Patent Application
20100290996
This invention generally relates to microbial culturing. More particularly, the present invention is directed to applying a low sedimental fluid shear environment to manipulate microorganisms. Microorganisms obtained from a low sedimental fluid shear culture, which exhibit modified phenotypic and molecular genetic characteristics, are useful for the development of novel and improved diagnostics, therapeutics, vaccines and bioindustrial products. Further, application of low sedimental fluid conditions to microorganisms permits identification of molecules uniquely expressed under these conditions, providing a basis for the design of new therapeutic targets.
Environmental conditions and crewmember immune dysfunction associated with spaceflight may increase the risk of infectious disease during a long-duration mission. Previous studies using the enteric bacterial pathogen, Salmonella enterica serovar Typhimurium, showed that growth in a ground-based spaceflight analog bioreactor, termed the rotating wall vessel (RWV), induced molecular genetic and phenotypic changes in this organism. Specifically, S. typhimurium grown in this spaceflight analog culture environment, described as low shear modeled microgravity (LSMMG), exhibited increased virulence, increased resistance to environmental stresses (acid, osmotic, and thermal), increased survival in macrophages, and global changes in gene expression at the transcriptional and translational levels. However, our knowledge of microbial changes in response to spaceflight or spaceflight analog conditions and the corresponding changes to infectious disease risk is still limited and unclear. Elucidation of such risks and the mechanisms behind any spaceflight or spaceflight analog-induced changes to microbial pathogens holds the potential to decrease risk for human exploration of space and provide insight into how pathogens cause infections in Earth-based environments.
The present invention is directed to applying a low sedimental shear environment to manipulate microorganisms, and to microorganisms and compositions obtained based on such manipulation.
In one aspect, the present invention provides a method for modifying or manipulating a microorganism by culturing the microorganism under low sedimental fluid shear conditions, and harvesting the cultured microorganism. The present method applies to microorganism including but not limited to bacteria, viruses, fungi, protozoa, protists, and worms (such as helminthes), among others. Examples of microorganisms contemplated by the present invention include Salmonella sp. (particularly Salmonella typhimurium), Streptococcus pneumoniae, Pseudomonas aeruginosa, Candida albicans, and Saccharomyces cerevisiae.
In one embodiment, the fluid shear level in the low sedimental shear environment in which the microorganism is being cultured is adjusted to be 100 dynes per cm2 or lower, preferably lower than 50 dynes per cm2, more preferably lower than 20 dynes per cm2, even more preferably 10 dynes per cm2 or lower, or even lower than 0.1 dynes per cm2.
In another aspect, the present invention provides methods of modifying a phenotypic characteristic of a microorganism by culturing the microorganism in low sedimental shear environments. Phenotypic characteristics that can be modified in accordance with the present invention include but are not limited to, virulence, immunogenicity, stress resistance (such as thermal, acid or oxidative stress resistance), resistance to drugs including anti-microbial compounds (e.g., resistance of a fungus to an anti-fungal compound), ability of a bacterium to form biofilm in culture, metabolic capabilities, among others.
In a specific embodiment, low sedimental shear conditions are applied to an attenuated vaccine strain of a microorganism to enhance the efficacy of the vaccine strain.
In a further aspect, the present invention is directed to the modulation of one or more ion concentrations to manipulate, e.g., to amplify or inhibit, responses of microorganisms to low sedimental shear environments. Ions which can be manipulated to achieve modification of microorganisms include but are not limited to phosphate, chloride, sulfate/sulfur, bromide, nitrate-n, o-phosphate, pH/hydrogen ion, calcium, chromium, copper, iron, lithium, fluoride, magnesium, manganese, molybdenum, nickel, potassium, sodium and zinc, among others.
In another aspect, the present invention provides microorganisms harvested from a low sedimental shear culture.
In still another aspect, the present invention provides a therapeutic composition, including a vaccine composition, comprised of a microorganism obtained from a low sedimental shear culture. Microorganisms suitable for use in the therapeutic composition of the present invention include, for example, Salmonella sp. (particularly Salmonella typhimurium), including an attenuated Salmonella vaccine strain, Streptococcus pneumonia, Pseudomonas aeruginosa, Candida albicans and Saccharomyces cerevisiae, harvested from a culture grown under low sedimental shear conditions.
In another aspect, the present invention provides other compositions formulated with a microorganism obtained from a low sedimental shear culture, useful for various bioindustrial applications.
In a further aspect, the present invention provides a method for identifying a gene of a microorganism which modulates the response of the microorganism to low sedimental shear environments. The method includes culturing the microorganism in a low sedimental shear environment, comparing expression of candidate genes in the microorganism in the low sedimental shear environment relative to control sedimental shear conditions, and identifying genes that exhibit differential expression. Functional categories of genes that have been or can be identified as differentially expressed in accordance with the present invention include, without limitation, virulence genes, iron metabolism genes, ion response or utilization genes, cell surface polysaccharide genes, protein secretion genes, flagellar genes, stress genes, genes coding for ribosomal proteins, genes coding for fimbrial proteins, transcriptional regulator genes, genes involved in extracellular matrix/biofilm synthesis, stress response genes, sigma factors, genes encoding RNA binding proteins, genes encoding small noncoding regulatory RNAs (small RNAs), DNA polymerase genes, RNA polymerase genes, plasmid transfer/conjugation genes, chaperone proteins, carbon utilization genes, Metabolic pathway genes, energy metabolism genes, chemotaxis genes, genes encoding heat shock proteins, genes encoding putative proteins, genes encoding recombination proteins, genes encoding transport system proteins, genes encoding membrane proteins, genes encoding cell wall components (including LPS), housekeeping genes, genes encoding structural proteins and enzymes, and plasmid genes.
In another aspect, the present invention is directed to the use of a host, including during space flight, to study interactions between the host and a microorganism pathogen or an attenuated vaccine strain when both are simultaneously placed in a low sedimental shear environment. The pathogen can, for this purpose, also have been manipulated in an RWV or similar analog. According to this aspect of the invention, hosts include animals, animal analogs, plants, insects, and cell and/or tissue cultures from animals, animal analogs or plants.
The present invention is predicated in part on the discovery of global changes in microorganisms which resulted from growth in spaceflight or spaceflight analogs which produce low sedimental shear environments around the microorganisms, including phenotypic (such as virulence and stress resistance) and molecular genetic (gene expression) changes. Specifically, during spaceflight aboard the Space Shuttle mission STS-115 and STS-123 and on the ground using a spaceflight analogue bioreactor, the Rotating Wall Vessel, changes were observed in gene expression (mRNA and protein), virulence, and stress resistance using the microorganism Salmonella typhimurium, for example. Further, a conserved global regulator, the Hfq protein, has been identified to be involved in the response to the environment of low sedimental shear stress during spaceflight and spaceflight analogue culture. Conventional culture conditions, which are currently available in the marketplace, do not have the capability to grow microorganisms in low sedimental shear environments, and therefore are unable to recapitulate low fluid shear levels found within an infected host. The recognition of the phenotypic and molecular genetic changes of microorganisms in response to low sedimental shear environments allows the development of modified microorganism with desirable and improved phenotypic characteristics, such as enhanced immunogenicity and protection against infection, altered stress resistance, altered metabolic capabilities, and altered ability to form biofilms. The modified microorganism can be used in formulating therapeutic and vaccine compositions, as well as bioindustrial products. Further, the use of low sedimental shear environments in accordance with the present invention permits identification of novel target molecules for vaccine and therapeutic development, which would not have been possible using conventional culture conditions.
In one aspect, the present invention provides a method for modifying or manipulating a microorganism by culturing the microorganism under low sedimental fluid shear conditions, and harvesting the cultured microorganism. This aspect of the invention excludes culturing a Salmonella sp., particularly a wild type (i.e., naturally occurring, unmodified Salmonella sp.) in a low sedimental fluid shear environment created by a rotating wall vessel bioreactor.
“Low sedimental fluid shear conditions” and “low sedimental fluid shear environments”, or in short, “low sedimental shear” conditions or environments, contemplated by the present invention include space flight and space flight analogs which produce low sedimental shear environments. Examples of space flight analog include commercial analog bioreactors such as rotating wall vessels (RWV), and other art-recognized low sedimental shear environments as understood by the skilled artisan. The RWV is a rotating bioreactor (
The term “microorganism” includes bacteria, viruses, fungi, protozoa, protists, and worms (such as helminthes), among others. Examples of microorganisms contemplated by the present invention include Salmonella sp. (particularly Salmonella typhimurium), Streptococcus pneumoniae, Pseudomonas aeruginosa, Candida albicans, and Saccharomyces cerevisiae.
In one embodiment, modification of the microorganism is achieved by altering the fluid shear levels in the low sedimental shear environment in which the microorganism is being cultured. The fluid shear level in the culture can be adjusted to 100 dynes per cm2 or lower, preferably lower than 50 dynes per cm2, more preferably lower than 20 dynes per cm2, even more preferably 10 dynes per cm2 or lower, or even lower than 0.1 dynes per cm2.
