland). The organism is classified as a Tier-1 select agent (Centers for Disease Control and Prevention. 17 Nov. 2008, posting date. Protecting the American public by ensuring safe and secure possession, use, and transfer of select agents and toxins that pose a threat to public health, CDC Select Agent Program, Centers for Disease Control and Prevention, Atlanta, Ga.; Inglesby et al., 2000, JAMA 283:2281-2290; Pearson et al., 1998, Biological Weapons Proliferation: Reasons for Concern, Courses of Action. Henry L. Stimson Center, Washington D.C.), and the progression of septicemic and pneumonic forms of plague is very rapidly fatal after first appearance of the symptoms (Inglesby et al., 2000, JAMA 283:2281-2290; Rosenzweig et al., 2011, Antimicrob Agents Chemother 55:5034-5042; Layton et al., 2011, PLoS Negl Trop Dis 5:e959; Peterson et al., 2010, Open Microbiol J 4:34-46). Alarmingly, antibiotic-resistant strains of Y. pestis have been isolated from plague patients and also engineered for bioweaponization (Inglesby et al., 2000, JAMA 283:2281-2290). Therefore, vaccination would be the optimal strategy for human protection against this deadly disease; however, there are currently no Food and Drug Administration (FDA)-licensed plague vaccines available in the United States (Smiley, 2008, Expert Rev Vaccines 7:209-221; CDC, Fed Regist 77:61083-61115; Alvarez et al., 2010, Biotechnol Adv 28:184-196).
Although a heat-killed plague vaccine composed of Y. pestis 195/P strain was in use in the United States until 1999, the production of this vaccine was discontinued because of its effectiveness only against the bubonic plague and not the pneumonic form, and it was highly reactogenic in humans (Rosenzweig et al., 2011, Appl Microbiol Biotechnol 91:265-286; Williams et al., 1980, Bull World Health Organ 58:753-756). Various live-attenuated Y. pestis EV76 vaccine strains, which lack the pigmentation locus (pgm) required for iron acquisition, provide protection against both bubonic and pneumonic plague and are being used in some parts of the world where plague is endemic (Smiley, 2008, Expert Rev Vaccines 7:209-221). However, these EV76-based vaccines are not genetically uniform and are also highly reactogenic (Cui et al., 2014, Infect Genet Evol 26:172-179), and, hence, do not meet the standards for FDA approval. In addition, the Δpgm mutants of Y. pestis (e.g., KIM/D27 strain) may not be safe because of a reported case of fatal infection in an individual with hemochromatosis (2011, Morb Mortal Wkly Rep 60:201-205; Quenee et al., 2012, J Infect Dis 206:1050-1058).
In an effort to search for a new live-attenuated plague vaccine, we recently constructed Δlpp ΔmsbB Δail triple mutant which was deleted for genes encoding Braun lipoprotein (Lpp), an acetyltransferase (MsbB), and the Attachment Invasion Locus (Ail) (Tiner et al., 2015, Infect Immun 83:1318-1338). Lpp activates Toll-like receptor-2, which leads to the production of pro-inflammatory cytokines and septic shock (Glauser et al., 1991, Lancet 338:732-736; Braun et al., 1974, Annu Rev Biochem 43:89-121; Neilsen et al., 2001, J Immunol 167:5231-5239; Aliprantis et al., 1999, Science 285:736-739). On the other hand, MsbB modifies lipopolysaccharide (LPS) by adding lauric acid to the lipid A moiety, thus, resulting in increased biological potency of LPS (Aliprantis et al., 1999, Science 285:736-739; Clementz et al., 1996, J Biol Chem 271:12095-12102; Clementz et al., 1997, J Biol Chem 272:10353-10360; Anisimov et al., 2007, J Med Microbiol 56:443-453; Oyston et al., J Med Microbiol 52:289-294; Perez-Gutierrez et al., 2010, Infect Immun 78:2768-2781; Sha et al., 2013, Infect Immun 81:815-828). Ail is a ˜17 kDa outer membrane protein with four extracellular loops, and the loop 2 (L2) has been reported to be mainly responsible for Ail-mediated bacterial serum resistance and adherence/invasion of the host cells (Tiner et al., 2015, Infect Immun 83:1318-1338; Bartra et al., 2008, Infect Immun 76:612-622; Felek et al., 2009, Infect Immun 77:825-836; Felek et al., 2010, Infect Immun 78:4134-4150; Hinnebusch et al., 2011, Infect Immun 79:4984-4989; Kolodziejek et al., 2007, Microbiology 153:2941-2951; Kolodziejek et al., 2010, Infect Immun 78:5233-5243; Tsang et al., 2010, Infect Immun 78:3358-3368; Tsang et al., 2012, J Biol Chem 287:16759-16767; Yamashita et al., 2011, Structure 19:1672-1682).
In this study, to further characterize the vaccine potential of the Δlpp ΔmsbB Δail triple mutant, we evaluated its effectiveness when administered by the most common subcutaneous (s.c) or the intramuscular (i.m.) route (CDC, 2011, MMWR Recomm Rep 60:1-64). Since Ail also has immunogenic potential in addition to its role as a virulence factor (Erova et al., 2013, Clin Vaccine Immunol 20:227-238), we aimed at mutating the corresponding nucleotides in the all gene that encodes essential amino acid (aa) residues required for virulence of L2 instead of deleting the whole all gene from the Δlpp ΔmsbB mutant of CO92 (Yamashita et al., 2011, Structure 19:1672-1682; Miller et al., 2001, Mol Microbiol 41:1053-1062). Indeed, the generated Δlpp ΔmsbB::ailL2 mutant was severely impaired in Ail-associated virulence traits, e.g., serum resistance, host cell adhesion and invasion. Most importantly, immunization of mice with either the Δlpp ΔmsbB Δail or the Δlpp ΔmsbB::ailL2 mutant via either the i.m. or the s.c. route, elicited robust humoral and cellular immune responses, which conferred up to 100% protection in animals at high pneumonic challenge doses of 70-92 LD50 with WT CO92. Therefore, Δlpp ΔmsbB Δail and Δlpp ΔmsbB::ailL2 mutants represent excellent plague vaccine candidates. In addition, such vaccines could be effectively administrated via different routes, providing flexibility during immunization.
Materials & Methods
Bacterial Strains and Plasmids.
All bacterial strains and plasmids used in this study are listed in Table 3. Y. pestis and recombinant Escherichia coli strains were grown as described by us previously (Tiner et al., 2015, Infect Immun 83:1318-1338; Sha et al., 2013, Infect Immun 81:815-828; Agar et al., 2009, Microbiology 155:3247-3259; van Lier et al., 2014, Infect Immun 82:2485-2503). All of our studies were performed in a Tier-1 select agent facility within the Galveston National Laboratory (GNL), UTMB. The molecular biological reagents were purchased from Promega (Madison, Wis.), Clontech (Palo Alto, Calif.), and Qiagen, Inc., Valencia, Calif.). HeLa cells were obtained from the American Type Culture Collection (ATCC, Manassas, Va.).
Y. pestis CO92
Orientalis, and naturally resistant to polymyxin B
Y. pestis CO92 intermediate lpp, msbB and ail triple gene deletion mutant that
E. coli DH5α λpir
aCDC = Centers for Disease Control and Prevention
Mutation of the ail Gene.
Four aa residues (Lysine-88, Aspartate-91, Aspartate-93, and Phenyalanine-94) in L2 of Ail were changed to alanine by using polymerase chain reactions (PCRs). Briefly, the primer pairs Aup5-mAup3 and mAdn5-Adn3 (Table 4) were used to introduce specific mutations within L2 region of the all gene, as well as to amplify the mutated all gene with its up- and down-stream DNA sequences, respectively. The mutated all gene, designated as ailL2, with its up- and down-stream flanking DNA sequences were joined together by PCR with the primer pair Aup5-Adn3 (Table 4). The above-mentioned PCR product was subsequently cloned into the suicide vector pDMS197 (Edwards et al., 1998, Gene 207:149-157), which generated the recombinant plasmid pDMS197-ailL2 (Table 3).
pestis CO92
aBold bases denote loop 2 region, underlined bases denote mutations and the lower case letter represent restriction enzyme sites.
Previously, we constructed an intermediate lpp, msbB, and all triple gene deletion mutant (Δlpp ΔmsbB Δail-Km) of Y. pestis CO92 (Table 3) that carried a kanamycin resistance gene (Kmr) cassette (Datsenko et al., 2000, Proc Natl Acad Sci USA 97:6640-6645) in place of the ail gene (Tiner et al., 2015, Infect Immun 83:1318-1338). Therefore, the recombinant suicide vector pDMS197-ailL2 was electroporated into the Δlpp ΔmsbB Δail-Km strain (Genepulser Xcell; Bio-Rad, Hercules, Calif.) (Tiner et al., 2015, Infect Immun 83:1318-1338). The transformants that were sensitive to kanamycin (Kms) and resistant to 5% sucrose were picked up and screened by PCR with the primer pair Up5-Dn3 (Table 4) (Tiner et al., 2015, Infect Immun 83:1318-1338) to ensure genomic replacement of the Kmr cassette with the ailL2 gene. Genomic DNA sequencing with the primer SqAil (Table 4) (Tiner et al., 2015, Infect Immun 83:1318-1338) was used to further confirm replacement and to ensure no alteration in the ailL2 surrounding regions in the Δlpp ΔmsbB::ailL2 mutant, when compared to that of the native all gene in the Δlpp ΔmsbB double mutant.
Production of AilL2 and plasminogen activator (Pla) protease in the Δlpp ΔmsbB::ailL2 mutant of Y. pestis CO92.
The Δlpp ΔmsbB, Δlpp ΔmsbB Δail triple and the Δlpp ΔmsbB::ailL2 mutant were grown overnight in Heart Infusion Broth (HIB) at 28° C. with shaking at 180 rpm, and the resulting bacterial cells (representing similar colony forming units [CFU]) were dissolved in SDS-PAGE sample buffer. An aliquot of the samples was then resolved by SDS-PAGE and the Western blots analyzed with polyclonal antibodies to Ail and Pla (Erova et al., 2013, Clin Vaccine Immunol 20:227-238). As a loading control, the presence of DnaK in the bacterial pellets was assessed by using anti-DnaK monoclonal antibodies (Enzo, Farmingdale, N.Y.).
Serum Resistance, Adherence, and Invasion of Y. pestis CO92 Mutants
Various Y. pestis strains were grown overnight, harvested, and then diluted in phosphate-buffered saline (PBS) to an OD600 of 0.2 (˜1×108 CFU/ml). A 50-μl volume of the diluted bacteria (˜5×106 CFU) was mixed with either 200 μl of undiluted normal (unheated) or heat-inactivated (56° C./30 min) human sera (Sigma-Aldrich, St. Louis, Mo.). After incubation at 37° C. for 2 h, the number of surviving bacteria (CFU) in each sample was determined by serial dilutions and plating on Sheep Blood Agar (SBA) plates (Tiner et al., 2015, Infect Immun 83:1318-1338; Sha et al., 2013, Infect Immun 81:815-828). Percent bacterial survival was calculated by dividing the average CFU in samples incubated in normal serum by the average CFU in samples incubated in the heat-inactivated serum and multiplying by 100.
