Mycobacterium tuberculosis (Mtb) has infected about two billion people and the lung of a single infected patient contains more than a billion bacilli. Poor treatment compliance leads to selection and increasing spread of multi- and extremely-drug resistant strains.
Provided are isolated polypeptides comprising the amino-terminal domain of Mycobacterium tuberculosis porin A (MtpA), wherein the polypeptide is a porin monomer. Also provided are isolated polypeptides comprising the carboxy-terminal domain of MtpA, wherein the polypeptide is a toxin.
Also provided are methods of treating or preventing a Mycobacterium tuberculosis (Mtb) infection in a subject with or at risk of developing a Mtb infection. The methods comprise administering to the subject a first agent that modulates the activity of a Mycobacterium tuberculosis porin (Mtp) and a second agent that treats or prevents the Mtb infection.
Provided herein are chimeric porin polypeptides. The chimeric porin polypeptides comprise a first polypeptide comprising an amino-terminal domain of a Mycobacterium tuberculosis porin and a second polypeptide comprising an antigen.
Also provided are methods of eliciting in a subject an immune response to an antigen. The methods comprise administering to the subject a modified Mycobacterium, wherein the modified Mycobacterium comprises a chimeric porin polypeptide described herein.
Mycobacterium tuberculosis porin (Mtp) oligomers are provided herein. The Mtp oligomers can comprise 2 to 12 isolated polypeptides comprising the amino-terminal domain of MtpA.
Also provided are nucleic acid sequences encoding a single-chain Mycobacterium tuberculosis porin (Mtp) oligomer. The nucleic acid sequences can comprise at least a first and a second porin encoding nucleotide sequence and a linker nucleic acid sequence encoding an amino acid linker. The first nucleotide sequence can encode a first Mtp monomer and the second nucleotide sequence can encode a second porin monomer.
Methods of detecting an analyte in a conductive liquid medium are provided. The methods comprise applying an electric field to the Mtp oligomers described herein, wherein the Mtp oligomers have a vestibule and a constriction zone that define a tunnel. The Mtp oligomers can be positioned between a first and second conductive liquid medium and the first or second conductive liquid medium can comprise the analyte.
Also provided are methods of inducing necrotic cell death in a subject. The methods comprise administering to the subject an isolated polypeptide comprising the carboxy-terminal domain of MtpA. The carboxy-terminal domain of MtpA can also be used in methods of treating or preventing excessive eye blinking, muscle pain disorders, hyperhidrosis, or cervical dystonia in a subject.
Further provided are methods of screening for an agent that modulates the activity of a Mtp porin. The methods comprise contacting a cell with the agent to be tested, wherein the cell comprises the Mtp, and determining the activity of the Mtp. An increase or decrease in the activity of the Mtp as compared to a control indicates that the agent modulates the activity of the Mtp.
Methods of screening for an agent that neutralizes a toxin produced by Mycobacterium tuberculosis are provided herein. The methods comprise contacting a cell with the agent to be tested, contacting the cell with the toxin, and detecting the level of cell death in the presence of the agent. A decrease in the level of cell death as compared to a control indicates the agent neutralizes the toxin.
The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.
Provided herein are isolated polypeptides comprising the amino-terminal domain of Mycobacterium tuberculosis porin A (MtpA). The polypeptide including the amino-terminal domain of MtpA can be a porin monomer. Optionally, the amino-terminal domain of MtpA comprises amino acids 1-443 of SEQ ID NO:1. Optionally, the isolated polypeptides comprise SEQ ID NO:1.
Also provided are isolated polypeptides comprising the carboxy-terminal domain of MtpA. The carboxy-terminal domain of MtpA can be a toxin. Optionally, the amino-terminal domain of MtpA comprises amino acids 650-846 of SEQ ID NO:1.
Optionally, in the polypeptides disclosed herein, the amino-terminal domain, the carboxy-terminal domain, or both the amino-terminal and carboxy-terminal domain of MtpA comprise one or more mutation(s).
Also provided are chimeric porin polypeptides. The chimeric porin polypeptides comprise a first polypeptide comprising the amino-terminal domain of a Mycobacterium tuberculosis porin (Mtp) and a second polypeptide comprising a selected antigen. The chimeric porin polypeptide comprises an antigen not naturally associated with the amino-terminal domain of Mtp. Optionally, the Mtp is MtpA. The amino-terminal domain of MtpA can, for example, comprise amino acids 1-443 of SEQ ID NO:1.