In accordance with the present invention, culturing a microorganism in low sedimental shear environments induces both phenotypic and molecular genetic changes. Accordingly, in a further aspect, the present invention provides methods of modifying a phenotypic characteristic of a microorganism by culturing the microorganism in low sedimental shear environments.
A “phenotypic characteristic” of a microorganism, as would be understood by those skilled in the art, include any observable or detectable physical or biochemical characteristics of a microorganism, including but not limited to, virulence, immunogenicity, stress resistance (such as thermal, acid or oxidative stress resistance), resistance to drugs including anti-microbial compounds (e.g., resistance of a fungus to an anti-fungal compound), ability of a bacterium to form biofilm in culture, among others.
Changes in phenotypic characteristics are often associated with or caused by molecular genetic changes. As used herein, the term “molecular genetic changes” refer to changes in gene expression, which manifest at any or a combination of mRNA, rRNA, tRNA, small non-coding RNA levels and protein levels.
The present inventors have demonstrated global changes in gene expression, virulence and stress resistance characteristics of Salmonella typhimurium, which resulted from growth in a spaceflight or spaceflight analog (RWV) which produces low sedimental shear environments around the cells. A conserved global regulator, the Hfq protein, has been identified to be involved in the response to the environment of low sedimental shear stress during spaceflight and spaceflight analogue culture. While Salmonella typhimurium has been used as an example to illustrate the phenotypic and genetic changes, due to the nature of the effect and the conservation of global regulators between different organisms, multiple organisms should display similar changes in characteristics in response to low sedimental shear environments.
In one embodiment, low sedimental shear conditions are applied to a microorganism to alter (increase or decrease) the virulence of the microorganism. In a specific embodiment, low sedimental shear conditions are applied to a microorganism to increase the virulence of the microorganism. By “virulence” it is meant the ability of a microorganism to cause disease. Virulence of a microorganism can be determined by any of the art-recognized methods, including suitable animal models. The ability of low sedimental shear conditions to increase virulence of a microorganism allows for the development of new therapeutic compositions. Without being bound to any particular theory, the global changes of a microorganism resulting from culturing in a low sedimental shear environment may include expression of antigens by the microorganism that would not be expressed under conventional culturing conditions but are possibly expressed during infection of a host by the microorganism. In addition, a microorganism exhibits enhanced stress resistance and improved ability of survival after being cultured in a low sedimental shear environment. As a result, a vaccine prepared using such microorganism is able to survive longer in a recipient host to induce desirable protective immunity.
In one embodiment, low sedimental shear conditions are applied to an attenuated vaccine strain of microorganism to enhance the efficacy of the vaccine strain. Enhanced vaccine efficacy includes, but is not limited to improved immunogenicity (i.e. ability of the vaccine strain to provoke immune response), and/or improved protection against subsequent challenges. Attenuated microbial vaccine strains are well-documented in the art and can be prepared by various well-known methods, such as serial passaging or site-directed mutagenesis.
In a specific embodiment, the present invention provides a method of enhancing the immunogenicity and/or protection of an attenuated Salmonella vaccine strain by culturing the attenuated Salmonella vaccine strain in a low sedimental shear environment and harvesting the cultured strain.
In another specific embodiment, the present invention provides a method of enhancing the immunogenicity and/or protection of a recombinant attenuated Salmonella vaccine strain expressing one or more antigens from other pathogens by culturing the attenuated recombinant Salmonella vaccine strain in a low sedimental shear environment and harvesting the cultured strain.
In another embodiment, low sedimental shear conditions are applied to a microorganism to enhance stress resistance of the microorganism. The resulting, more resilient microorganism is particularly useful for the development of biomedical products like vaccines and bioindustrial products, such as biofuels. Enhanced performance and robustness of consortia of microorganisms are also useful for bioremediation.
In still another embodiment, low sedimental shear conditions are applied to a microorganism to modify the ability of the microorganism to form biofilm. In a specific embodiment, low sedimental shear conditions are applied to a microorganism to enhance the ability of the microorganism to form biofilm. As illustrated hereinbelow, S. typhimurium strain X3339, which does not form biofilm when cultured in the LB medium in ground, is able to form biofilm after grown in spaceflight. On the other hand, an attenuated S. typhimurium strain vaccine strain X9558pYA4088, which forms biofilm in 1×g culture in the RWV, showed reduced ability to form biofilm after grown in LSMMG. Accordingly, one could employ low sedimental shear conditions to manipulate the ability of the microorganism to form biofilm, either to increase such ability in order to develop bioindustrial products useful for sewage treatment and pollution control, or to decrease the ability to form biofilm. Altered biofilm production could be important for enhanced efficacy and robustness of microbial consortia for bioremediation, sewage treatment, microbial fuel cells, and possibly vaccines.
In a further aspect, the present invention is directed to the modulation of one or more ion concentrations to manipulate, e.g., to amplify or inhibit, responses of microorganisms to low sedimental shear environments.
The present inventors have discovered that the environmental ion concentration during microbial growth strongly influences the intensity of changes in virulence and gene expression profiles in response to low sedimental shear conditions. For example, higher concentrations of phosphate ions altered the ability of S. typhimurium to respond to spaceflight and minimized its pathogenic-related effects. The term “ions” as used herein is not limited to one particular type of ion, and includes, e.g., phosphate, chloride, sulfate/sulfur, bromide, nitrate-n, o-phosphate, pH/hydrogen ion, calcium, chromium, copper, iron, lithium, fluoride, magnesium, manganese, molybdenum, nickel, potassium, sodium and zinc, among others.
In one embodiment, one or more ion concentrations are modulated to inhibit pathogenic responses of microorganisms to low sedimental shear environments. Such modulations are useful in human spaceflight to mitigate the adverse effects of microorganisms necessarily present and undergoing the subject pathogenic responses during and because of such flight. Such modulations are also useful to counteract pathogenic responses of microorganisms to low sedimental shear environments encountered during infection of a host, in which case, modulation of ion concentrations can be achieved by oral administration to the host with compositions containing one or more ions, or ion chelators.
In one embodiment, one or more ion concentrations are modulated to modulate, i.e., to amplify or decrease, the responses of microorganisms to low sedimental shear environments. Such modulations are useful, e.g., to enhance the immunogenicity of a strain for the development of vaccine or other therapeutic products, to enhance the stress resistance of a microorganism for the development of bioindustrial products.
In a further aspect, the present invention provides microorganisms harvested from a low sedimental shear culture.
In one embodiment, the present invention provides Salmonella sp. obtained from a culture grown in spaceflight. In a preferred embodiment the microorganism is Salmonella typhimurium. Salmonella sp., particularly wild type (native) Salmonella sp., obtained from a culture grown under low sedimental shear conditions provided by the RWVs is excluded from the scope of the present invention. In another embodiment, the present invention provides Streptococcus pneumonia harvested from a culture grown under low sedimental shear conditions. In still another embodiment, the present invention provides Pseudomonas aeruginosa harvested from a culture grown under low sedimental shear conditions. In yet another embodiment, the present invention provides a fungus, such as Candida albicans and Saccharomyces cerevisiae, harvested from a culture grown under low sedimental shear conditions.
In another aspect, the present invention provides a therapeutic composition comprised of a microorganism obtained from a low sedimental shear culture. The therapeutic composition can be a vaccine composition with improved efficacy as compared to a vaccine made of the same microorganism grown in a control (normal) sedimental shear culture. In one embodiment, the present invention provides a vaccine composition containing Salmonella sp. obtained from a culture grown under low sedimental shear conditions. In a preferred embodiment the microorganism is Salmonella typhimurium. In another preferred embodiment, the present invention provides a vaccine composition containing a recombinant attenuated Salmonella anti-pneumococcal vaccine strain harvested from a culture grown under low sedimental shear conditions. In another embodiment, the present invention provides a vaccine containing Streptococcus pneumonia harvested from a culture grown under low sedimental shear conditions. In still another embodiment, the present invention provides a vaccine containing Pseudomonas aeruginosa harvested from a culture grown under low sedimental shear conditions. In yet another embodiment, the present invention provides a therapeutic composition containing a fungus, such as Candida albicans and Saccharomyces cerevisiae, harvested from a culture grown under low sedimental shear conditions.
Other compositions formulated with a microorganism obtained from a low sedimental shear culture, useful for various bioindustrial applications, are also included within the scope of the present invention.
In a further aspect, the present invention provides a method for identifying a gene of a microorganism which modulates the response of the microorganism to low sedimental shear environments.
Conventional culture conditions, which are currently available in the marketplace, do not have the capability to grow microorganisms in low sedimental shear environments, and therefore are unable to recapitulate low fluid shear levels found within an infected host. Thus, many of the genes that could be expressed or proteins that could be functional are not documented or investigated. These genes are critical to understanding microbial responses during growth in many unique conditions, such as spaceflight, and in many common conditions encountered during the course of microbial natural lifecycles, such as locations in the host during microbial infection. Low sedimental shear environments are useful to identify classes of genes (including regulatory RNAs) and proteins that have heretofore not been recognized, characterized or understood from microorganisms cultured in standard culture conditions.