Human HeLa cervical epithelial cells (4×105) were seeded in 12-well plates as described previously (Tiner et al., 2015, Infect Immun 83:1318-1338) and infected with various mutants of Y. pestis CO92 at a multiplicity of infection (MOI) of 100. The plates were centrifuged at 1200 rpm for 10 min to facilitate bacterial contact with the HeLa cells. After 2 h of incubation, the adherence and invasion of bacteria were evaluated as we recently described (Tiner et al., 2015, Infect Immun 83:1318-1338; Cowan et al., 2000, Infect Immun 68:4523-4530).
Animal Studies
Six-to-eight-week old, female Swiss-Webster mice (17 to 20 g) were purchased from Taconic Laboratories (Germantown, N.Y.). All of the animal studies were performed in the Animal Biosafety Level (ABSL)-3 facility under an approved Institutional Animal Care and Use Committee protocol.
i) Immunization. Mice were immunized by the i.m. route with one or two doses of 2×106 CFU/100 microliter of the Δlpp ΔmsbB Δail triple or the Δlpp ΔmsbB::ailL2 mutant. The mutants were administered in a 50 microliter volume in each of the hind legs. When two doses of each vaccine strain were administered, they were injected twenty-one days apart (on days 0 and 21). Mice receiving only one dose were injected on the same day when the other animals received their second vaccine dose (on day 21). Another set of mice was immunized by the s.c. route with two doses of 2×106 CFU/100 microliter of the Δlpp ΔmsbB Δail triple or the Δlpp ΔmsbB mutai(L2) mutant strains. Subcutaneous doses were injected at one site twenty-one days apart (on days 0 and 21). Mice were assessed for morbidity and/or mortality over the duration of vaccination.
ii) Antibody responses. Blood was collected by the retro-orbital route from vaccinated mice two weeks after each immunization (on days 14 and 35). Pre-immunization blood samples served as a control. Sera were separated and filtered by using Costar 0.1-μm centrifuge tube filters (Corning Inc., Corning, N.Y.). ELISA plates were coated with the F1-V fusion protein (1 ng/microliter, BEI Resources, Manassas, Va.) (Tiner et al., 2015, Infect Immun 83:1318-1338; Sha et al., 2013, Infect Immun 81:815-828; van Lier et al., 2014, Infect Immun 82:2485-2503). Total IgG and antibody isotypes against F1-V (capsular antigen F1 and a type 3 secretion system [T3SS] component low calcium response V antigen [LcrV]) in the sera (1:5 serially diluted) of animals immunized with the Δlpp ΔmsbB Δail triple or the Δlpp ΔmsbB::ailL2 mutant were then determined as we previously described (Tao et al., 2013, PLoS Pathog 9:e1003495).
iii) Challenge. Twenty-one days after the last immunization (day 42), the immunized mice were anesthetized with a mixture of xylazine-ketamine and then exposed by the i.n. route to 3.5×104 to 4.6×104 CFU/40 microliter (70 to 92 LD50) of the bioluminescent WT Y. pestis CO92 luc2 strain (WT CO92 luc2), which contains the luciferase operon (luc or lux) allowing in vivo imaging of mice for bacterial dissemination in real time (Tiner et al., 2015, Infect Immun 83:1318-1338; Sha et al., 2013, Microb Pathog 55:39-50). Naive mice of the same age were used as controls. On days 3 and 7 post infection (p.i.), the animals were imaged by using an in vivo imaging system (IVIS) 200 bioluminescent and fluorescence whole-body imaging workstation (Caliper Corp. Alameda, Calif.) in the AB SL-3 facility.
iv) Histopathological Analysis. Immunized mice (n=2) which received two doses of the vaccine by either the i.m. or the s.c. route were euthanized three weeks after the second vaccine dose (on day 42). Similarly, three mice from each of the immunized groups that survived pneumonic challenge with 70 LD50 of WT CO92 luc2 strain were sacrificed on day 54 post challenge. Age-matched naïve uninfected mice (n=2) were also euthanized as a control. Lungs, liver, and the spleen were harvested from these mice, fixed in 10% neutral buffered formalin (Agar et al., 2009, Microbiology 155:3247-3259; Sha et al., 2008, Infect Immun 76:1390-1409), and tissues processed and sectioned at 5 The samples were mounted on slides and stained with hematoxylin and eosin (H&E). Tissue lesions were scored on the basis of a severity scale, which correlated with estimates of lesion distribution and the extent of tissue involvement (minimal, 2 to 10%; mild, >10 to 20%; moderate, >20 to 50%; severe, >50%), as previously described (Agar et al., 2009, Microbiology 155:3247-3259; Sha et al., 2008, Infect Immun 76:1390-1409). The histopathological evaluation of the tissue sections was performed in a blinded fashion.
v) Progression of Infection. To monitor progression of infection in real time, various bioluminescent stains of Y. pestis CO92 were constructed by using the Tn7-based system (Agar et al., 2009, Microbiology 155:3247-3259). The Tn7-based system integrates the target gene in a site-specific manner downstream of the conserved glmS (glucosamine-6-phosphate synthase) gene on the bacterial chromosome (Craig, 1996, Curr Top Microbiol Immunol 204:27-48; Peters et al., 2001, Nat Rev Mol Cell Biol 2:806-814). Briefly, the lux operon was removed from the plasmid pUC18-mini-Tn7T-lux-Gm by SpeI/KpnI restriction enzyme digestion and sub-cloned into plasmid pUC18R6KT-mini-Tn7T (Choi et al., 2005, Nat Methods 2:443-448) resulting in a derivative, designated as pUC18R6KT-mini-Tn7T-lux (Table 3).
Subsequently, the Kmr cassette from the pUC4K plasmid (BamHI digestion) was inserted into pUC18R6KT-mini-Tn7T-lux, thus, resulted in the creation of the pUC18R6KT-mini-Tn7T-lux-Km plasmid (Table 3). The electrocompetent cells of WT CO92, Δlpp ΔmsbB Δail triple, and the Δlpp ΔmsbB::ailL2 mutant strains were electroporated with mixed (2 to 1) pTNS2 and pUC18R6KT-mini-Tn7T-lux-Km plasmids (Agar et al., 2009, Microbiology 155:3247-3259; Sha et al., 2013, Microb Pathog 55:39-50; Choi et al., 2005, Nat Methods 2:443-448), and selected for Kmr and luminescence. The insertion of lux at the attTn7 region was confirmed by PCR using primer pair P1-P2 (Table 4) which specifically amplified the region between Y. pestis glmS gene and the Tn7 insertion cassette (Agar et al., 2009, Microbiology 155:3247-3259). Luminescence intensity of each strain was determined by relative luminescence unit (RLU) measurement (Spectramax M5e, Molecular Devices, Sunnyvale, Calif.) (Sha et al., 2013, Microb Pathog 55:39-50).
Mice (n=3) were then infected with 2×106 CFU/100 microliter of the above generated bioluminescent stains: Δlpp ΔmsbB Δail-lux, Δlpp ΔmsbB::ailL2-lux, or the WT CO92-lux (Table 3) by the i.m. route. The IVIS images were taken immediately after challenge and then every 12 h until 48 h p.i. After 48 h, mice were euthanized and the muscles, lungs, liver, and the spleen were removed immediately following animal sacrifice. The tissues were homogenized in 1 ml of PBS, and serial dilutions of the homogenates were spread on the SBA plates to assess dissemination of the bacteria to peripheral organs (Sha et al., 2013, Infect Immun 81:815-828). Portions of each organ from 3 mice at each time point were also removed for histopathological analysis (Agar et al., 2009, Microbiology 155:3247-3259; Sha et al., 2008, Infect Immun 76:1390-1409).
vi) T-cell proliferative responses and cytokine production. Mice (n=5) were infected by the i.m. or the s.c. route with Y. pestis KIM/D27 (pgm locus minus strain) (Table 3), the Δlpp ΔmsbB Δail triple, or the Δlpp ΔmsbB::ailL2 mutant strains of Y. pestis CO92 at a dose of 1×103 CFU/100 microliter. The T-cell proliferation in response to heat-killed WT CO92 antigens (pulsed) was measured on day 21 p.i., as we previously described (Sha et al., 2013, Infect Immun 81:815-828; van Lier et al., 2014, Infect Immun 82:2485-2503). T-cells from uninfected mice as well as un-pulsed T cells served as negative controls. The T-cell culture supernatants were collected at 48 h to measure cytokine/chemokine production by using a mouse 6-plex assay kit (Bio-Rad Laboratories Inc.). After 72 h of incubation, 1 μCi of [3H] thymidine was added into each well, and the cells harvested 16 h later using a semi-automated sample harvester, FilterMate Harvester (PerkinElmer, Waltham, Mass.), followed by the measurement of radioactive counts (TopCount NXT, PerkinElmer).
Statistical Analysis.
For majority of the experiments, one-way analysis of variance (ANOVA) was used with the Tukey's post hoc test for data analysis except for the serum resistance assay which was examined using the Student's t-test. We used Kaplan-Meier survival estimates for animal studies, and p values of ≤0.05 were considered significant for all of the statistical tests used. The standard deviations were derived from three independently performed experiments with three replicates per experiment for in vitro assays.
Results
Evaluation of protection provided by intramuscular immunization of mice with the Δlpp ΔmsbB Δail triple mutant in a pneumonic plague model.
Mice were i.m. vaccinated with one or two doses of the Δlpp ΔmsbB Δail triple mutant at 2×106 CFU/dose. As shown in
No disease symptoms were observed in mice during above immunizations. All of the mice immunized with two doses of the triple mutant survived the i.n. challenge with 92 LD50 of WT CO92 luc2 strain when administered on day 21 after vaccination (
The above infected mice were also imaged on day 3 p.i. to monitor progression of infection. As shown in
In the immunized group of mice receiving two doses of the vaccine, none of the animals were positive for bioluminescence after pneumonic challenge (
In Vitro Characterization of the Δlpp ΔmsbB::ailL2 Mutant of Y. pestis CO92.
The replacement of native ail with the ailL2 gene in the Δlpp ΔmsbB double mutant of Y. pestis CO92 was confirmed by PCR analysis. Further genomic DNA sequencing revealed no unexpected alterations in the ailL2 gene as well as in its adjacent regions on the chromosome when compared to that of its parental strain (data not shown). In addition, we examined expression of the ailL2 gene by Western blot analysis. As shown in
Due to Ail's ability to impart serum resistance to Y. pestis (Tiner et al., 2015, Infect Immun 83:1318-1338; Bartra et al., 2008, Infect Immun 76:612-622; Hinnebusch et al., 2011, Infect Immun 79:4984-4989; Kolodziejek et al., 2007, Microbiology 153:2941-2951; Kolodziejek et al., 2010, Infect Immun 78:5233-5243), the WT CO92 and its Δlpp ΔmsbB double, Δlpp ΔmsbB Δail triple, and Δlpp ΔmsbB::ailL2 mutants were evaluated for their ability to be killed by the complement cascade. Although all tested strains survived similarly in heat-inactivated sera after 2 h incubation with CFU in the range of 1.6 to 2.0×104/ml; less than 10% of the Δlpp ΔmsbB Δail triple mutant survived when exposed to the normal sera (
Since Ail also functions in adherence and subsequent invasion of bacteria in the host cells, these virulence phenotypes of WT CO92 and its various mutants were examined in HeLa cells. Both the adherence (
Evaluation of protection provided by intramuscular or subcutaneous immunization of mice with the Δlpp ΔmsbB Δail triple or the Δlpp ΔmsbB::ailL2 mutant in a pneumonic plague model.