Optionally, the chimeric porin polypeptide can further comprise a third polypeptide comprising an amino acid linker. The amino acid linker can comprise a cleavage sequence.
The antigen can, for example, be selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a prion antigen, and a parasitic antigen. An antigen is a molecule recognized by the immune system. A viral antigen can, for example, include any viral antigenic molecule or inactivated or attenuated virus (e.g., an envelope protein, a structural protein, and/or a capsid protein). Optionally, the viral antigen is envelope glycoprotein GP 120 of HIV. A bacterial antigen can, for example, include any bacterial antigenic molecule or inactivated or attenuated bacterium. Optionally, the bacterial antigen can be, for example, CFP-10 or ESAT-6 of Mycobacterium tuberculosis. A fungal antigen can, for example, include any fungal antigenic molecule or inactivated or attenuated fungus. Optionally, the fungal antigen is glucanase Crfl of Aspergillus fumigatus. A prion antigen can, for example, comprise any prion antigenic molecule or inactivated or attenuated prion. A parasitic antigen can, for example, include any parasitic antigenic molecule or inactivated or attenuated parasite. Optionally, the parasitic antigen is 19 kDa-merozoite surface protein-1 (MSP-1(19) of Plasmodium falciparum. As a general rule, surface antigens are most useful for provoking an immune response and can include cellular lipids proteins, proteoglycans or portions thereof.
Optionally, the antigen of interest comprises a cancer antigen. Cancer antigens can, for example, include any antigenic molecule associated with the cancer. An antigenic molecule associated with the cancer can, for example, include an antigenic portion of a polypeptide that is overexpressed in the cancer, an antigenic portion of a polypeptide that is expressed on the cell surface of cancer cells, or an antigenic portion of the proteoglycans or lipids on the cell surface of the cells of the cancer. Optionally, the cancer antigen is human prostate stem cell antigen (PSCA). Cancer antigens are known in the art, see, e.g., Sonpavde et al., Urol. Oncol. 25:451-9 (2007); Suri, Expert Opin. Biol. Ther. 6:379-89 (2006); Saleh et al., Curr. Pharm. Des. 11:3461-73 (2005), Bensalah et al., Prostate Cancer Prostatic Dis. 11:112-20 (2008); Disis et al., Breast Dis. 20:3-11 (2004); McNeel, Cancer Chemother. Biol. Response Modif. 22:247-61 (2005); Jager et al., Curr. Opin. Immunol. 14:178-82 (2002); Obata et al., Breast Cancer 6:305-11 (1999), which are incorporated herein for cancer antigens and methods of making and using them.
Also provided are Mycobacterium tuberculosis porin (Mtp) oligomers. The Mtp oligomers can, for example, comprise 2 to 12 of the disclosed isolated polypeptides comprising the amino-terminal domain of MtpA, the carboxy-terminal domain of MtpA, or both. For example, the isolated polypeptide comprises amino acids 1-443 of SEQ ID NO:1.
Also provided are nucleic acid sequences encoding the chimeric polypeptides or a single-chain Mycobacterium tuberculosis porin (Mtp) oligomer. The nucleic acid sequences encoding the single-chain Mtp oligomer comprise at least a first and a second porin encoding nucleotide sequence and a linker nucleic acid sequence encoding an amino acid linker. The first nucleotide sequence can, for example, encode a first Mtp monomer. The second nucleotide sequence can, for example, encode a second porin monomer, wherein the second encoded porin monomer is a Mycobacterium porin monomer. Optionally, the nucleic acid can further comprise a third, fourth, fifth, sixth, seventh, and eighth nucleotide sequence. The third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences can, for example, encode a third, fourth, fifth, sixth, seventh, and eighth porin monomer, wherein the porin monomer is a Mycobacterium porin monomer.
Optionally, at least one of the encoded porin monomers comprises a wild-type Mycobacterium tuberculosis porin A (MtpA) monomer. Optionally, all of the encoded porin monomers of the single-chain oligomer comprise a wild-type MtpA monomer. The encoded wild-type MtpA monomer can comprise SEQ ID NO:1. Optionally, one or more of the encoded porin monomers can comprise amino acids 1-443 of SEQ ID NO:1.
The encoded amino acid linker can, for example, comprise 10 to 20 amino acids. Optionally, the encoded amino acid linker comprises 15 amino acids. Optionally, the encoded amino acid linker comprises a (GGGGS)3 (SEQ ID NO:4) peptide sequence.
As used herein, the terms peptide, polypeptide, or protein are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein interchangeably to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more.