As demonstrated by the present inventors, the space-traveling Salmonella had changed expression of 167 genes, as compared to bacteria that remained on Earth. A conserved global regulator, the Hfq protein, has been identified to be involved in the response to the environment of low sedimental shear stress during spaceflight and spaceflight analogue culture. Bacteria that lack the
Hfq gene did not respond to the low sedimental shear conditions. These results highlight Hfq as a therapeutic target. In addition, a number of genes have been identified in accordance with the present invention to respond in the same direction in both RWV microarray analysis and spaceflight analysis, including dps, fimA, hfq, ptsH, rplD, and yaiV. Several genes have been identified to be regulated in different directions in the two conditions (i.e. up in RWV, but down in flight or vice versa), including ppiB, sipD and frdC.
According to the present invention, microbial genes that modulate the response of a microorganism to low sedimental shear environments can be identified by culturing the microorganism in a low sedimental shear environment, and comparing expression (at mRNA or protein level) of candidate genes in the microorganism in the low sedimental shear environment relative to ground control conditions. Those that exhibit differential expression can be identified from candidate genes. The embodiment of identifying modulator genes by culturing a Salmonella species in a low sedimental shear environment provided by the RWV is excluded from this aspect of the invention.
Gene expression can be determined by a variety of art-recognized techniques, including but not limited to, microarray analysis of mRNA, rRNA, tRNA, or small non-coding RNA, RT-PCR or qRT-PCR, Western blot, and proteomics analysis. By “differential expression” it is meant that the ratio of the levels of expression under two different conditions is at least 1.5, preferably at least 2.0, more preferably at least 3.0, even more preferably 5.0 or more. After microorganisms are harvested from a spaceflight or spaceflight analog (such as the RWV bioreactor described above), cells are processed so as to retain the expression profile from a low sedimental shear culture, prior to a specific target identification assay is being performed. For example, for microarray analysis, cells are fixed immediately in RNA Later™ or other relevant fixative. Total RNA is isolated from cells, labeled with fluorescent dyes (such as Cy3 and Cy 5), and used to hybridize to microarrays with genomic DNA. Two assays are performed, one for LSS (low sedimental shear) and one for CSS (control sedimental shear) cultured cells, respectively. After quantitation, the ratio of expression of LSS to CSS is determined. Genes with ratios of 2 or greater (or 0.5 or less) (either up or down-regulated in LSS, respectively) can be identified, for example. For proteomics, cells are fixed using RNA Later or similar fixative, or fixed by flash freezing and storage at −80 degrees C. Cells are lysed and proteins are precipitated with acetone. After digestion with trypsin, the protein samples are subjected to a proteomic assay of choice: MudPIT, LC/MS-MS, 2-D gels followed by MALDI, for example. Proteins that are present under LSS conditions and not in CSS (or vice-versa) can be identified. For Western blotting, cells are fixed using RNA Later or similar fixative, or by flash freezing and storage at −80 degrees C. Fixed cells are resuspended in a protein sample buffer for SDS-PAGE and run on gel, followed by Western blot analysis using antisera from patients/animals or immunizations against prominent antigens. Prominent proteins bands in LSS samples as compared to CSS samples will correspond to proteins that are recognized by the patient/animal and up-regulated under LSS conditions. Protein bands can be cut out and are subjected to MALDI to identify the molecular nature of the underlying protein(s).
Functional categories of genes that have been or can be identified as differentially expressed in accordance with the present invention include, without limitation, virulence genes, iron metabolism genes, ion response or utilization genes, cell surface polysaccharide genes, protein secretion genes, flagellar genes, stress genes, genes coding for ribosomal proteins, genes coding for fimbrial proteins, transcriptional regulator genes, genes involved in extracellular matrix/biofilm synthesis, stress response genes, sigma factors, genes encoding RNA binding proteins, genes encoding small noncoding regulatory RNAs (small RNAs), DNA polymerase genes, RNA polymerase genes, plasmid transfer/conjugation genes, chaperone proteins, carbon utilization genes, Metabolic pathway genes, energy metabolism genes, chemotaxis genes, genes encoding heat shock proteins, genes encoding putative proteins, genes encoding recombination proteins, genes encoding transport system proteins, genes encoding membrane proteins, genes encoding cell wall components (including LPS), housekeeping genes, genes encoding structural proteins and enzymes, and plasmid genes.
In certain instances, the functions of identified genes may have been already documented. In other cases, the functions are unknown, or their unique expression in LSS conditions is unknown. The functions of differentially expressed genes identified from LSS cultures can be further characterized by making a mutant microorganism in which a particular gene of interest is mutated (e.g., completely knocked out), and assessing whether the mutant microorganism exhibits any change in virulence, stress resistance or any other phenotypic characteristics, and therefore determining whether this gene is involved in establishing infection, for example. Alternatively, the expression of a gene of interest, which has been identified from LSS cultures, can be altered by mutating its promoter, or completing replacing its promoter with a heterologous promoter, to increase or decrease its expression in order to determine the role of the gene in establishing infection.
If a gene differentially expressed in LSS conditions is determined to be involved in establishing infection, such gene makes a good target, because a loss of function in this gene will be expected to decrease the ability of the microorganism to cause infection. This will provide a basis for intelligent design of pharmaceutical compounds for treating and preventing infection by this microorganism.
Further, proteins identified as uniquely expressed in LSS conditions can be used as antigen for immunizations.
In still a further aspect of the present invention, LSS cultures are used for screening for new drugs against infection by a microorganism. This is achieved by culturing the microorganism in a LSS environment, contacting the microorganism in the culture with a candidate compound, and determining the inhibitory effect of the compound on the growth of the microorganism as indicative of the therapeutic efficacy of the compound. This method of the present invention has the advantage to be able to select compounds that are effective against the microorganism in an in vivo LSS environment during infection.
In another aspect, the present invention is directed to the use of a host, including during space flight, to study interactions between the host and a microorganism pathogen or an attenuated vaccine strain when both are simultaneously placed in a low sedimental shear environment. The pathogen can, for this purpose, also have been manipulated in an RWV or similar analog.
According to this aspect of the invention, hosts include animals, animal analogs, plants, insects, and cell and/or tissue cultures from animals, animal analogs or plants.
In one embodiment, the invention is directed to the use of animal models, including during space flight, as hosts to study interactions between the host and a microorganism pathogen in a low sedimental shear environment. Animal models include those typically used by the art, and include without limitation, animals of the class Mammalia; preferably rodents such as mice, rats and the like.
In another embodiment, the present invention is directed to the use of so-called animal model analogs as hosts, including during spaceflight, to examine the host-pathogen interaction. Animal model analogs as hosts include those known in the art, such as without limitation, invertebrates, e.g. from the class Nematoda and the like, for the purpose herein.
In still another embodiment, the invention contemplates the use of plants as hosts, including during space flight, to examine the effect of space flight on the host-pathogen interaction, e.g., that leads to infection and disease.
In yet another embodiment, the invention is directed to the use of cell and/or tissue cultures from animals (including mammals), animal analogs (e.g. invertebrates such as nematodes and the like) and/or plants as hosts, including during space flight, to examine the effect of space flight on the host-pathogen interaction.
This invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. All the publications mentioned in the present disclosure are incorporated herein by reference.
This example describes experiments conducted with the bacterial pathogen Salmonella typhimurium which was grown aboard Space Shuttle mission STS-115 and compared to identical ground control cultures. Global microarray and proteomic analyses revealed 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to the spaceflight environment. Hfq involvement was confirmed with a ground based microgravity culture model. Spaceflight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm.
The virulent, mouse-passaged Salmonella typhimurium derivative of SL1344 termed χ3339 was used as the wild type strain in all flight and ground-based experiments (5). Isogenic derivatives of SL1344 with mutations Δhfq, hfq 3′Cm, and invA Km were used in ground-based experiments (13, 22). The Δhfq strain contains a deletion of the hfq open reading frame (ORF) and replacement with a chloramphenicol resistance cassette, and the hfq 3′Cm strain contains an insertion of the same cassette immediately downstream of the WT hfq ORF. The invA Km strain contains a kanamycin resistance cassette inserted in the invA ORF. Lennox broth (LB) was used as the growth media in all experiments (5) and phosphate buffered saline (PBS) (Invitrogen, Carlsbad, Calif.) was used to resuspend bacteria for use as inoculum in the FPAs. The RNA fixative RNA Later II (Ambion, Austin, Tex.), glutaraldehyde (16%) (Sigma, St. Louis, Mo.), and formaldehyde (2%) (Ted Pella Inc., Redding, Calif.) were used as fixatives in flight experiments.