Since our data presented in
The bioluminescence images further showed that the organism disseminated from the lungs to the whole body of all naïve but infected control mice (10/10) by day 3 p.i. (
To gauge immunogenicity of the vaccine strains via different routes of immunization, sera were collected from all mice 14 days after each immunization. We noted a boost in antibody titers between the first and the second dose when vaccination was performed via the s.c. route. However, this phenomenon was not observed via the i.m. route of immunization, as the peak antibody titers were achieved after only one vaccine dose (data not shown). Both of the above-mentioned mutants triggered higher level of antibody responses (IgG titers of 1:46,875) to F1-V antigen when vaccination occurred via the i.m. route over the s.c. route of immunization, which showed IgG titers of 1:18,000 (
Histopathological analysis of mouse tissues after intramuscular immunization with the Δlpp ΔmsbB Δail triple and the Δlpp ΔmsbB::ailL2 mutant and post exposure to WT Y. pestis CO92 in a pneumonic plague model.
Prior to WT CO92 challenge and after two doses of vaccination, organs (muscles, lungs, liver, and the spleen) were harvested from mice (n=2) in each group for histopathological analysis. Muscles from mice immunized with the Δlpp ΔmsbB::ailL2 mutant were within the normal limits histopathologically, similar to that for muscles obtained from naive, unimmunized mice (
As all i.m.-immunized animals survived exposure to 70 LD50 of WT CO92 luc2 strain (
When comparing histopathological changes, essentially similar data were obtained when immunization occurred via the s.c. route with either of the two mutants, and after challenge of the immunized mice by the pneumonic route with WT CO92 (data not shown).
Progression of infection and histopathological lesions in mice intramuscularly infected with the Δlpp ΔmsbB Δail triple and the Δlpp ΔmsbB::ailL2 mutant of Y. pestis CO92.
Mice (n=3) were either challenged with 2×106 CFU of WT CO92-lux, the Δlpp ΔmsbB Δail-lux, or the Δlpp ΔmsbB::ailL2-lux strain. Using IVIS, animals were imaged at 0, 12, 24, 36, and 48 h p.i. At 0 h, all mice were positive for bioluminescence that was localized to the injection site in the muscle (
However, mice infected with the WT CO92-lux strain had increased bioluminescence localized to the muscle at 12 h (
To further examine bacterial load in mice, after in vivo imaging at 48 h p.i., all of the animals were sacrificed, and the organs (muscles, lungs, liver, and the spleen) were harvested and subjected to bacterial count determination. As shown in
All of the animals infected with the WT CO92-lux strain had severe plague symptoms and were at the verge of death. In contrast, mice infected with either the Δlpp ΔmsbB Δail-lux triple or the Δlpp ΔmsbB::ailL2-lux mutant had minimal-to-no detectable bacterial load in any of the organs examined at 48 h p.i. (
Activation of T cells by the Δlpp ΔmsbB Δail triple and the Δlpp ΔmsbB::ailL2 mutant of Y. pestis CO92 after intramuscular or subcutaneous immunization of mice.
To investigate T-cell responses, mice were i.m. or s.c. infected/immunized with 1×103 CFU dose of either KIM/D27, the Δlpp ΔmsbB Δail triple, or the Δlpp ΔmsbB::ailL2 mutant strain. Y. pestis KIM/D27 is a pgm-locus minus mutant with similar characteristics as the live-attenuated Y. pestis EV76 vaccine strain (You et al., 2012, Biomed Environ Sci 25:440-448), and, thus, served as an appropriate control. No clinical symptoms were observed in mice immunized with either the Δlpp ΔmsbB Δail triple or the Δlpp ΔmsbB::ailL2 mutant strain. Although none of KIM/D27-infected mice succumbed to infection at this low dose (1×103 CFU), they all had ruffled fur and were lethargic up to 7 days post-immunization. On day 21 p.i., T-cells isolated from these mice were re-stimulated with the heat-killed WT Y. pestis CO92 ex-vivo, and T-cell proliferation (in terms of cpm) as well as cytokine production were evaluated.
As shown in
Supernatants collected from the above-mentioned T-cells were then assessed for cytokine production by using a Bio-Rad mouse 6-plex assay kit. Robust cytokine/chemokine production (i.e., IFN-γ TNF-α, IL-6, IL-1□, IL-10, and IL-17A) was observed in T-cells obtained from mice immunized intramuscularly across all the above-mentioned Y. pestis mutant strains tested in response to re-stimulation with the heat-killed Y. pestis CO92 (
Discussion
The resurgence of plague in many parts of the world, the existence of antibiotic-resistant strains naturally or generated intentionally, and the lack of a current FDA-approved plague vaccine in the United States necessitate the development of an effective vaccine.
Currently, the most promising and undergoing clinical trials are recombinant subunit vaccines consisting of F1 and LcrV antigens. These F1-V-based vaccines are efficacious against pneumonic plague in rodents and macaques (Fellows et al., 2015, Clin Vaccine Immunol 22:1070-1078; Agar et al., 2009, Microbes Infect 11:205-214; Quenee et al., 2011, Am J Pathol 178:1689-1700; Quenee et al., 2011, Vaccine 29:6572-6583; Williamson et al., 2011, Vaccine 29:4771-4777); however, protection was variable in African green monkeys (Smiley, 2008, Expert Rev Vaccines 7:209-221; Rosenzweig et al., 2011, Appl Microbiol Biotechnol 91:265-286; Quenee et al., 2011, Vaccine 29:6572-6583; Smiley, 2008, Immunol Rev 225:256-271; FDA, 2012, African Green monkey (Chlorocebus aethiops) animal model development to evaluate treatment of pneumonic plague). Further, F1 capsular antigen is dispensable for virulence (Sha et al., 2011, J Clin Microbiol 49:1708-1715; Quenee et al., 2008, Infect Immun 76:2025-2036) and the LcrV amino acid (aa) sequence has diverged among Y. pestis strains (Perry et al., 1997, Clin Microbiol Rev 10:35-66; Huang et al., 2006, Clin Med Res 4:189-199). Therefore, the F1-V-based subunit vaccines most likely will not provide optimal protection across all plague-causing strains in humans, specifically those that have been intentionally modified for possible use in terrorist attacks (Anisimov et al., 2010, Infect Genet Evol 10:137-145; Anisimov et al., 2007, Adv Exp Med Biol 603:23-27).
The live-attenuated vaccines which promote both humoral- and cell-mediated immune responses may represent a better option to overcome the above-mentioned shortcomings of the subunit vaccines (Smiley, 2008, Expert Rev Vaccines 7:209-221; Rosenzweig et al., 2011, Appl Microbiol Biotechnol 91:265-286). Recently, others and our laboratory reported development of mutant strains of Y. pestis that have shown vaccine potential (Tiner et al., 2015, Infect Immun 83:1318-1338; Sha et al., 2013, Infect Immun 81:815-828; van Lier et al., 2014, Infect Immun 82:2485-2503). For example, a single dose of our Δlpp ΔmsbB Δail triple mutant conferred dose-dependent protection in mice against developing subsequent pneumonic plague when immunization occurred by the intranasal route (Tiner et al., 2015, Infect Immun 83:1318-1338). Our data showed that up to 3.4×106 CFU dose of this vaccine strain was unable to kill mice, and the animals developed balanced Th1- and Th2-antibody responses which provided subsequent protection (70%) to mice when challenged with 28 LD50 of WT CO92 (Tiner et al., 2015, Infect Immun 83:1318-1338). Although the experiments were not performed in parallel, our intramuscular immunization data with a single dose of this mutant (
It is generally believed that intranasal immunization has an advantage as it results in the development of both mucosal immunity and the systemic immune response. However, during mucosal immunization, the vaccine must be able to penetrate the epithelial barrier and to survive luminal host innate defenses. On the contrary, intramuscular immunization enables the vaccine to easily access blood vessels to reach blood circulation to directly stimulate the immune system (Nicolas et al., 2008, Expert Rev Vaccines 7:1201-1214). However, irrespective of the vaccination route, the Δlpp ΔmsbB Δail triple mutant (at doses of 2×106-3.4×106 CFU) was unable to confer 100% protection to immunized mice in a single dose against developing subsequent pneumonic plague (Tiner et al., 2015, Infect Immun 83:1318-1338) (
Ail protein has eight transmembrane domains with four extracellular loops, and 8 aa residues in loops 2 and 3 of Y. enterocolitica being responsible for imparting serum resistance and microbe's ability to adhere/invade host cells (Miller et al., 2001, Mol Microbiol 41:1053-1062). Likewise, 3 aa residues within loop 1 have been predicted to play an important role in bacterial adherence (Yamashita et al., 2011, Structure 19:1672-1682). Loop 2 of Ail in Y. enterocolitica and Y. pestis share 70% homology, and mutations in 4 aa residues resulted in drastically altering Δlpp ΔmsbB::ailL2 mutant's ability to adhere, invade, and or to exhibit serum resistance when compared to WT CO92 and other tested mutants (
A slight increase in serum resistance of the Δlpp ΔmsbB::ailL2 mutant when compared to that of the Δlpp ΔmsbB Δail triple mutant was possibly related to the contribution of other Ail loops in serum resistance that were intact in the Δlpp ΔmsbB::ailL2 mutant. Indeed loops 1 and 3 of Ail in Y. pestis are important for adherence and invasion, with up to 50% reduction in the above mentioned phenotypes when these two loops were mutated (Tsang et al., 2013, PLoS One 8:e83621). However, a similar adherence and invasion of Δlpp ΔmsbB::ailL2 and Δlpp ΔmsbB Δail triple mutants in HeLa cells signified an important role of loop 2 in Ail-associated virulence phenotypes and also suggested that a conformational association between various loops of Ail might be necessary for efficient adherence and invasion of bacteria to the host cells.
A comparable level of AilL2 production in the Δlpp ΔmsbB::ailL2 mutant and its parental Δlpp ΔmsbB strain (
A biased Th2 antibody response was observed in mice when vaccinated with the Δlpp ΔmsbB::ailL2 mutant by the s.c. route which provided a slightly lower protection rate in immunized mice (67%) during subsequent pneumonic challenge. In comparison, 88% protection rate with a balanced Th1 and Th2 antibody response and a higher T-cell proliferation were noticed in mice when vaccinated by the s.c. route with the Δlpp ΔmsbB Δail triple (
The reason for this phenomenon is not clear; however, additional animal models, such as rat or nonhuman primate (NHP), may be needed to fully evaluate immunogenic potential of the Δlpp ΔmsbB::ailL2 mutant. Indeed studies have shown that Ail plays even a more important role in the pathogenesis of Y. pestis infection in a rat model of pneumonic plague when compared to the mouse model (Bartra et al., 2008, Infect Immun 76:612-622; Hinnebusch et al., 2011, Infect Immun 79:4984-4989; Kolodziejek et al., 2007, Microbiology 153:2941-2951; Kolodziejek et al., 2010, Infect Immun 78:5233-5243). In addition, a correlation between distinct IgG antibody subclasses and the Th1/Th2 profile seen in mice may differ in humans (Nicolas et al., 2008, Expert Rev Vaccines 7:1201-1214).
Of the two vaccination routes examined, and based on the protection rates, antibody and T-cell responses generated by the Δlpp ΔmsbB Δail triple and Δlpp ΔmsbB::ailL2 mutants in the mouse model, i.m. route was certainly optimal when compared to the s.c. route (
Recently, it was reported that intradermal inoculation of Y. pestis in C57BL/6J mice resulted in faster kinetics of infection when compared to subcutaneous route of inoculation due to organisms' greater ability to access the vascular and lymphatic vessels in the dermis (Gonzalez et al., 2015, Infect Immun 83:2855-2861). Studies have shown that dermis of the skin is enriched in terminal lymphatic vessels which facilitate antigen uptake as well as infiltration of immune cells to mount a stronger immune response as compared to the subcutaneous layer (Combadiere et al., 2011, Hum Vaccin 7:811-827; Shayan et al., 2006, Carcinogenesis 27:1729-1738; Teunissen et al., 2012, Curr Top Microbiol Immunol 351:25-76). Therefore, future studies examining intradermal route of immunization with our mutants will be undertaken.
IL-17A is a signature cytokine of Th17 cells which has recently been shown to provide an antibody-independent heterogonous protection and has also been implicated in protecting the host against many pathogenic bacterial infections, including Y. pestis (Kumar et al., 2013, Curr Opin Immunol 25:373-380; Lin et al., 2011, J Immunol 186:1675-1684). Interestingly, production of IL-17A from T-cells was also observed in our previous study with the Δlpp ΔmsbB double mutant of WT CO92 when mice were intranasally immunized (Sha et al., 2013, Infect Immun 81:815-828). Similarly, IL-17A was induced by the intranasal immunization of mice with the Y. pestis strain D27-pLpxL KIM/D27 engineered to express E. coli LpxL (Szaba et al., 2009, Infect Immun 77:4295-4304), which contributed significantly to the cell-mediated defense against pulmonary Y. pestis Infection (Lin et al., 2011, J Immunol 186:1675-1684). Therefore, the induction of Th17 response in addition to the Th1 and Th2 responses provided by the Δlpp ΔmsbB Δail triple and Δlpp ΔmsbB::ailL2 mutants might be beneficial in live-attenuated plague vaccines, and need to be further studied.
The vaccine dose we used for both of our mutants (2×106 CFU/dose) was considerably lower compared to 8×108 CFU/dose of the live-attenuated EV76 vaccine strain given to humans by the i.m. route in some countries, and 1×107 CFU/dose that has been used in murine studies (Zhang et al., 2014, Scand J Immunol 79:157-162; Qi et al., 2010, Vaccine 28:1655-1660; Wang et al., 2010, Clin Vaccine Immunol 17:199-201). In addition, up to 3×109 CFU/dose of EV76 has been used to immunize humans by the cutaneous route (Feodorova et al., 2014, Emerg Microbes Infect 3:e86). EV76 vaccine strain causes severe local and systemic reactions in both animals and human (Russell et al., 1995, Vaccine 13:1551-1556; Meyer et al., 1974, J Infect Dis 129:Suppl:S13-18; Meyer et al., 1974, J Infect Dis 129:Suppl:S85-12; Hallett et al., 1973, Infect Immun 8:876-881), and more seriously, deaths have been reported in NHPs (Meyer et al., 1974, J Infect Dis 129:Suppl:585-12). In addition, a similarpgm-minus strain of Y. pestis retains virulence in mice and NHPs when administered by the intranasal (i.n.) and intravenous (i.v.) routes (Smiley, 2008, Immunol Rev 225:256-271; Meyer et al., 1974, J Infect Dis 129:Suppl:585-12; Une et al., 1984, Infect Immun 43:895-900), raising serious questions about their suitability as a human vaccine (Sun et al., 2011, J Infect Dev Ctries 5:614-627). Indeed, a fatal laboratory-acquired infection with the pgm-minus KIM/D27 strain in an individual with hemochromatosis was reported recently (2011, Morb Mortal Wkly Rep 60:201-205), and mice infected intramuscularly with 103 CFU of KIM/D27 in our study also showed ruffled fur and lethargy up to 7 days post infection. However, mice immunized with up to 2-3.4×106 of either the Δlpp ΔmsbB Δail triple or Δlpp ΔmsbB::ailL2 mutant via various immunization routes (i.n., i.m. and s.c.) did not display any local or systemic reactions as well as any adverse histopathological lesions (
In summary, both of our Δlpp ΔmsbB Δail triple and Δlpp ΔmsbB::ailL2 mutants have rationally designed in-frame deletions, and, therefore, trigger minimal inflammatory response. Most importantly, T-cells isolated from mice immunized with either the Δlpp ΔmsbB Δail triple or the Δlpp ΔmsbB::ailL2 mutant via the i.m route displayed stronger proliferative responses than the KIM/D27-vaccinated mice (
Identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 LD50 of wild-type (WT) CO92. From this screen, 118 clones showing impairment in disseminating to spleen were obtained based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD50 when tested in a mouse model of bubonic plague, with 10/20 aforementioned mutants providing 40% or higher survival rates at an infectious dose of 40 LD50. Upon sequencing, six of the attenuated mutants carried interruptions in genes encoding hypothetical proteins or proteins with putative functions. In-frame deletion mutation of two of the genes identified from the screen, namely rbsA that codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11-12 LD50 in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40-100%) at 12 LD50 in a pneumonic plague mouse model. Earlier, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the Δlpp single or the Δlpp ΔmsbB double mutant augmented the attenuation to provide 90-100% survivability to mice in a pneumonic plague model at 20-50 LD50s. The Δlpp ΔmsbB ΔrbsA triple mutant-infected mice at 50 LD50 were 90% protected upon subsequent challenge with 12 LD50 of WT CO92, suggesting that this mutant or others carrying combinational deletion of genes identified through our screen could potentially be further tested and developed into a live attenuated plague vaccine(s). This Example is also available as Ponnusamy et al., 2015, Infect Immun., 83:2065-2081.
Introduction
Yersinia pestis is a Tier-1 select agent and leads to three pathodynamic manifestations in humans, namely bubonic, septicemic, and pneumonic plague (Perry et al., 1997, Clin Microbiol Rev 10:35-66). Although the disease is endemic in certain regions of the globe (Anonymous, 1999, Wkly Epidemiol Rec 74:447), the use of this organism as a biological warfare agent is a significant worldwide concern. Particularly, aerosolized droplets charged with Y. pestis can lead to primary pneumonic plague and subsequent person-to-person spread, with a narrow window for antibiotic intervention (Rollins et al., 2003, Am J Clin Pathol 119 Suppl:S78-85; Kumar et al., 2011, J Bioterrorism & Biodefense 2:112; Inglesby et al., 2000, JAMA 283:2281-2290). Consequently, an ideal strategy to combat the disease is to have a vaccine offering long-lasting immunity.
Until 1999, a heat-killed plague vaccine composed of Y. pestis 195/P strain was available for use in the United States; however, the production of this vaccine was discontinued because of its reactinogenicity and effectiveness only against bubonic but not the pneumonic plague (Williams et al., 1980, Bull World Health Organ 58:753-756; Rosenzweig et al., 2011, Appl Microbiol Biotechnol 91:265-286). As a live-attenuated vaccine, a pigmentation locus (required for iron acquisition) minus strain, designated as EV76, of Y. pestis is currently used in China and the states of the former Soviet Union where plague is endemic. Although protective against both bubonic and pneumonic plague, its reactinogenicity and the possibility that EV76 could behave like a virulent wild-type (WT) strain in individuals with underlying diseases, e.g., hemochromatosis (Anonymous, 2011, MMWR Morb Mortal Wkly Rep 60:201-205), precludes Food and Drug Administration (FDA) approval of such vaccines for human use.
Consequently, significant efforts have been made in recent years to formulate recombinant subunit and DNA-based vaccines to combat Y. pestis infections (Rosenzweig et al., 2011, Appl Microbiol Biotechnol 91:265-286; Feodorova et al., 2012, Emerg Microbes & Infect 1: p. e36). However, a majority of these vaccines are composed of two dominant antigens of Y. pestis: F1 capsular antigen and V antigen (a structural component of the type 3 secretion system [T3SS]) (Rosenzweig et al., 2011, Appl Microbiol Biotechnol 91:265-286). One of the concerns associated with such vaccines is that F1 capsular antigen is dispensable for the bacterial virulence (Sha et al. 2011, J Clin Microbiol 49:1708-1715) and the gene encoding V antigen is not fully conserved among various virulent strains of Y. pestis (Anisimov et al., 2010, Infect Genet Evol 10:137-145). Thus, F1-V antigen-based vaccines may provide minimal cross protection. Further, both humoral and cell-mediated immune responses play roles during protection of the host from plague, and hence, subunit vaccines may not be optimal (Smiley, 2007, Adv Exp Med Biol 603:376-386; Lin et al., 2011, J Immunol 186:1675-1684). Consequently, serious consideration should be given to develop live-attenuated plague vaccines.
Identification and characterization of novel virulence factors of Y. pestis to rationally design a better live-attenuated vaccine and also to formulate effective new therapeutics are of significant importance. Various virulence factors of Y. pestis have been identified and are primarily of plasmid origin, e.g., the T3SS is carried by the pCD1 plasmid, plasminogen activator (Pla) protease and pesticin genes are harbored on the pPCP1 plasmid, and the F1 capsular antigen-encoding gene is located on the pMT1 plasmid (Deng et al., 2002, J Bacteriol 184:4601-4611; Parkhill et al., 2001, Nature 413:523-527; Cornelis, 2002, J Cell Biol 158:401-408; Sing et al., 2002, J Exp Med 196:1017-1024; van Lier et al., 2014, Infect Immun 82:2485-2503). Apart from these well-known virulence factors of Y. pestis, very limited information is available on other virulence factors/mechanisms that contribute to the extreme virulent phenotype of the plague bacterium. More recently, Braun lipoprotein (Lpp) and an acyltransferase (MsbB) that modifies lipid A moiety of lipopolysaccharide (LPS), were shown to contribute to Y. pestis virulence during both bubonic and pneumonic plague, and currently, mutants devoid of these genes are being exploited for developing live-attenuated plague vaccines (van Lier et al., 2014, Infect Immun 82:2485-2503; Sha et al., 2013, Infect Immun 81:815-828; Sha et al., 2008, Infect Immun 76:1390-1409). Similarly, an outer membrane protein Ail (Attachment Invasion Locus) which provides serum resistance to Y. pestis plays an important role during septicemic plague, allowing the plague bacterium to resist host complement-mediated killing (Bartra et al., 2008., Infect Immun 76:612-622). Since Y. pestis is a facultative intracellular pathogen, during its intracellular life cycle, the bacterium up-regulates the expression of various virulence genes, including those that code for F1 capsular antigen and pH 6-antigen (PsaA), the latter of which is an adherence factor (Li et al., 2008, Infect Immun 76:1804-1811; Du et al., 2002, Infect Immun 70:1453-1460).
Recently, a number of genome-wide functional studies have been performed, mainly utilizing array-based approaches to identify other possible virulence factors of Y. pestis. During mammalian host infection, Y. pestis increases expression of genes associated with insecticidal-toxin synthesis, iron acquisition and storage, metabolite transportation, amino acid biosynthesis, and proteins that provide Y. pestis a survival advantage against neutrophil generated reactive nitrogen species (Sebbane et al., 2006, Proc Natl Acad Sci USA 103:11766-11771; Vadyvaloo et al., 2010, PLoS Pathog 6:e1000783; Chauvaux et al., 2007, Microbiology 153:3112-3124; Lathem et al., 2005, Proc Natl Acad Sci USA 102:17786-17791). Although efforts have been made to further explore these targets to comprehend their underlying pathophysiological mechanisms in the disease process, the knowledge accumulated in this area is still limited (Perry et al., 1997, Clin Microbiol Rev 10:35-66; Yang et al., 2010, Front Cell Infect Microbiol 2:157). In the same vein, we performed these studies to identify novel virulence factors that are critical during infection and dissemination of Y. pestis in a mouse model. We employed a high-throughput signature-tagged mutagenesis (STM) approach, and subsequently screened the mutants for attenuation in vivo models of bubonic and pneumonic plague.