An FPA consists of a glass barrel that can be divided into compartments via the insertion of rubber stoppers and a lexan sheath into which the glass barrel is inserted. Each compartment in the glass barrel was filled with a solution in an order such that the solutions would be mixed at specific timepoints in flight via two actions: (1) downward plunging action on the rubber stoppers and (2) passage of the fluid in a given compartment through a bevel on the side of the glass barrel such that it was released into the compartment below. Glass barrels and rubber stoppers were coated with a silicone lubricant (Sigmacote, Sigma, St. Louis, Mo.) and autoclaved separately before assembly. A stopper with a gas exchange membrane was inserted just below the bevel in the glass barrel before autoclaving. FPA assembly was performed aseptically in a laminar flow hood in the following order: 2.0 ml LB media on top of the gas exchange stopper, one rubber stopper, 0.5 ml PBS containing bacterial inoculum (approximately 6.7×106 bacteria), another rubber stopper, 2.5 ml of either RNA fixative or LB media, and a final rubber stopper. Syringe needles (gauge 25⅝) were inserted into rubber stoppers during this process to release air pressure and facilitate assembly. To facilitate group activation of FPAs during flight and to ensure proper containment levels, sets of 8 FPAs were loaded into larger containers termed group activation packs (GAPs).
Six to eight week old female Balb/c mice (housed in the Animal Facility at the Space Life Sciences Lab at Kennedy Space Center) were fasted for approximately 6 hours and then per-orally infected with increasing dosages of S. typhimurium harvested from flight and ground FPA cultures and resuspended in buffered saline gelatin (5). Ten mice per infectious dosage were used, and food and water were returned to the animals within 30 minutes post-infection. The infected mice were monitored every 6-12 hours for 30 days. The LD50 value was calculated using the formula of Reed and Muench (23).
A portion of cells from the viable, media-supplemented cultures from flight and ground FPAs were fixed for scanning electron microscopic analysis using 8% glutaraldehyde and 1% formaldehyde and were processed for SEM as described previously (24).
Total cellular RNA purification, preparation of fluorescently-labeled, single stranded cDNA probes, probe hybridization to whole genome S. typhimurium microarrays, and image acquisition was performed as previously described (7) using three biological and three technical replicates for each culture condition. Flow cytometric analysis revealed that cell numbers in flight and ground biological replicate cultures were not statistically different (using SYTO-BC dye per manufacturer's recommendations; Invitrogen, Carlsbad, Calif.). Data from stored array images were obtained via QuantArray software (Packard Bioscience, Billerica, Mass.) and statistically analyzed for significant gene expression differences using the Webarray suite as described previously (25). GeneSpring software was also used to validate the genes identified with the Webarray suite. To obtain the genes comprising the spaceflight stimulon as listed in Table 1, the following parameters were used in Webarray: a fold increase or decrease in expression of 2 fold or greater, a spot quality (A-value) of greater than 9.5, and p-value of less than 0.05. For some genes listed in Table 1, the following parameters were used: a fold increase or decrease in expression of value greater than 1.6 or less then 0.6 respectively, an A-value of 8.5 or greater, and p-value of less then 0.1. The vast majority of genes listed in Table 1 had an A-value of greater than 9.0 (with most being greater than 9.5) and a p-value of 0.05 or less. The exceptions were as follows: sbmA (p-value=0.06), oxyS (A-value=8.81), rplY (A-value=8.95), cspD (A-value=8.90), yfiA (p-value=0.08), ompX (p-value=0.09), has (p-value=0.08), rmf (A-value=8.82), wcaD (p-value 0.09), and fliE (A-value=8.98). To identify spaceflight stimulon genes also contained in the Hfq regulon, proteins or genes found to be regulated by Hfq or RNAs found to be bound by Hfq as reported in the indicated references were scanned against the spaceflight microarray data for expression changes within the parameters above (8, 12, 13, 16, 26).
Acetone-protein precipitates from whole cell lysates obtained from flight and ground cultures (representing three biological replicates) were subjected to MudPIT analysis using the LC-LC-MS/MS technique as described previously (27, 28). Tandem MS spectra of peptides were analyzed with TurboSEQUEST™ v 3.1 and XTandem software, and the data were further analyzed and organized using the Scaffold program (29, 30). See Table 2 for the specific parameters used in Scaffold to identify the proteins in this study.
S. typhimurium cultures were grown in rotating wall vessels in the LSMMG and 1×g orientations and assayed for resistance to pH=3.5 and survival inside J774 macrophages as described previously (5), except that the RWV cultures were grown for 24 hours at 37 degrees C. For acid stress assays, the percentage of surviving bacteria present after 45-60 minutes acid stress (compared to the original number of bacteria before addition of the stress) was calculated. A ratio of the percent survival values for the LSMMG and 1×g cultures was obtained (indicating the fold difference in survival between these cultures) and is presented as the acid survival ratio in
To determine which genes changed expression in response to spaceflight, total bacterial RNA was isolated from the fixed flight and ground samples, qualitatively analyzed to ensure lack of degradation via denaturing gel electrophoresis, quantitated, and then reversed transcribed into labeled, single-stranded cDNA. The labeled cDNA was co-hybridized with differentially-labeled S. typhimurium genomic DNA to whole genome S. typhimurium microarray slides. The cDNA signal hybridizing to each gene spot was quantitated, and the normalized, background-subtracted data was analyzed for statistically-significant, 2-fold or greater differences in gene expression between the flight and ground samples. 167 genes were found to be differentially-expressed in flight as compared to ground controls from a variety of functional categories (69 up-regulated and 98 down-regulated) (Table 1). The proteomes of fixed cultures were also obtained via multi-dimensional protein identification (MudPIT) analysis. Among 251 proteins expressed in the flight and ground cultures, 73 were present at different levels in these samples (Table 2). Several of the genes encoding these proteins were also identified via microarray analysis. Collectively, these gene expression changes form the first documented bacterial spaceflight stimulon indicating that bacteria respond to this environment with widespread alterations of expression of genes distributed globally throughout the chromosome (
The data indicated that a pathway involving the conserved RNA-binding regulatory protein Hfq played a role in this response (Table 3). Hfq is an RNA chaperone that binds to small regulatory RNA and mRNA molecules to facilitate mRNA translational regulation in response to envelope stress (in conjunction with the specialized sigma factor RpoE), environmental stress (via alteration of RpoS expression), and changes in metabolite concentrations, such as iron levels (via the Fur pathway) (8-12). Hfq is also involved in promoting the virulence of several pathogens including S. typhimurium (13), and Hfq homologues are highly conserved across species of prokaryotes and eukaryotes (14). The data strongly supported a role for Hfq in the response to spaceflight: (1) The expression of hfq was decreased in flight, and this finding matched previous results in which S. typhimurium hfq gene expression was decreased in a ground-based model of microgravity (7); (2) Expression of 64 genes in the Hfq regulon was altered in flight (32% of the total genes identified), and the directions of differential changes of major classes of these genes matched predictions associated with decreased hfq expression (see subsequent examples); (3) several small regulatory RNAs that interact with Hfq were differentially regulated in flight as would be predicted if small RNA/Hfq pathways are involved in a spaceflight response; (4) The levels of OmpA, OmpC, and OmpD mRNA and protein are classic indicators of the RpoE-mediated periplasmic stress response which involves Hfq (15). Transcripts encoding OmpA, OmpC, and OmpD (and OmpC protein level) were up-regulated in flight, correlating with hfq down-regulation; (5) Hfq promotes expression of a large class of ribosomal structural protein genes (12), and many such genes exhibited decreased expression in flight; (6) Hfq is a negative regulator of the large tra operon encoding the F plasmid transfer apparatus (16), and several tra genes from related operons on two plasmids present in S. typhimurium χ3339were up regulated in flight; (7) Hfq is intimately involved in a periplasmic stress signaling pathway that is dependent on the activity levels of three key proteins, RpoE, DksA, and RseB: differential expression of these genes was observed in flight (8, 12); (8) Hfq regulates the expression of the Fur protein and other genes involved in the iron response pathway, and several iron utilization/storage genes were found to have altered expression in flight (9, 11). This finding also matched previous results in which iron pathway genes in S. typhimurium changed expression in a ground-based model of microgravity, and the Fur protein was shown to play a role in stress resistance alterations induced in the same model (7).
Experiments were performed to verify a role for Hfq in the spaceflight response using a cellular growth apparatus that serves as a ground-based model of microgravity conditions termed the rotating wall vessel (RWV) bioreactor (
Wild type and isogenic hfq mutant strains of S. typhimurium were grown in the RWV in the LSMMG and 1×g positions and assayed for the acid stress response and macrophage survival. While the wild type strain displayed a significant difference in acid resistance between the LSMMG and 1×g cultures, this response was not observed in the hfq mutant, which contains a deletion of the hfq gene and replacement with a Cm-r cassette (
Increased Virulence of S. typhimurium Grown in Spaceflight as Compared to Ground Controls.