STM is a powerful genome manipulation technique in both prokaryotes and eukaryotes and has been successfully used to identify virulence factors of many pathogens, such as Salmonella Typhimurium, Mycobacterium tuberculosis, Vibrio cholerae, and Yersinia enterocolitica (Mazurkiewicz et al., 2006, Nat Rev Genet 7:929-939). In this approach, multiple mutants can be combined together and subjected to a screening process to determine competitive value of each of the mutants. A recent study by Palace et al., focusing on factors essential for deep tissue growth, revealed that various amino acid and sugar transporters are necessary during the deep tissue survival of Y. pestis (Palace et al., 2014, mBio 5:e01385-14). Notably, a branched-chain amino acid importer gene (brnQ) was identified as essential in evoking bubonic plague in a mouse model (Palace et al., 2014, mBio 5:e01385-14). The use of this approach in other Yersinia species helped in identifying genes related to the biosynthesis of LPS, T3SS, and other metabolic pathways as necessary virulence factors during infection of the host (Darwin et al., 1999, Mol Microbiol 32:51-62; Karlyshev et al., 2001, Infect Immun 69:7810-7819; Mecsas et al., 2001, Infect Immun 69:2779-2787).
In this study, by using STM approach with 53 unique signature tags, 5,088 mutants of Y. pestis CO92 were created and screened for impairment in disseminating to the spleen in a mouse model of pneumonic plague. Among 118 clones that failed to disseminate to the spleen, 15 mutants were either attenuated in a mouse model of bubonic plague at a higher infectious dose and/or in a pneumonic mouse model with an infectious dose equivalent to 12 LD50 of WT CO92. Subsequently, the role of rbsA that codes for a putative sugar transport system ATP-binding protein; vasK, a component of the type VI secretion system; and ypo0498 (a gene within another T6SS cluster with a putative function) in the pathogenesis of Y. pestis infection was studied by in-frame deletion of these genes from WT- or the Δlpp single and Δlpp ΔmsbB double mutant background strains of CO92.
Materials and Methods
Bacterial strains, plasmids, and culture conditions.
Bacterial strains and plasmids used in this study are provided in Table 5. E. coli cultures were grown overnight at 37° C. with 180 rpm shaking in Luria-Bertani (LB) broth or grown on LB agar plates for 18-20 h. Y. pestis strains were cultured overnight at 28° C., unless specifically noted, with shaking at 180 rpm in heart infusion broth (HIB) (Difco, Voigt Global Distribution Inc., Lawrence, Kans.) or grown for 48 h on 5% sheep blood agar (SBA) (Teknova, Hollister, Calif.) or HIB agar plates. As appropriate, the organisms were cultivated in the presence of antibiotics such as ampicillin, kanamycin, and polymyxin B at concentrations of 100, 50, and 35 μg/ml, respectively. All of the experiments with Y. pestis were performed in the Centers for Disease Control and Prevention (CDC)-approved select agent laboratory in the Galveston National Laboratory (GNL), UTMB.
Y. pestis CO92
A. hydrophila*
Aeromonas hydrophila* human diarrheal isolate
E. coli
E. coli strain S17-1, recA pro hsdR RP4-2-Tc::Mu-
Construction of Y. pestis CO92 signature-tagged transposon mutant library.
A total of 5,088 transposon mutants of WT CO92 were created, which included 96 mutants for each of the 53 unique 40 by long signature tags (Hensel et al., 1995, Science 269:400-403). As a source of the tags, 53 E. coli S-17 strains, each harboring the plasmid pUTminiTn5Km2STM with a unique tag, were used as donor strains and conjugated with WT CO92 (Silver et al., 2007, J Bacteriol 189:6763-6772). Initially, 56 tags were chosen as previously described (Silver et al., 2007, J Bacteriol 189:6763-6772) and were tested for their cross-hybridization. Three out of the 56 tags showed cross-reaction under our tested conditions, and, therefore, were excluded from the study. For each of the 53 signature tags, the following procedures were carried out. The E. coli S17-1 strain (Table 5) carrying the transposon with a unique signature tag was grown overnight, sub-cultured, and then further grown for 4 h (OD600˜0.6). Separately, WT CO92 was grown overnight and mixed in a 4 to 1 ratio with the above-mentioned donor E. coli strains. An aliquot of the mixture was spread on LB agar plates and incubated at 30° C. for 24 h. Subsequently, the cultures from the LB plates were collected in sterile phosphate-buffered saline (PBS), and a portion of the mixture was spread on HIB agar plates containing polymyxin B and kanamycin for 48 h at 28° C. Following the incubation period, separate trans-conjugant colonies were tested for resistance to polymyxin B (WT CO92 is naturally resistant to this antibiotic) (Table 5) and kanamycin, but sensitive to ampicillin. Finally, 96 trans-conjugants, which did not show any obvious growth defects, with each tag were randomly picked and individually inoculated in the wells of a 96-well microtiter plate. After 24 h of growth, the plates were stored at −80° C. after the addition of glycerol to a final concentration of 15% (
Preparation of input mutant pools of Y. pestis CO92 and collection of corresponding output mutant pools from the spleen in a mouse model of pneumonic plague.
Each of the 96 mutant pools prepared above was individually tested in female Swiss-Webster mice (Taconic Biosciences, Inc., Hudson, N.Y.) after infecting them via the intranasal (i.n.) route. The animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC)-approved protocol in the Animal Biosafety Level (ABSL)-3 facility located in the GNL.
DNA hybridization-based screening of input and output mutant pools of Y. pestis CO92.
The DNA hybridizations were performed for each of the input and output pools separately, as previously described (Silver et al., 2007, J Bacteriol 189:6763-6772). Briefly, 53 signature tags were polymerase chain reaction (PCR) amplified (Phusion® High-Fidelity PCR Kit, New England Biolabs, Inc., Ipswich, Mass.) using primers P2 and P4 from the respective transposon plasmids pUTminiTn5Km2STM (
With P2 and P4 primers (Table 6), the tag sequences from each of the input and the corresponding output DNA pools were PCR amplified and gel purified as described above, and digoxigenin (DIG) (Roche Applied Science, Indianapolis, Ind.) labeled by PCR using P2 and P4 primers as described previously (Silver et al., 2007, J Bacteriol 189:6763-6772). The labeled tags were digested with the HindIII restriction enzyme to remove the primer sequences, and denatured at 95° C. for 5 min before proceeding to hybridization. The membranes prepared as mentioned above were pre-hybridized with the DIG hybridization solution (Roche Applied Science), and finally, the labeled tags were added to the membrane in a fresh-hybridization solution and incubated overnight at 42° C.
Following hybridization, the membranes were subjected to washing, blocking, and developing at room temperature (RT), unless otherwise stated, as follow: i) twice for 5 min each in 2×SSC plus 0.1% sodium dodecyl sulfate (SDS), ii) twice for 5 min each in 0.1×SSC plus 0.1% SDS at 65° C., and once for 5 min in 0.1 M maleic acid, 0.15 M NaCl [pH 7.5], 0.3% [wt/vol] Tween 20 (MNT) buffer. Then, the membranes were placed in 1× blocking solution (Roche Applied Science) for 30 min and were probed with monoclonal anti-DIG antibody in 1× blocking solution for 30 min. The membranes were washed twice for 5 min each in MNT solution, and equilibrated for 5 min in 0.1 M Tris-HCl, 0.1 M NaCl [pH 9.5] solution. Finally, ready-to-use CDP-Star solution (Roche Applied Science) was applied to each membrane, incubated for 5 min, and the positive hybridization signals were visualized on luminescent image analyzer (Imagequant LAS4000, GE Healthcare Life Sciences). All of the hybridization steps were performed in a hybridization oven.
Testing individual signature-tagged transposon mutants of Y. pestis CO92 in bubonic and pneumonic plague mouse models.
Mutant clones that exhibited either complete or partial loss in virulence in terms of their ability to disseminate to the spleen, as determined by the hybridization reactions, were selected for further study. Each of the mutants was individually used to infect a group of five Swiss-Webster mice via the subcutaneous (s.c.) route at a dose equivalent to 8 LD50 of WT CO92 (1 LD50 by the s.c. route is 50 CFU) (van Lier et al., 2014, Infect Immun 82:2485-2503). The attenuated mutants after the first screen by bubonic infection were subjected to a stringent second screen in mice (n=10 to 20) at a higher infectious dose of 40 LD50. The animals were observed for mortality over a period of 21-28 days. The mutant clones that were attenuated to show at least 40% animal survival, were selected for genomic characterization of the transposon insertion sites.
Transposon mutants that showed promising results during the first s.c. screening were further tested for their level of attenuation in a pneumonic plague mouse model. Each selected mutant was used to infect a group of five Swiss-Webster mice at an infection dose equivalent to 12 LD50 of WT CO92. The animals were observed for mortality over a period of 9 days.
Genomic characterization of transposon insertion sites in the signature-tagged mutants of Y. pestis CO92.
Inverse PCR was used to amplify DNA fragment flanking the mini-Tn5 insertion as described previously (Silver et al., 2007, J Bacteriol 189:6763-6772; Silver et al., 2007, Proc Natl Acad Sci USA 104:9481-9486). Briefly, genomic DNA from the above selected signature-tagged transposon mutants was extracted by using a DNeasy blood and tissue kit (Qiagen, Inc.). An aliquot (2 μg) of the genomic DNA was digested with the restriction enzymes BamHI, PstI, SalI or XbaI (New England Biolabs), and the resulting fragments were ligated using T4 DNA ligase (Promega, Madison, Wis.). Inverse PCR was performed using outward-facing primers P1 and P3 annealing to mini-Tn5 sequence (Table 6). Subsequently, a nested PCR amplification was carried out on each of the inverse PCR products using primers P5 and P6 (Table 6). The primers P5 and P6 annealed downstream to the primer pair P1 and P3. Then, the resulting PCR products were gel purified and sequenced using the primer P6 (Table 6). Based on the sequence information, transposon insertion sites were identified in the genome of Y. pestis CO92 (Parkhill et al., 2001, Nature 413:523-527).
Construction of in-frame deletion mutants and testing in mouse models of bubonic and pneumonic plague.
To construct in-frame deletion mutants of Y. pestis CO92, λ-phage recombination system was used (Datsenko et al., 2000, Proc Natl Acad Sci USA 97:6640-6645). Initially, the WT CO92 strain was transformed with plasmid pKD46 (Table 5) and grown in the presence of 1 mM L-arabinose to induce the expression of λ-phage recombination system. The above-mentioned Y. pestis culture was processed for the preparation of electroporation competent cells (Sha et al. 2011, J Clin Microbiol 49:1708-1715; Datsenko et al., 2000, Proc Natl Acad Sci USA 97:6640-6645). The latter were then transformed with 0.5 to 1.0 μg of the linear dsDNA constructs carrying the kanamycin resistance (Kmr) gene cassette that was immediately flanked by bacterial FRT (flippase recognition target) sequence followed by on either side by 50 by of DNA sequences homologous to the 5′ and 3′ ends of the gene to be deleted from WT CO92. The plasmid pKD46 from the mutants that had successful Kmr gene cassette integration at the correct location was cured by growing the bacteria at 37° C. The latter mutants were transformed with plasmid pFlp2 (Table 5) to excise the Kmr gene cassette (Agar et al., 2009, Microbiology 155:3247-3259). Eventually, the plasmid pFlp2 was also cured from the kanamycin sensitive (Kms) clones by growing them in a medium containing 5% sucrose (Agar et al., 2009, Microbiology 155:3247-3259). To confirm the in-frame deletion, mutants showing sensitivity to kanamycin and ampicillin were tested by PCR using appropriate primer pairs (Table 6) and sequencing of the PCR products.
To construct double or triple in-frame deletion mutants of CO92, a similar procedure was followed using selected single (Δlpp) or double (Δlpp ΔmsbB) in-frame deletion mutants that existed in the laboratory (Table 5). To construct a recombinant plasmid for complementation studies, complete open reading frame of the gene of interest along with 200 by upstream DNA sequence corresponding to the promoter region of that gene from WT CO92 was PCR amplified using Phusion® High-Fidelity PCR Kit (New England Bioloabs). Then, the DNA construct was cloned in plasmid pBR322 in place of the tetracycline resistance (TO conferring gene cassette (Table 5).
Single, double, and triple isogenic mutants, and their complemented strains, were then tested in both bubonic and pneumonic plague mouse models along with the WT CO92 strain as a control. For re-challenge experiments, after 28 days p.i. with the selected mutants, the bioluminescent WT CO92 carrying luciferase gene operon, luxCDABE (Table 5), was used to infect mice as described previously (Sha et al., 2013, Microb Pathog 55:39-50). Also in vivo imaging was performed on re-challenged animals using IVIS 200 bioluminescent and fluorescence whole-body imaging workstation (Caliper Corp. Alameda, Calif.).
Western blot analysis for detecting a T6SS effector, hemolysin-coregulated protein (Hcp), in the isogenic mutants of Y. pestis CO92
Overnight grown cultures of various Y. pestis and Aeromonas hydrophila strains (the latter was reclassified as A. dhakensis (Grim et al., 2014, Appl Environ Microbiol 80:4162-4183) were harvested and the supernatants mixed with 20% trichloroacetic acid (v/v). The resulting precipitates were dissolved in the SDS-PAGE buffer by boiling and subjected to SDS 4-15% gradient polyacrylamide gel electrophoresis. The proteins from the gel were then transferred to a Hybond™-ECL™ nitrocellulose membrane (GE Healthcare) by following the standard procedure (Suarez et al., 2010, Microbiology 156:3678-3688). The membrane was blocked with 1% bovine serum albumin [BSA] or 5% skim milk, and, subsequently, incubated with anti-Hcp antibodies specific for Y. pestis (1:1000) followed by incubation with the secondary antibodies (Goat anti-mouse IgG [1:10000]) (Southern Biotechnology Associates, Inc., Birmingham, Ala.). The membrane was washed with TBS (Tris Buffered Saline: 20 mM Tris-base, 136 mM NaCl [pH 7.4])/0.05% Tween 20, and the blot was developed using SuperSignal® West Dura Extended Duration Substrate (Pierce, Rockford, Ill.). Finally, the positive signal was detected by using ImageQuant LAS4000 platform (GE Healthcare). Polyclonal antibodies raised in mice against Hcp of Y. pestis were used for immunoblot analysis. The hcp gene (YPO3708) of Y. pestis CO92 was over-expressed in E. coli using the pET30a vector system as a His-tag recombinant protein and purified by using Ni2+ chromatography (Suarez et al., 2010, Microbiology 156:3678-3688). As a loading control for immunoblot analysis, we used monoclonal antibodies against DnaK (Enzo, Farmingdale, N.Y.), a member of conserved Hsp70 chaperone family.
Growth Kinetics of WT Y. pestis CO92, its ΔrbsA Mutant, and the Complemented Strain.
Overnight cultures of various Y. pestis strains were washed in PBS and normalized to the same absorbance by measuring optical density at 600 nm (OD600). These bacterial cultures were then inoculated separately (with approximately 1×107 CFU) in 20 mL of the modified M9 medium (1×M9 salts [22 mM KH2PO4, 33.7 mM Na2HPO4, 8.55 mM NaCl, 9.35 mM NH4Cl], 1 mM MgSO4, 2.5 mM CaCl2, 0.001 mg/mL FeSO4, 0.0001% thiamine, 0.1% casamino acids) (all chemicals were obtained from Sigma-Aldrich, St. Louis, Mo.) contained in 125 mL polycarbonate Erlenmeyer flasks with HEPA-filtered tops. The medium either did not contain any sugar or supplemented with 0.4% glucose or 0.4% ribose, and the cultures were incubated at 28° C. with shaking at 180 rpm. Samples were taken by removing 100 μL of the culture from each of the flasks at the indicated time points. Each of the samples was serially diluted, plated on SBA agar plates, and incubated at 28° C. for 48 h to determine CFU/mL.
Statistical Procedures.
Animal survival rate was statistically analyzed using Kaplan-Meier survival estimates with Bonferonni post-hoc test. In vitro growth of WT CO92, its ΔrbsA mutant or the complemented strain, under different nutritional conditions was analyzed by one-way ANOVA followed by Tukey post-hoc test. Wherever applicable, the p-values were reported, and a p value of <0.05 was considered significant.
Results
Y. pestis CO92 signature-tagged transposon mutant library and its primary screen in a mouse model of pneumonic plague.
We generated a library of mutants with 53 unique DNA tags from WT CO92 by using a transposon Tn5-based system (
The infectious dose of input 96 mutant pools in mice given by the i.n. route was 5 LD50 equivalent of the wild-type (WT) CO92 (van Lier et al., 2014, Infect Immun 82:2485-2503). Three days p.i., ˜60% of the animals died due to developing pneumonic plague and the remaining animals had clinical symptoms of plague, such as lethargy and ruffled fur. The excised spleens from these mice, irrespective of their survival status, had high bacterial counts in the range of 1×107 to 1×108 CFU per organ. Under the conditions of hybridization and washing optimized for the study, the nylon membranes harboring purified 53 unique signature tags hybridized with the 96 input pool DNA probes (obtained from each transposon mutant pool with 53 signature tags), and showed a clear pattern of positive reactions without any background (a representative blot is shown in
By this hybridization approach, we identified a total of 118 potential mutant candidates; among these 108 had no detectable signal on the output DNA pool membranes, and the remaining had very weak signals when compared to the corresponding input DNA pool membranes (a representative blot is shown in
Second screen of selected signature-tagged transposon mutants of Y. pestis CO92 in a mouse model of bubonic plague.
We performed a second screen with the above-generated 118 mutant clones by injecting mice with individual mutants via the s.c. route at an aimed 8 LD50 equivalent of WT CO92 (van Lier et al., 2014, Infect Immun 82:2485-2503). Of 118 mutant clones tested, 20 of them (˜17%) showed attenuation to the level of at least 20% or more animal survival (up to 100%) when compared to the WT CO92 on day 21 p.i. (Table 7). One of our long term goals is to identify candidate genes that could be deleted from the WT CO92 to develop a novel live-attenuated plague vaccine. Therefore, we also tested some of the surviving animal groups after infection with representative transposon mutants to withstand re-challenge with the WT CO92. The re-challenge occurred by a more stringent i.n. route, which evokes pneumonic plague, to gauge immunogenicity of the transposon mutant clones. As noted in
15-F2
pla
85
85
85
Plasminogen activator
44-F11
vasK (ypo3603)
60
60
60
Type-6 secretion system component-
VasK
Genetic characteristics of signature-tagged transposon mutants of Y. pestis CO92 that are attenuated in a mouse model of bubonic plague
Identification of the disrupted genes or genetic regions in the transposon mutants was accomplished using inverse PCR followed by confirmation of the mutation location by sequencing of the PCR products. Genomic characterization of 18 out of 20 attenuated mutants identified during the first screen by s.c. challenge was listed in Table 7, while genomic locations of transposon integration for mutant clones 19-F7 and 47-F10 were not confirmed. Of these confirmed 10 mutants showing attenuation under high stringency in a mouse model of bubonic plague, transposon insertion was identified in one of six uncharacterized genes (Table 8). For example, clones 52-B1 and 2-H3 encode putative surface-exposed and membrane protein, respectively. Clones 44-B5, 53-C3, 54-F6, and 39-G4 code for hypothetical or proteins with putative functions. In clone 47-G5, the transposon interruption occurred in the cytochrome o ubiquinol oxidase subunit II (Table 8). Two of the genes encoded previously characterized virulence factors and included the pla protease gene located on the pCP1 plasmid of Y. pestis (van Lier et al., 2014, Infect Immun 82:2485-2503; Lathem et al., 2007, Science 315:509-513). The pla protease gene was interrupted in the mutant clone (15-F2) (Table 8), adding credibility to our screening process. This mutant was highly attenuated, as 85% of the animals survived the challenge in a bubonic plague model on day 21 p.i. (Table 8).
Mutant clone 44-F11, with 60% of the mice surviving the challenge, was identified as having a disruption in a gene whose product has homology to VasK of A. dhakensis SSU, a key component of the T6SS (Suarez et al., 2008, Microb Pathog 44:344-361) (Table 8). Clone number 42-B8 was identified as having a transposon insertion within the gene referred to as rbsA, and 85, 70, and 60% of the animals survived after challenge on days 7, 14, and 21 p.i., respectively, in a mouse model of bubonic plague (Table 8). This gene is part of the rbs operon that codes for a putative ribose transport system and has been described in orthologous systems (Barroga et al., 1996, Protein Sci 5:1093-1099; Zaitseva et al., 1996, Protein Sci 5:1100-1107).
Likewise, the clone 44-B5 in which the transposition occurred in a hypothetical gene was highly attenuated, with 100, 85, and 85% survival of animals on days 7, 14, and 21 p.i., respectively (Table 8).
Pathodynamics of bubonic plague infection for the isogenic mutants of Y. pestis CO92 deleted for genes rbsA, vasK or Ypo0498 in a mouse model.
Transposon mutagenesis does not always provide a true estimate of bacterial attenuation during infection due partly to possible polar effects, and, therefore, we created isogenic mutants for three of the genes rbsA, vasK, and ypo0498 (Table 8). Gene rbsA was targeted as it had the most functional information available from orthologs in other bacterial species (Barroga et al., 1996, Protein Sci 5:1093-1099; Zaitseva et al., 1996, Protein Sci 5:1100-1107). Similarly, the gene ypo3603 (vasK) was targeted due to the virulence attributes associated with orthologous genes (Suarez et al., 2008, Microb Pathog 44:344-361). Another candidate gene, ypo0498, which encodes a hypothetical protein and is part of another T6SS locus, was also selected. These mutants were then used to challenge mice by the s.c. route to replicate data obtained during the transposon mutant screening.
Animals infected with the ΔrbsA or the ΔvasK isogenic mutant by the s.c. route showed a statistically significant attenuation, as 40% (p=0.042) and 70% (p=0.002), respectively, of the mice survived when challenged with 10 LD50 equivalent of WT CO92 (
Characterization of the ΔvasK mutant of Y. pestis CO92 in a mouse model of pneumonic plague.