Since growth during spaceflight and potential global reprogramming of gene expression in response to this environment could alter the virulence of a pathogen, we compared the virulence of S. typhimurium spaceflight samples to identical ground controls as a second major part of our study. Bacteria from flight and ground cultures were harvested and immediately used to inoculate female Balb/c mice via a per-oral route of infection on the same day as Shuttle landing. Two sets of mice were infected at increasing dosages of either flight or ground cultures, and the health of the mice was monitored every 6-12 hours for 30 days. Mice infected with bacteria from the flight cultures displayed a decreased time to death (at the 107 dosage), increased percent mortality at each infection dosage and a decreased LD50 value compared to those infected with ground controls (
To determine any morphological differences between flight and ground cultures, scanning electron microscopic (SEM) analysis of bacteria from these samples was performed. While no difference in the size and shape of individual cells in both cultures was apparent, the flight samples demonstrated clear differences in cellular aggregation and clumping that was associated with the formation of an extracellular matrix (
Salmonella typhimurium genes differentially-regulated during spaceflight mission STS-115
Salmonella typhimurium proteins identified in flight and ground total cell
This example describes experiments designed to test the hypothesis that ion concentrations could be manipulated to prevent the enhanced Salmonella virulence imparted during flight. Salmonella cultured in varying media conditions aboard STS-115 and STS-123 were analyzed. These experiments allowed the identification of a) media ion composition that prevents spaceflight-induced increases in Salmonella virulence, and b) commonalities and differences in Salmonella gene expression between growths of the same pathogen in different media during spaceflight. As with spaceflight growth in LB, Salmonella grown in M9 media during flight displayed differential expression of many genes, including those associated with either the regulation of, or regulation by the Hfq protein and small regulatory RNAs. Salmonella grown in various media demonstrated that ion concentrations had a direct effect on the virulence of the cultures. Moreover, higher concentrations of phosphate ions present in M9 medium during spaceflight analogue culture altered its pathogenic-related effects, thus providing the first evidence of a mechanism behind this response.
Strains and Media. The virulent, mouse-passaged Salmonella typhimurium derivative of SL1344 termed F3339 was used in all experiments18. Lennox broth (LB) (10 g tryptone, 5 g yeast extract, 5 g NaCl)19, M9 medium (0.4% glucose)9, or LB-M9 salts medium were used as the growth media in all experiments. Phosphate buffered saline (PBS) (Invitrogen, Carlsbad, Calif.) was used to resuspend bacteria for use as inoculum in the flight and ground hardware. The LB-M9 salts medium consisted of LB medium supplemented with the following amounts of ions: 8.54 mM NaCl, 25.18 mM NaH2PO4, 18.68 mM NH4Cl, 22 mM KH2PO4, and 2 mM MgSO4. The RNA fixative RNA Later II (Ambion, Austin, Tex.), was used to preserve nucleic acid and protein. Bacterial cell culture, microarray analysis, MudPIT proteomics, murine infections, and acid stress assays were performed as described previously1. qRT-PCR analysis was performed with primers hybridizing to the indicated genes as described previously using the 16S rRNA gene to normalize samples20. Data from three to nine separate technical replicate reactions was used for each gene in
Bacterial cell culture. Spaceflight and ground cultures were grown in specialized hardware termed fluid processing apparatus (FPA) as described previously1. Briefly, an FPA consists of a glass barrel that can be divided into compartments via the insertion of rubber stoppers and a lexan sheath into which the glass barrel is inserted. Each compartment in the glass barrel was filled with a solution in an order such that the solutions would be mixed at specific time points in flight via two actions: (1) downward plunging action on the rubber stoppers and (2) passage of the fluid in a given compartment through a bevel on the side of the glass barrel such that it was released into the compartment below. Glass barrels and rubber stoppers were coated with a silicone lubricant (Sigmacote, Sigma, St. Louis, Mo.) and autoclaved separately before assembly. A stopper with a gas exchange membrane was inserted just below the bevel in the glass barrel before autoclaving. FPA assembly was performed aseptically in a laminar flow hood in the following order: 2.0 ml media (either LB, M9 or LB-M9) on top of the gas exchange stopper, one rubber stopper, 0.5 ml PBS containing bacterial inoculum (approximately 6.7×106 bacteria), another rubber stopper, 2.5 ml of either RNA fixative (for gene expression analysis) or media (either LB, M9 or LB-M9 for virulence studies), and a final rubber stopper. Syringe needles (gauge 25⅝) were inserted into rubber stoppers during this process to release air pressure and facilitate assembly. To facilitate group activation of FPAs and to ensure proper containment levels, sets of 8 FPAs were loaded into larger containers termed group activation packs (GAPs). All ground control cultures were incubated in the Orbital Environmental Simulator (OES) room at the Kennedy Space Center, which is linked in real-time to the Shuttle and maintains identical temperature and humidity conditions. After activation, cultures were grown for 25 hours in either spaceflight or ground until either fixation or media supplementation. Upon landing, cultures were received for processing approximately 2.5 hours after Shuttle touchdown.
Microarray analysis. Total cellular RNA purification from cultures grown in M9 media, preparation of fluorescently labeled, single stranded cDNA probes, probe hybridization to whole genome S. typhimurium microarrays, and image acquisition was performed as previously described1,8 using three biological and three technical replicates for each culture condition. Direct microscopic cell counting and spectrophotometric readings indicated that cell numbers in flight and ground biological replicate cultures differed by less than 2-fold. Data analysis was performed using software as described previously1. To obtain the genes comprising the spaceflight stimulon in M9 media, the following parameters were used in Webarray software22: an expression ratio of flight to ground of 1.8 fold or greater or 0.6 or less; a spot quality (Avalue) of greater than 9.5, and p-value of less than 0.05. To identify spaceflight stimulon genes also contained in the Hfq regulon, proteins or genes found to be regulated by Hfq or RNAs found to be bound by Hfq as reported in the indicated references were scanned against the spaceflight microarray data for expression changes within the parameters above11-14.
Multidimensional protein identification (MudPIT) analysis via tandem mass spectrometry coupled to dual nano-liquid chromatography (LC-LC-MS/MS). Acetone-protein precipitates from whole cell lysates obtained from flight and ground cultures grown in M9 media (representing three biological replicates) were subjected to MudPIT analysis using the LC-LC-MS/MS technique (three technical replicates) as described previously1,23,24. Tandem MS spectra of peptides were analyzed with TurboSEQUEST™ v 3.1 and XTandem software, and the data were further analyzed and organized using the Scaffold program1,23,24. Table 6 describes the specific parameters used in Scaffold to identify the proteins in this study.
Murine infection assay. Six to eight week old female Balb/c mice (housed in the Animal Facility at the Space Life Sciences Lab at Kennedy Space Center) were deprived of food and water for approximately 6 hours and then per-orally infected with increasing dosages of S. typhimurium harvested from either flight or ground FPA cultures and resuspended in buffered saline gelatin1. Infectious dosages increasing ten-fold in a range between approximately 1×104 and 1×109 bacteria (thus comprising six infectious dosages per bacterial culture) were used in the infections. Ten mice per infectious dosage were used, 20 μl per dose, and food and water were returned to the animals within 30 minutes post-infection. The infected mice were monitored every 6-12 hours for 30 days. The LD50 value was calculated using the formula of Reed and Muench25.
Ground based RWV cultures and acid stress assays. S. typhimurium cultures were grown in rotating wall vessels (RWVs) for 24 hours at 37 degrees C. in the LSMMG and 1×g orientations in LB, M9, or LB media supplemented with the indicated ions from M9 salts (LB-M9 salts media) and assayed for resistance to pH 3.5 as described previously1,15. The percentage of surviving bacteria present after 45-60 minutes acid stress (compared to the original number of bacteria before addition of the stress) was calculated via serial dilution and CFU plating. A ratio of the percent survival values for the LSMMG and 1×g cultures in all three growth media was obtained (indicating the fold difference in survival between these cultures) and is presented as the acid survival ratio in
Media and virulence in spaceflight±LB. Previous flight experiments aboard STS-115 indicated that S. typhimurium cultured during spaceflight exhibited increased virulence in a murine model of infection1. Briefly, bacteria cultured in LB during spaceflight and identical ground control cultures were harvested and immediately used to inoculate female Balb/c mice via a per-oral route of infection on the same day as Shuttle landing. Mice were infected at increasing dosages of either flight or ground cultures (10 mice per dose), and the health of the mice was monitored every 6-12 hours for 30 days. Previous results showed that mice infected with S. typhimurium grown in LB media in spaceflight aboard STS-115 displayed a decreased time to death and a 2.7 fold decrease in LD50 value compared with those infected with ground control cultures1. To confirm these findings, the identical flight experiment was performed again aboard STS-123. In agreement with the previous experiment, mice infected with S. typhimurium grown in spaceflight aboard STS-123 displayed a decreased time to death and a 6.9 fold decrease in LD50 value compared with those infected with ground control cultures (
Media and virulence in spaceflight±M9. Because of the strong association between nutrient composition of the growth media and the extent of changes observed in S. typhimurium responses in ground-based studies in the RWV, we evaluated S. typhimurium virulence using cultures grown in M9 minimal media in separate experiments aboard Space Shuttle missions STS-115 and STS-123. The procedures were otherwise identical to those described for LB media growth. However, M9 cultures from both missions displayed dramatically different virulence characteristics from those observed with LB cultured bacteria (
To further elucidate the effect of media composition on the virulence characteristics of S. typhimurium grown during spaceflight, an additional growth medium was used that consisted of LB media supplemented with specific salts used in the preparation of M9 media. These specific salts were chosen because our quantitative trace elemental analysis showed them to be at significantly different levels in the two media. Specifically, the elemental analysis indicated that the M9 medium had dramatically higher concentrations of phosphate (61-fold higher than the LB media) and magnesium (18-fold higher than the LB media). Other notable differences in the M9 medium included higher levels of sulfate (3.6-fold higher than the LB media), chloride (3-fold higher than the LB media), and potassium (2.4-fold higher than the LB media). Thus, as follow-up flight experiment aboard STS-123, S. typhimurium virulence was evaluated using cultures grown in LB media supplemented with 25.18 mM NaH2PO4, 22 mM KH2PO4, 18.68 mM NH4Cl, 8.54 mM NaCl, and 2 mM MgSO4 (designated as LB-M9 salts media), thereby bringing the levels of these salts in LB media to the same as those in M9 media. Interestingly, Salmonella cultured in LB-M9 salts media displayed virulence characteristics similar to those observed when only the M9 media was used (
Transcriptional and proteomic analysis. To determine which Salmonella genes changed expression in response to spaceflight culture in M9 minimal media, total bacterial RNA was isolated from fixed flight and ground samples, qualitatively analyzed to ensure lack of degradation, quantified, and then reversed transcribed into labeled, single-stranded cDNA. The labeled cDNA was co-hybridized with differentially-labeled S. typhimurium genomic DNA to whole genome S. typhimurium microarray slides. Statistically-significant differences in gene expression between the flight and ground M9 samples (above 1.8-fold increase and below 0.6-fold decrease in expression) were obtained (see Materials and Methods for details). 38 genes were found differentially-expressed in flight M9 cultures as compared to identical ground controls under these conditions (Table 5). Most notably, several genes involved in motility (9 genes: flgA, flgC, flgF, flgG, cheY, fliC, fliT, fliM, fljB), the formation of the Hyc hydrogenase (4 genes: hydN, hycF, hycD, hyB), and the Suf membrane transporter (3 genes: sufA, sufC, yhnA/sufE) were identified as differentially expressed. In addition, several genes encoding small regulatory RNA molecules (THI, csrB, rnpB, tke1) were also identified. The proteomes of fixed cultures from M9 flight and ground samples were also obtained via multi-dimensional protein identification (MudPIT) analysis. 173 proteins were identified as expressed in the flight and ground cultures, with 81 being present at statistically different levels in these samples (Table 6) indicating differential expression or stability. Notably, several proteins involved iron utilization and uptake (Fur, cytoplasmic ferritin, F—S cluster formation, bactoferrin, siderophore receptor TonB, iron transport protein, iron-dependent alcohol dehydrogenase, and ferric enterobactin receptor) and ribosome structure (L7, L32, S20, S13, S11 S19, L14, L33, S4, L4) were identified as differentially expressed. Collectively, these transcriptional and proteomic gene expression changes form the first documented bacterial spaceflight stimulon in minimal growth media.
The LB and M9 spaceflight stimulons. The S. typhimurium gene expression data from the analysis above in M9 medium were compared with the results from our previous gene expression analysis in LB medium for spaceflight and RWV cultures. Genes from each data set were cross-compared to each other to identify common genes that were present as differentially-expressed in both media. After this analysis, 15 genes (including adjacent genes) of the 38 identified as transcriptionally altered in response to spaceflight in M9 medium were also identified as differentially expressed in either spaceflight or ground-based microgravity analogue RWV culture in LB medium. This represents 39% ( 15/38) of the total genes found in the M9 transcriptional analysis.
This analysis was subsequently extended to include genes that also belong to the same directly-related functional or regulatory gene group (i.e. not necessarily the same gene or operon, but genes that function or are regulated as part of the same mechanism such as motility), and discovered that the percentage of common genes between analysis in M9 and LB media was 73% ( 28/38) (Table 5). The functional groups of genes that we identified as regulated by spaceflight or ground-based spaceflight analogue culture in both M9 and LB media included those involved in flagellar-based motility, Hyc hydrogenase formation, Suf transporter formation and other ABC transporters, and small regulatory RNA molecules (genes indicated in the section above). Additionally, there are also 8 “stand alone” genes that are believed to be not co-regulated with these gene groups and include four genes encoding putative, uncharacterized proteins (yaiA, trpD, yfiA, yhcB, grxB, acpP, yfiD, STM4002). Several genes encoding proteins identified in the spaceflight and ground proteomic analysis of M9 cultures were also identified in the gene expression analysis of M9 and LB cultures as well (Table 6).
Results from our previous studies indicated that 32% of the S. typhimurium genes identified as differentially regulated in spaceflight in LB medium belonged to a regulon of genes controlled by the conserved RNA-binding protein Hfq1. A requirement of hfq for alterations in Salmonella acid resistance and macrophage survival was demonstrated in response to a ground-based microgravity analogue model1. Therefore, the results of our spaceflight M9 microarray and proteomic analysis were scanned for members of a regulon of genes whose expression and activity is regulated by or regulates Hfq, or whose protein products form a functional regulatory complex with Hfq11-14. Consistent with the previous observations in LB, four small non-coding regulatory RNA genes (THI, csrB, rnpB, tke1) and three mRNA transcripts (rpoS, sufE, fliC) regulated by Hfq were observed in the microarray analysis in M9 media (7 of 38 or 18%).
When the hits from the proteomic analysis were scanned for relationships to the Hfq regulon, 28 of the 81 proteins (34%) found to be differentially expressed in response to spaceflight in M9 media belonged to the Hfq regulon, or are part of a directly related functional group of proteins that are regulated by Hfq. Several observations led to the Hfq regulon members being highlighted in our M9 proteomic analysis: 1) Hfq promotes the expression of a large class of ribosomal structural proteins, and we found differential expression of several of these genes in spaceflight (L7/L12, L32, S20, S13, S11, S19, SA, L14, L33, S4, L4); 2) Hfq regulates the expression of the Fur protein and other genes involved in iron metabolism, and we found that Fur and other iron-related genes are differentially regulated by spaceflight in M9 medium (Fur, Dps, NifU, FepA); 3) Several other proteins encoded by genes belonging to the Hfq regulon were also found in this analysis: NmpC, Tpx, PtsI, PtsH, SucC, LeuB, CysP, DppA, OppA, RpoZ, CsrA, RpoB, NlpB. This data, taken together with the microarray data, indicates the commonalities of the spaceflight response in Salmonella in both LB and M9 media, and represents the first common genes that have been identified to be regulated by spaceflight and/or ground based spaceflight analogue culture in both rich and minimal media.
Real time PCR analysis. To further confirm the commonalities observed in global gene expression analysis in response to spaceflight in both LB and M9 media, targeted quantitative real time PCR assays were performed using cDNA synthesized from total RNA harvested from spaceflight and ground cultures in LB and M9 media as templates (
Role of phosphate ion. Salmonella was previously demonstrated to consistently and reproducibly alter its acid tolerance response when grown in the RWV using LB medium15. To support findings from spaceflight that the supplementation of LB media with selected M9 salts disrupts S. typhimurium responses to this environment, cultures containing LB media, M9 media, and LB-M9 salts media were grown in the RWV at low shear modeled microgravity (LSMMG) and control orientations and evaluated for changes in acid tolerance. As demonstrated previously, cultures of S. typhimurium grown in LB media in the RWV (LSMMG) displayed altered acid resistance as compared to control cultures. However, no difference in acid tolerance was observed with cultures grown in M9 media or in LB-M9 salts media (
It was found that the increased S. typhimurium virulence observed with cultures grown in spaceflight in LB medium as compared to identical ground controls is not exhibited with cultures grown in M9 medium. Based upon the quantified differences in ion concentrations between LB and M9 media, LB medium was supplemented with inorganic ions to the same levels as those found in M9 medium. This supplementation was sufficient to prevent the enhanced Salmonella virulence imparted during flight. Subsequent testing in ground-based spaceflight analogue culture conditions indicted that the altered acid tolerance exhibited by Salmonella during culture in LB alone was prevented with the addition of inorganic phosphate. These results demonstrate a direct correlation between phosphate ion concentration and the phenotypic response of Salmonella to the environment of spaceflight analogue culture. The spaceflight-induced molecular genetic responses of S. typhimurium cultured in different growth media (LB versus M9) were also compared using whole genome transcriptional and proteomic analyses. Despite the multiple phenotypic differences in response to spaceflight between the two media, several common genes and gene families were altered in expression in both media during spaceflight culture. Identification of these genes whose expression is commonly regulated by the low fluid shear environment of spaceflight provides key targets whose expression can be manipulated to control microbial responses, including use for development of vaccines and therapeutics. As identified in this study, these targets include gene systems involved in flagellar-based motility, Hyc hydrogenase formation, Suf transporter formation and other ABC transporters, ribosomal structure, iron utilization, and small regulatory RNA molecule expression and function. Many of the genes that were found differentially expressed during spaceflight culture of S. typhimurium in M9 media were also consistent with those reported in LB culture for this same organism under identical conditions. In both cases, many of these genes are found in regulons that are controlled by or regulate the activity of the Hfq protein. The findings further highlight Hfq as a global regulator to target for further study to understand the mechanism used by Salmonella to respond to spaceflight, spaceflight analogue systems, and other physiological low fluid shear environments.