Following evaluation by the s.c. route of the disease (bubonic plague), the ΔvasK mutant was then assessed for attenuation in a mouse model of pneumonic plague. At a dose equivalent to 12 LD50 of WT CO92, the mice exhibited a 20 percent survival rate (p=0.031) by day 21 p.i., with no survival of animals challenged with a similar dose of WT CO92 (
In addition to generating a ΔvasK single mutant, we also deleted the vasK gene from the Δlpp background strain of CO92. Braun lipoprotein (Lpp) has previously been shown in our laboratory to provide attenuation in mouse models of pneumonic and bubonic plague through decreased intracellular survival in macrophages (Sha et al., 2013, Infect Immun 81:815-828; Sha et al., 2008, Infect Immun 76:1390-1409; Agar et al., 2009, Microbiology 155:3247-3259). The rationale for deleting the ΔvasK gene from the Δlpp background strain of CO92 was to delineate whether additive or synergistic attenuation could be achieved with the Δlpp ΔvasK double mutant in a mouse model of pneumonic plague.
Infection by the i.n. route with the Δlpp ΔvasK double mutant resulted in 90% survival (p<0.001) of mice at a dose equivalent to 12 LD50 when compared to only 5% survival rate of animals after challenge with the Δlpp single mutant by day 21 p.i. at a comparable challenge dose (
To determine if the attenuating effect of the vasK gene deletion from WT CO92 or its Δlpp mutant in mice was related to the inhibition of secretion of a T6SS effector, Hcp, a Western blot analysis was performed on the culture supernatants of WT CO92 and its Δlpp single, ΔvasK single, and Δlpp ΔvasK double mutants. We have shown earlier that the secretion of Hcp was blocked when the vasK gene was deleted from a diarrheal isolate SSU of A. dhakensis (Suarez et al., 2008, Microb Pathog 44:344-361). As seen in
The gene rbsA is required for the full virulence of Y. pestis CO92 in a pneumonic plague mouse model and in the utilization of ribose.
In a pneumonic plague model, the ΔrbsA mutant was attenuated with 30% of the mice (p=0.0098) having survived the challenge, while the animals infected with the WT CO92 or those infected with the rbsA complemented strain died by day 6 at a similar infectious dose of 11 LD50 (
Earlier studies suggested that domains with sequence homology to the RbsA protein function as a ribose transport system (Barroga et al., 1996, Protein Sci 5:1093-1099; Zaitseva et al., 1996, Protein Sci 5:1100-1107). To explore the role of ribose utilization, the ΔrbsA mutant was grown in a modified minimal medium that was restricted for the carbon source. In a minimal medium supplemented with 0.4% ribose, the ΔrbsA mutant displayed a delayed growth pattern similar to when no ribose was added to the medium (
In a minimal medium, WT CO92 without any carbon source grew poorly after 16 h of incubation; however, the addition of either 0.4% glucose or ribose resulted in luxuriant bacterial mass (p<0.001) (
To assess the potential of rbsA deletion as a component of the live attenuated vaccine and to further characterize its attenuating characteristics, we constructed double and triple isogenic mutants in which the rbsA gene was deleted from the Δlpp and Δlpp ΔmsbB background strains of CO92. The msbB gene encodes an acylytansferase that attaches lauric acid to the lipid A moiety to increase biological potency of LPS (Karow et al., 1992, J Bacteriol 174:702-710). The Δlpp ΔmsbB double mutant exhibited increased attenuation compared to respective single mutants alone (Sha et al., 2013, Infect Immun 81:815-828). The resulting double (Δlpp ΔrbsA) or the triple (Δlpp ΔmsbB ΔrbsA) isogenic mutant showed a synergistic reduction in virulence (
While none of the Δlpp ΔmsbB double mutant-infected mice survived day 5 p.i. at 20 LD50, 90% of the mice challenged with 15 LD50 of the WT CO92 died (
As part of the evaluation for its inclusion as a component in a live-attenuated vaccine candidate, we were interested in testing the immune-protective potential of this strain. To accomplish this, we re-challenged the groups of mice that received a 20 LD50 of the Δlpp ΔrbsA double mutant or the Δlpp ΔmsbB ΔrbsA triple mutant strain with a fully virulent bioluminescent Y. pestis CO92 strain on day 28 post initial challenge. Likewise, the group receiving a 50 LD50 of Δlpp ΔmsbB ΔrbsA triple mutant strain was subsequently challenged with bioluminescent Y. pestis CO92 strain (
Only 2 of the 10 mice (20%) from the initial 20 LD50 challenge group with the Δlpp ΔmsbB ΔrbsA triple mutant strain showed bacterial burden and later succumbed to infection due to WT CO92 (
Interestingly, animals initially challenged with the Δlpp ΔrbsA double mutant strain (75% protected) showed only a 50% survival after re-challenge (
Third screen of selected signature-tagged transposon mutants of Y. pestis CO92 in a mouse model of pneumonic plague.
During the initial stage of pneumonic plague, Y. pestis suppresses the host immune system to down-regulate the inflammatory response, and, thereby, creating a highly permissive niche for the bacterium to multiply in an unrestrictive manner (Bubeck et al., 2007, Infect Immun 75:697-705; Price et al., 2012, Proc Natl Acad Sci USA 109: 3083-3088). Subsequently, this accumulation of proliferating bacteria leads to the induction of a massive inflammatory reaction and that causes lung edema and death of the infected animals (Bubeck et al., 2007, Infect Immun 75:697-705; Price et al., 2012, Proc Natl Acad Sci USA 109: 3083-3088; Agar et al., 2008, Microbiology 154:1939-1948). Taken these findings together, it is likely that a Y. pestis mutant which is attenuated for dissemination to the peripheral organs after intranasal infection could still cause inflammatory changes in the lung tissue. Consequently, infected animals would succumb to infection due to pneumonia rather than the septicemic dissemination as noted during bubonic plague. Therefore, we further evaluated the extent of attenuation in causing pneumonic plague by the remaining 18 (ΔvasK and ΔrbsA isogenic mutants were already characterized) signature-tagged mutants (Table 7) identified during the screening process, and the animals were infected by the i.n. route at a dose equivalent to 12 LD50 of WT CO92 (Table 7).
In general, a delayed pattern of death was noted for all the mutants on day 3 after infection (Table 7). Nine out of 18 mutants exhibited between 40-100% survivals on day 9. The mutants 45-B9, 49-B1, 53-C3, and 53-G5 were unable to kill any mice, while 80% of mice survived challenge with mutants 52-B1, and 52-B5. These data implied that some mutants identified during the initial bubonic plague screen were attenuated in causing primary pneumonic plague as well and will be further characterized.
Discussion
Knowledge on the virulence factors of Y. pestis is crucial to developing a new plague vaccine or to design a better therapeutic intervention. As no FDA approved plague vaccine is available for humans and the antibiotics have limited role when the disease progresses to a clinical stage, search for novel virulence factors of the organism becomes a compulsive need to combat plague in the future. Here, we chose high-throughput STM approach, because this technique offers a power of analyzing multiple mutants simultaneously for attenuation in virulence either in vitro or in vivo assays (Mazurkiewicz et al., 2006, Nat Rev Genet 7:929-939; Hensel et al., 1995, Science 269:400-403).
In this study, more than 5,000 transposon mutants of Y. pestis CO92, an isolate originally from a human case of pneumonic plague in the United States (Williamson et al., 2011, Vaccine 29:4771-4777), were screened for impairment to disseminate to internal organs (e.g., spleen) in a mouse model of pneumonic plague (
The complexity of the mutant pools during STM is a crucial parameter and has to be carefully considered for obtaining a high quality screen in the animal models (Hensel et al., 1995, Science 269:400-403). Although an increased pool complexity would enable more mutants to be screened simultaneously, one might also enhance the probability that some virulent mutants would not be present in sufficient numbers in the organs of an infected animal, and, thus, leading to false positive data. In addition, the quantity of a labeled tag for each transposon is inversely proportional to the complexity of the tag pool during hybridization analysis (Hensel et al., 1995, Science 269:400-403). In our study, the number of signature tags was reduced from 56 to 53 due to cross-reaction noted for three tags during the prescreening step (Silver et al., 2007, J Bacteriol 189:6763-6772). The elimination of such tags is a pre-requisite in performing STM-based screens (Mazurkiewicz et al., 2006, Nat Rev Genet 7:929-939). At a challenge dose of 5 LD50 (used in our study), the inocula for the mutant pools with a complexity of 53 tags would provide ˜50 cfu of the each tested mutant to ensure adequate bacterial number at the initial infection site, i.e., the lungs.
Unlike in other STM studies, we neither opted for a second round of animal infection-hybridization screening process nor used an in vitro assay to narrow down the number of selected mutants for further studies (Silver et al., 2007, J Bacteriol 189:6763-6772; Flashner et al., 2004, Infect Immun 72:908-915). We rather chose bubonic plague mouse model and tested each of the 118 mutants individually and animals examined for mortality. While 20/118 mutants exhibited an attenuated phenotype at 8 LD50, only 10 showed promising level of attenuation at an infectious dose of 40 LD50 (Tables 3&4). The false positive clones appeared in our initial screen were most likely due to technical artifacts associated with the STM technique. Indeed, hybridization signals for 3 tags (2, 9 and 19) were consistently missing from the blots of our input pools (
Our genomic characterization of the 10 mutants showed transposon insertion largely in uncharacterized genes (in 6 mutants) encoding hypothetical or putative proteins (Table 4). In the remaining four mutants (15-F2, 42-B8, 44-F11, and 52-B1), the interrupted genes encoded or were assigned some functions. In clone 15-F2, the interruption occurred in a well-known virulence gene pla located on the pPCP1 plasmid of the plague bacterium. Pla plays an important role during bubonic plague, particularly in facilitating Y. pestis to disseminate systemically (van Lier et al., 2014, Infect Immun 82:2485-2503; Lathem et al., 2007, Science 315:509-513), thus validating our screening process.
STM technique has previously been used to identify virulence factors in several pathogenic bacteria, including Y. pestis (Flashner et al., 2004, Infect Immun 72:908-915; Leigh et al., 2005, Microb Pathog 38:259-266). These studies employed either Y. pestis C099-3015 strain lacking the pCD1 plasmid with a cell culture-based in vitro screen or Y. pestis Kimberley53 strain in a bubonic plague mouse model (infection dose of 100 cfu) as the initial screen (Flashner et al., 2004, Infect Immun 72:908-915; Leigh et al., 2005, Microb Pathog 38:259-266). Strain Kimberley53 was obtained by infecting mice by the s.c. route with Y. pestis Kimberley (Flashner et al., 2004, Infect Immun 72:908-915; Ben-Gurion et al., 1958, J Gen Microbiol 19:289-297). More recently, a laboratory reconstructed version of Y. pestis KIM10 strain, KIM1001, was used for transposon mutagenesis with a high-throughput sequencing (Tn-seq) to systematically probe the Y. pestis genome for elements contributing to fitness during infection. An intravenous route with an infection dose of 2.3×107 cfu of KIM1001 was used in order to preserve the diversity of the mutant pools (Palace et al., 2014, mBio 5:e01385-14). Although all of the above-mentioned studies yielded a set of genes associated with the virulence of Y. pestis, several of them belonged to the category of uncharacterized genes. Importantly, none of these genes were shared when compared with the interrupted genes identified in our mutant clones. This could be attributed to different strains and infection routes used as well as stringency of the screening procedures, and the threshold for accepting attenuated clones.
Two attenuated mutant clones were identified during our screens (44-B5 and 44-F11, Table 4) in which transposition occurred in the T6SS. Although the role of T6SS has been demonstrated in some pathogenic bacteria (Boyer et al., 2009, BMC Genomics 10:104; Pukatzki et al., 2009, Curr Opin Microbiol 12:11-17), its involvement in the pathogenesis of Y. pestis infections has not been elucidated. Based on in silico analysis, six T6SS clusters have been predicated in the Y. pestis genome (Boyer et al., 2009, BMC Genomics 10:104). Mutant 44-B5 was interrupted in an uncharacterized gene ypo0498 and it is within the ypo0495-ypo0518 locus, one of the six T6SS clusters found in Y. pestis CO92 (Boyer et al., 2009, BMC Genomics 10:104; Robinson et al., 2009, Microb Pathog 47:243-251). Interestingly, the expression of the genes (ypo0499-ypo0516) in this T6SS locus was found to be up regulated at 26° C. compared to that at 37° C., and, therefore, its role in bacterial lifecycle in fleas has been speculated (Robinson et al., 2009, Microb Pathog 47:243-251). However, deleting a portion (ypo0499-ypo0516) of this T6SS locus from WT CO92 did not affect the ability of the bacterium to infect the oriental rat flea, Xenopsylla cheopis, as well as its associated disease dynamics in both bubonic and pneumonic plague mouse models, although a decreased uptake by murine macrophage-like J774.A1 cells was noticed for the deletion mutant (Boyer et al., 2009, BMC Genomics 10:104; Robinson et al., 2009, Microb Pathog 47:243-251).
Similarly, the expression of the ypo0498 gene was up regulated approximately 3.7 folds when Y. pestis CO92 strain was exposed to human plasma, and was 14 folds higher at the mid-phase of the exponential growth compared to that at the stationary phase of bacterial growth. Interestingly, switching the growth temperature from 28° C. to 37° C. down-regulated the expression of the ypo0498 gene to approximately 9 folds (Chauvaux et al., 2007, Microbiology 153:3112-3124). Further, during infection in mice, the expression of the Δypo0498 gene was down regulated (2-fold) in the lung tissues when compared to bacterial growth in the BHI broth at 37° C. (Lathem et al., 2005, Proc Natl Acad Sci USA 102:17786-17791). Although we have shown a delayed time to death in mice infected with the Δypo0498 isogenic mutant in a bubonic plague model, it is as virulent as the WT CO92 in a pneumonic plague mouse model (data not shown). Therefore, the role of YP00498 and its associated T6SS locus in the pathogenesis of Y. pestis infections is still uncertain and needs further investigation.
In mutant 44-F11, the transposition occurred in the ypo3603 gene which is homologous to the vasK gene and encodes a key component of the T6SS (Suarez et al., 2008, Microb Pathog 44:344-361). The ypo3603 gene belongs to another T6SS locus (ypo3588-ypo3615) in Y. pestis CO92 (Boyer et al., 2009, BMC Genomics 10:104). Our earlier study showed that VasK mediated the secretion of T6SS effectors (e.g., Hcp) in A. dhakensis SSU, as the deletion of this gene led to a failure of the mutant to secrete Hcp to the extracellular medium and the mutant was attenuated in a speticemic mouse model of infection (Suarez et al., 2008, Microb Pathog 44:344-361). Although six possible T6SS gene clusters have been predicted, BLAST search of the hcp gene sequence against the other predicted protein-encoding genes on Y. pestis CO92 genome revealed only one copy of the hcp gene. Amino acid sequence of this Y. pestis hcp gene shares 82% and 81% homology with the Hcp of Vibrio cholerae and A. dhakensis strain SSU, respectively (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Surprisingly, deletion of the ypo3603 gene (ΔvasK) from Y. pestis CO92 did not prevent secretion of Hcp (
In our screen, we did not identify so far any genes related to the T3 SS which is an important virulence mechanism for Y. pestis (Cornelis, 2002, J Cell Biol 158:401-408). We would like to emphasize that our screening process for identifying Y. pestis mutants defective for dissemination to peripheral mouse organs in a pneumonic plague mouse model has not been completed as yet, and, thus far, only 50% of the total output pools have been successfully screened for this study. We expect that further screening of the remaining output pools would likely identify mutant clones related to the T3SS and its effectors as well as other known virulence factors of Y. pestis.
In addition to the above T6SS related mutants, clone 42-B8 was identified as having transposon insertion in the putative sugar transport system, ATP-binding protein, which is referred to as the rbsA gene. This gene is a part of the ribose transport (rbs) operon encoding ribose transport and modification system. The rbs operon consists of genes rbsDACBK, in which genes rbsD and rbsK are involved in phosphorylation of the ribose sugar. Based on the genomic composition, the rbsACB genes are organized in a polycistronic transcript and form the ribose transportation channel (Horazdovsky et al., 1987, J Mol Biol 197:27-35; Park et al., 1999, Embo J 18:4149-4156). RbsA carries an ATP binding domain and possesses nucleotide-binding property, while RbsB is a ribose-binding protein in the periplasmic space, and RbsC is a hydrophobic transmembrane protein (Barroga et al., 1996, Protein Sci 5:1093-1099; Zaitseva et al., 1996, Protein Sci 5:1100-1107; Park et al., 1999, J Bacteriol 181:1039-1042).
In addition to transport ribose, RbsA was shown to mediate chemotaxis of ribose sugar for E. coli, and when this gene was mutated, the chemotactic activity as well as the ribose transportation across the bacterial membrane was significantly affected (Barroga et al., 1996, Protein Sci 5:1093-1099). We have shown for the first time that deletion of the rbsA gene attenuated the bacterium in both bubonic and pneumonic plague mouse models and clearly demonstrated the role of RbsA in the pathogenesis of Y. pestis infection. Although the underlying mechanism of attenuation is currently not clear, however, considering the primary role of RbsA in sugar transportation, it is most likely that failure to utilize ribose by the ΔrbsA mutant would have a negative effect on bacterial fitness and survival inside the hostile environment of the host.
Interestingly, the rbs operon has been reported to be regulated by quorum sensing (QS) AI-2 system, and RbsB shares structural resemblance with the sensor protein LuxP (Pukatzki et al., 2009, Curr Opin Microbiol 12:11-17; Bladergroen et al., 2003, Mol Plant Microbe Interact 16:53-64). Therefore, RbsA may regulate bacterial virulence through QS. In addition, RbsB has been reported as a putative effector of T6SS in Rhizobium leguminosarum (Pukatzki et al., 2009, Curr Opin Microbiol 12:11-17). In line with this finding, it is possible that the secretion of RbsB may be affected by the deletion of the ypo3603 gene, a homolog of vasK. There could possibly be an interplay between RbsA and VasK which would constitute part of our future studies. Similar to RbsA, a glucose importer (PtsG) from Y. pestis strain KIM1001 was recently reported as required for in vivo growth in mouse spleen tissues, although deletion of this gene did not change the pathodynamics of bubonic plague from its parental strain (Palace et al., 2014, mBio 5:e01385-14). Likewise, the chvE-gguAB operon in Agrobacterium tumefaciens encodes a glucose and galactose importer. Sugar binding to ChvE triggers a signaling response that results in virulence gene expression (Davidson et al., 2008, Microbiol Mol Biol Rev 72:317-364). These studies highlighted the importance of sugar transporter in bacterial pathogenesis.
We noted that the level of attenuation of the isogenic mutants (
Although the transposon DNA constructs used in this study did not carry any known prokaryotic transcription termination sites, the level of mRNA transcription from the neighboring genes or from other genes of an operon (e.g., rbs operon) and their stability and secondary structure could be altered when the transposon insertion occurred. However, these transcriptional alternations would be minimal in isogenic mutants, and thus such mutants showed less attenuation than the corresponding transposon mutants.
Despite the reduced level of attenuation observed with each of the single isogenic mutants, they would be invaluable in the construction of a live-attenuated plague vaccine strain carrying deletion for multiple virulence genes. Evidently, when the rbsA gene was deleted from the Δlpp mutant of CO92 strain, the level of attenuation increased synergistically in a mouse model of pneumonic plague (
Lipoproteins play varying roles in different bacterial species such as their involvement in host cell colonization and adhesion, bacterial cell division, protein folding and signal transduction (Kovacs-Simon et al., 2011, Infect Immun 79:548-561). In Y. pestis KIM/D27 strain (deleted for the pigmentation [pgin] locus), we reported that deletion of the lpp gene led to increased production of cytokines, such as interferon (IFN)-γ and interleukin (IL)-2 from mouse splenic T-cells, and IL-12 from macrophages. Further, this mutant caused less apoptotic changes and increased NF-kappa B signaling in both mouse splenocytes and macrophages (Liu et al., 2010, Microb Pathog 48:42-52). Similarly, we showed that the splenic T-cells from mice infected with the Δlpp Δpla double mutant of CO92 showed increased tumor necrosis factor (TNF)-α production when such immune cells were exposed ex vivo to heat-killed WT CO92 antigens (van Lier et al., 2014, Infect Immun 82:2485-2503). Further, the Δlpp mutant of CO92 was defective in intracellular survival in RAW 264.7 murine macrophages, and it was attenuated in evoking both bubonic and pneumonic plague in a mouse model (Sha et al., 2013, Infect Immun 81:815-828).
Therefore, it is likely that the deletion of rbsA and vasK genes from the Δlpp background strain of CO92, or the deletion of the rbsA gene from the Δlpp ΔmsbB background strain would augment the host immune system to increase cytokine production. Both IFN-γ and TNF-α contribute to host survival by inhibiting bacterial multiplication in vivo and increasing bacterial clearance by macrophages (Nakajima et al., 1993, Infect Immun 61:23-31). However, further detailed studies are needed to fully understand this cytokine interplay in synergistic attenuation of virulence by the double (Δlpp ΔvasK, Δlpp ΔrbsA) or triple (Δlpp ΔmsbB ΔrbsA) isogenic mutants during mouse models of plague infection.
In summary, we have identified 20 potential targets that could be associated with the full virulence of Y. pestis CO92 strain by using the STM approach. Among them, 15 mutants were either attenuated in a mouse model of bubonic plague at a higher infectious dose of 40 LD50 and/or in a pneumonic mouse model with an infectious dose equivalent to 12 LD50 of WT CO92. For the first time, we have demonstrated the role of VasK and RbsA in the pathogenesis of Y. pestis infections. The generated double mutants, Δlpp ΔvasK and Δlpp ΔrbsA, as well as the triple mutant Δlpp ΔmsbB ΔrbsA showed promising potential in their further development as live-attenuated vaccines. Our future study will continue to characterize the remaining genes that have been identified during this study and their roles in causing plague.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
This application claims the benefit of U.S. Provisional Application Ser. No. 62/103,432, filed Jan. 14, 2015, and 62/121,760, filed Feb. 27, 2015, each of which is incorporated by reference herein.
This invention was made with government support under AI064389 and AI071634, awarded by the National Institutes of Health. The government has certain rights in the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20160199475 A1 | Jul 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62103432 | Jan 2015 | US | |
| 62121760 | Feb 2015 | US |