Salmonella typhimurium genes altered in expression during growth in M9 minimal media
Salmonella typhimurium proteins identified via MudPit analysis as present
This example describes a general protocol for culturing a live attenuated Salmonella enterica serovar Typhimurium vaccine strain under low sedimental shear conditions, and to evaluate the immunogenicity of the vaccine strain cultured in this manner in a mouse model. A recombinant attenuated Salmonella enterica serovar Typhimurium anti-pneumococcal vaccine strain x9558 (Δpmi-2426 Δ(gmd-fcl)-26 ΔPfur33::TTaraCPBADfur ΔPcrp527::TTaraCPBADcrp δasdA27::TTaraCPBADc2 ΔaraE25 ΔaraBAD23 ΔrelA198::araCPBADlacITT ΔsopB1925 ΔagfBAC811 ΔfliC180 ΔfljB217) encoding pneumococcal antigen (pspA capsular gene) on plasmid pYA4088 is used in this example as illustration. However, any live attenuated bacterial vaccine strain can be used that carries one or more attenuating mutations of interest—including heterologous recombinant vaccine strains that express foreign antigens to elicit innate humoral and cellular immune responses. Moreover, Lennox broth is used for Salmonella strain culture in this example, any growth media and incubation conditions required to cultivate the strain of interest can be used. In addition, while the Rotating Wall Vessel bioreactor is used as the culture modality to achieve low sedimental shear stress, other culture environments that achieve this environment can also be used (including the spaceflight environment).
Live attenuated bacterial vaccine strain growth conditions. The attenuated Salmonella vaccine strain is first grown in Lennox broth (L-broth) as a static or aerated overnight culture at 37° C. Cultures are then inoculated at a dilution of 1:200 into 50 ml of L broth and subsequently introduced into the RWV bioreactor. Care is taken to ensure that the reactor is completely filled with culture media and no bubbles are present (i.e. zero headspace). The reactor vessel is oriented to grow cells under conditions of low sedimental shear or control sedimental shear. Two different RWV bioreactors, one in each physical orientation (low sedimental shear or control sedimental shear, respectively), should be simultaneously inoculated with the bacterial strain. Incubations in the RWV are at 37° C. or room temperature with a rotation rate of 25 rpm. Culture times are for 10 hours (which corresponds to mid-log phase growth) or 24 hours (which corresponds to stationary phase). Cell density is measured as viable bacterial counts plated on L agar for colony forming units per ml (CFU/ml). This is done to ensure that low sedimental shear and control sedimental shear-grown Salmonella are in the same phase of growth for use in subsequent experiments.
Modulations in low shear sedimental culture conditions. Bacterial strains can be grown under the identical conditions above with the exception that the manipulations of the low sedimental shear environments are made within physiological ranges encountered by pathogens in the mammalian host. This can be done by the inclusion of inert beads of different sizes in the RWV bioreactor during cell culture, but other approaches are also possible.
Oral immunization of mice with attenuated Salmonella vaccine strains and protection against challenge with a virulent wild-type strain. Protective immunity elicited by attenuated Salmonella strains cultured under low shear sedimental and control shear sedimental conditions will determined in BALB/c mice following peroral (p.o.) inoculation. Six-to-ten-week-old female BALB/c mice (Charles River Laboratories, Wilmington, Mass.) will be immunized by peroral (p.o.) administration of serial dilutions of a low sedimental shear or control sedimental shear grown attenuated Salmonella vaccine strain. While this example focuses on oral infection of mice, other immunization methods can also be used, including peroral, intraperitoneal, nasal, vaginal administration, among others. Likewise, other hosts can be used for infection, including but not limited to, other animals, animal analogues, plants, insects, nematodes, and cell and tissue cultures from animals, animal analogues and plants. In addition, infections can be administered while both the host and pathogen are simultaneously in a low shear sedimental environment, including spaceflight. Mice are housed in autoclavable micro-isolator cages with free access to standard laboratory food and water for one week before use to allow acclimation. Bacteria for use in these studies are grown in the RWV under the conditions described above, harvested from the bioreactor by dispensing into a 50 ml polypropylene conical tube, and immediately harvested by centrifugation at room temperature for 10 minutes at 7,974×g. Bacteria are immediately resuspended in 1.0 ml buffered saline with gelatin (BSG).
Specifically, mice to be used in p.o. immunization with attenuated live vaccine strains or inoculation with challenge strains are deprived of food and water for 4-6 h. An attenuated Salmonella vaccine strain is grown simultaneously in the RWV bioreactors in the low shear sedimental conditions and control shear sedimental conditions and harvested as described above. Appropriate dilutions of the bacteria (low shear sedimental or control shear sedimental) will be prepared for p.o. inoculation of mice. Results will be obtained from ten mice/inoculum dose. Specifically, ten mice per group will be perorally inoculated with 106, 107, 108, and 109 CFU of the attenuated Salmonella vaccine strain grown under low shear sedimental or control shear sedimental conditions, respectively. Challenge with fully virulent wild-type Salmonella is given orally 30 days after immunization and mice are observed for four weeks thereafter. (Other routes of challenge may also be used). (In the case of recombinant attenuated Salmonella vaccine strain encoding heterologous antigen against another pathogen, challenge will also be with the fully virulent pathogen for which Salmonella carries the heterologous antigen. For example, for the recombinant attenuated Salmonella anti-pneumococcal vaccine strain, challenge would be with fully virulent Streptococcus pneumoniae.) Following challenge, mice will be monitored for signs of disease at least twice daily. These include a hunched posture, scruffy coat, and unwillingness to open eyes or move around. Mortality of the mice will be observed for 30 days. The median lethal dose will be determined by the method of Reed and Muench.
Enumeration of bacteria in mouse tissues. The effect of low sedimental shear on the tissue distribution and persistence of Salmonella in mice will be assessed in vivo by peroral inoculation into six-to-ten-week-old female BALB/c mice. Bacteria are grown and harvested as described above. Quantitation of viable Salmonella in tissues and organs will be performed as described previously from two groups of five mice each in two independent trials. The mice will be euthanized by CO2 asphyxiation at 3, 5, and 7 days postinfection for subsequent harvesting of tissues and enumeration of bacteria to determine colonization of Salmonella. Thereafter, to determine persistence of Salmonella in mice, tissues will be harvested from mice weekly through through 60 days. Fecal pellets will also be collected to monitor shedding of Salmonella throughout the entire duration of the study. The number of Salmonella present in the tissues will be determined by viable counting of serial dilutions of the homogenates on MacConkey agar (Difco, Detroit, Mich.) supplemented with lactose at 1% final concentration. Murine tissues that will be analyzed include Peyer's patches, intestinal epithelium (minus Peyer's patches), liver, spleen and mesenteric lymph nodes.
Measurement and duration of antibody responses by ELISA following infection of animals with live attenuated recombinant bacterial vaccine strains carrying heterologous antigens. Groups of eight mice each will be immunized orally with different doses of a live attenuated Salmonella recombinant vaccine strain carrying a foreign antigen of interest and grown and harvested as described above. The live attenuated recombinant S. typhimurium vaccine strain used for the teaching the claims in this application express the pneumococcal PspA capsular antigen, however, any antigen(s) from any pathogen of interest could be used in these studies. Animal immunizations will be carried out perorally as described above. Booster immunizations may be given to enhance antibody responses to the foreign antigen. Serum samples (retroorbital puncture) and vaginal washings will be collected 2, 4, 6, and 8 weeks after immunization as described previously. Humoral, mucosal and cellular immune responses can be measured against Salmonella and/or to the heterologous antigen that it encodes.
The levels of antibodies present in mouse sera against the pneumococcal PspA capsular antigen and S. typhimurium LPS will be determined using enzyme-linked immunosorbent assay (ELISA) as follows. Ninety-six well Immulon plates (Dynatech, Chantilly, Va.) will be coated with 10 μg of a recombinant pneumococcal PspA capsular surface protein (rPspA) in 0.2 M bicarbonate/carbonate buffer (pH 9.6) at 4° C. overnight. Nonspecific binding sites will be blocked with 1% BSA in phosphate buffered saline (PBS)+0.1% Tween20 (pH 7.4) (blocking buffer) at room temperature for 1 h. Serum samples and vaginal washings will be diluted 1:100 and 1:10, respectively, in blocking buffer. One hundred microliters of the diluted samples will be added in duplicate to the plates and incubated at 37° C. for 2 h. The plates are then washed with PBS+0.1% Tween20 three times. One hundred microliters of biotin-labeled goat anti-mouse IgA or IgG will be added, respectively, and incubated at 4° C. overnight. Alkaline phosphatase-labeled ExtrAvidin (Sigma, St. Louis, Mo.) is added to the plates and incubated at room temperature for 1 h. Substrate solution (0.1 ml) containing p-nitro-phenylphosphate (1 mg/ml) in 0.1 M diethanolamine buffer (pH 9.8) will be added and the optical density of the resulting substrate reaction is read at 405 nm with an automated ELISA reader (BioTech, Burlington, Vt.).
Measurement of central memory T cells following infection of animals with live attenuated recombinant bacterial vaccine strains carrying heterologous antigens. The induction of memory responses is critical for the long-term protective efficacy of vaccines. In particular, the CD4+ CD44high CD62Lhigh and CD8315 +CD44highCD62Lhigh central memory T cells play a central role in the recall response (Krishnan, L., K. Gurnani, C. J. Dicaire, H. van Faassen, A. Zafer, C. J. Kirschning, S. Sad, and G. D. Sprott. 2007. Rapid clonal expansion and prolonged maintenance of memory CD8+ T Cells of the effector (CD44highCD62Llow) and central (CD44highCD62Lhigh phenotype by an archaeosome adjuvant independent of TLR2. J. Immunol. 178:2396-2406). Thus, the effect of low sedimental shear cultivation of the vaccine strain on stimulation of a memory T cell response will be evaluated. Eight weeks after immunization, spleens will be isolated from mice and compared to control (unimmunized and mock infected) mice. Splenic cells will be stimulated with antigen (rPspA) and then examined by FACS analysis for T cell markers indicative of memory cells.
Measurement of innate immune responses/cytokines following infection of animals with live attenuated recombinant bacterial vaccine strains carrying heterologous antigens. Six weeks after immunization, sera from immunized and control mice will be subjected to Bio-Plex Protein Array System (Bio-Rad, Hercules, Calif.) or ELISPOT analysis to determine antigen stimulation of cytokine production as described previously (Li Y, Wang S, Xin W, Scarpellini G, Shi Z, Gunn B, Roland K L, Curtiss R III. A sopB Deletion Mutation Enhances the Immunogenicity and Protective Efficacy of a Heterologous Antigen Delivered by Live Attenuated Salmonella enterica Vaccines. Infect Immun. 2008 Sep. 2. Epub ahead of print). The cytokine secretion profiles from splenic lymphocytes will be compared (other tissues may also be utilized). Both Th1 and Th2 cytokines will be profiled. Briefly, samples will be incubated with antibody-coupled beads for 1 h with shaking. Beads will be washed 3× with wash buffer to remove unbound protein and subsequently incubated with biotinylated detection cytokine-specific antibody for 1 h with shaking. The beads will then be washed once more followed by incubation for 10 min with streptavidin-phycoerythrin. After this incubation, beads will be washed and resuspended in assay buffer, and the contents of each well will be subjected to the flow-based Bio-Plex Suspension Array System, which identifies each different color bead as a population of protein and quantifies each protein target based on secondary antibody fluorescence. Cytokine concentrations will be calculated by Bio-Plex Manager software using a standard curve derived from a recombinant cytokine standard.
Immunoblotting for detection and quantiation of heterologous antigens carried by attenuated Salmonella vaccine strains from serum of infected animals. For immunoblotting, the S. typhimurium recombinant attenuated strain x9558 that carries the pneumococcal capsular antigen on plasmid pYA4088 will be grown with aeration overnight at 37° C. Five hundred microliters of each culture will be pelleted and resuspended in 2× sample loading buffer and boiled for 5 min. Protein preparations will be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (12.5% polyacrylamide) and prepared for Coomassie brilliant blue staining or Western blot analysis. In addition to heterologous antigen detection, detection of Salmonella antigens can also be performed using Salmonella outer membrane protein antigens or LPS.
This example describes the stress response phenotypes observed for a recombinant attenuated Salmonella anti-pneumococcal vaccine strain, X9558 pYA4088 (Δpmi-2426 Δ(gmd-fcl)-26 ΔPfur33::TTaraCPBADfur ΔPcrp527::TTaraCPBADcrp ΔasdA27::TTaraCPBADc2 ΔaraE25 ΔaraBAD23 ΔrelA198::araCPBADlacITT ΔsopB1925 ΔagfBAC811 ΔfliC180 ΔfljB217) during low fluid shear culture in the RWV bioreactor. The results indicate that low fluid shear culture in the RWV confers increased protection to the strain to survive virulence-related stress responses, including an increased ability to survive acid stress, thermal stress, and oxidative stress. In addition, low fluid shear culture of this strain decreased its biofilm formation. These low fluid shear-conferred phenotypes are all important features that could significantly enhance the immunogenicity and protection of this vaccine strain.
Differences in the O.D. readings before and after the RWVs were stopped:
There is a marked difference in the O.D. reading for the 1×G condition before and after the RWV was stopped. This difference in OD was due to the enhanced biofilm formation in the 1×g condition as compared to the LSMMG condition, and not differences in cell numbers—as the cells were in the same phase of growth under the two conditions, however, most of the cells were sessile (attached to the membrane) in the 1×g RWV, leaving fewer cells in the supernatant (planktonic cells) for cell counting. When the RWVs were taken apart, a biofilm like substance was observed on the membrane with the 1×G culture. No such biofilm formation was observed for the LSMMG cultures.
The data show that the cells can withstand thermal stress in the LSMMG condition much better as compared to the 1×G condition at 55° C.
In this experiment, LSMMG and 1×g cultures were subjected to acid stress and assayed for resistance to pH 3.5 (by addition of an amount of concentrated citrate buffer that has been previously determined to give this pH value) as described previously (Nickerson et al., Infection and Immunity, 2000; Wilson et al, Applied and Environmental Microbiology, 2002). The pH level during the assay was monitored using pH strips, and then confirmed with a pH electrode at the end of the assay. The percentage of surviving bacteria present after 30, 45, 60 and 90 minutes of acid stress (compared to the original number of bacteria before addition of the stress) was calculated via serial dilution and CFU plating. These results were compared identical control cultures that were not subjected to acid stress (no citrate buffer was added), but instead were allowed to sit on the bench top statically and at time points 30, 60 and 90 minutes about 100 ul were taken out and added to 9.9 ml of PBS. It can be seen that the LSMMG cells survive acid stress better than 1×G grown cells
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In a separate experiment, oxidative stress (in the form of hydrogen peroxide) was applied to cells.
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It can be seen that the LSMMG cells survive the oxidative stress much better than the 1×G grown cells after 30 min.
The above results were obtained using the recombinant attenuated Salmonella anti-pneumococcal vaccine strain X9558 that carries the pneumococcal capsular antigen on plasmid pYA4088. The S. typhimurium UK-1 wild-type parent strain showed similar results.
Cultures of P. aeruginosa flew in the same flight experiment with S. typhimurium aboard STS-115 to determine changes in gene expression compared to otherwise identical ground controls. Preliminary results from microarray analysis indicate that of the 226 P. aeruginosa genes that were differentially regulated during spaceflight, 59 (˜23%) are regulated by Hfq—including those encoding ribosomal proteins, iron metabolic pathways, carbon metabolic pathways, cytoplasmic and periplasmic sigma factors, and ion response pathways. See Tables 7 and 8. In addition, it has been shown that LSMMG-cultured P. aeruginosa demonstrated an increased sensitivity to acid stress as compared to the control orientation. Collectively, these data supports an association between the gene expression and phenotypic response of P. aeruginosa during flight and LSMMG culture and the Hfq regulon.
Scanning electron microscopy (SEM) (
Initial studies were performed on S. aureus N315 to determine phenotypic responses to culture in LSMMG and 1×G. When grown in LSMMG, S. aureus displayed a distinct decrease in the golden carotenoid pigmentation compared to growth in the control orientation, based upon a colorimetric assay for the primary carotenoid, staphyloxanthin. Notably, the addition of phosphate ion (25 mM Na2HPO4) to the media increased both the pigmentation of the LSSMG and 1×G control cultures. This latter finding is in agreement with the finding described above that environmental ions modulate the LSMMG acid stress response in Salmonella.
This invention was made with Government support under Contract No. NASA grant NCC2-1362 awarded by the United States NASA. The Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US08/75818 | 9/10/2008 | WO | 00 | 7/26/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60993208 | Sep 2007 | US | |
| 61079160 | Jul 2008 | US | |
| 61032252 | Feb 2008 | US | |
| 61045419 | Apr 2008 | US |