As discussed above, the polypeptides provided herein have a desired function. The polypeptide comprising the amino-terminal domain of MtpA functions as a porin monomer. The polypeptide comprising the carboxy-terminal domain of MtpA functions as a toxin. The chimeric porin polypeptide functions to elicit an immune response in a subject by providing an antigenic polypeptide to the subject.
As with all peptides, polypeptides, and proteins, including fragments thereof, it is understood that additional modifications in the amino acid sequence of amino-terminal or carboxy-terminal domains of MtpA or the chimeric porin polypeptide can occur that do not alter the function of the peptides, polypeptides, or proteins. Such modifications include conservative amino acid substitutions and are discussed in greater detail below. Thus, the polypeptides described herein can be modified so long as the desired function is maintained. It is understood that one way to define any known modifications and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the modifications and derivatives in terms of identity to specific known sequences. Specifically disclosed are polypeptides which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the amino-terminal domain, carboxy-terminal domain, SEQ ID NO:1, or the chimeric porin polypeptide are provided herein. For example, provided are polypeptides which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to SEQ ID NO:1. Those of skill in the art readily understand how to determine the identity of two polypeptides. For example, the identity can be calculated after aligning the two sequences so that the identity is at its highest level.
Another way of calculating identity can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection.
The same types of identity can be obtained for nucleic acids by, for example, the algorithms disclosed in Zuker, Science 244:48-52 (1989); Jaeger et al., Proc. Natl. Acad. Sci. USA 86:7706-10 (1989); Jaeger et al. Methods Enzymol. 183:281-306 (1989), which are herein incorporated by reference for at least material related to nucleic acid alignment. It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein.
Protein modifications include amino acid sequence modifications. Modifications in amino acid sequence may arise naturally as allelic variations (e.g., due to genetic polymorphism), may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences), or may arise due to environmental influence (e.g., exposure to ultraviolet light), such as induced point, deletion, insertion and substitution mutants. These modifications can result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations. Amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional, or deletional modifications. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional modifications are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table 1 and are referred to as conservative substitutions.
Modifications, including the specific amino acid substitutions, are made by known methods including the methods described in the Examples below. By way of example, modifications are made by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the modification, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis.
Nucleic acids that encode the polypeptide sequences, variants, and fragments thereof are disclosed. These sequences include all degenerate sequences related to a specific protein sequence, i.e., all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequences.
Also provided are methods of treating or preventing a Mycobacterium tuberculosis (Mtb) infection in a subject with or at risk of developing a Mtb infection. A subject with Mtb includes subjects diagnosed with an Mtb infection. A subject at risk includes a subject with a known exposure or with a potential exposure to a Mtb source, include, for example, at a prison or medical facility. The methods comprise administering to the subject a first agent that modulates the activity of a Mycobacterium tuberculosis porin (Mtp) and a second agent that treats or prevents the Mtb infection. The Mtp can, for example, comprise Mycobacterium tuberculosis porin (MtpA).
The first agent can, for example, target the amino-terminal domain of MtpA. By targeting the amino-terminal domain of MtpA it is meant that the first agent can bind directly or indirectly to block the function of the amino-terminal domain. Optionally, the first agent targets amino acids 1-443 of SEQ ID NO:1. Optionally, the first agent modulates the activity of the Mtp by increasing the uptake of the second agent by the Mtp as compared to uptake in the absence of the first agent. Optionally, the first agent modulates the activity of the Mtp by decreasing the uptake of one or more nutrients or carbon sources by the Mtp as compared to the uptake in the absence of the agent.
Optionally, the one or more nutrients can be selected from the group consisting of carbon, oxygen, hydrogen, nitrogen, phosphorous, sulfur, potassium, magnesium, calcium, iron and trace elements. Optionally, the carbon source can be selected from the group consisting of sugars, amino acids, glycerol, fatty acids, lipids, and detergents. Nutrients and carbon sources for bacteria are known in the art, see, e.g., Lim, Microbiology, 3rd Ed., Kendall/Hunt Publishing (2003).
The methods for treating or preventing a Mtb infection in a subject with or at risk of developing a Mtb infection can, for example, comprise administering to the subject a first agent that neutralizes a toxin produced by Mycobacterium tuberculosis and a second agent that treats or prevents the Mtb infection. By neutralizing a toxin, it is meant that the first agent eliminates or reduces the function of the toxin. Optionally, the toxin comprises the carboxy-terminal domain of Mycobacterium tuberculosis porin A (MtpA). The first agent can, for example, target a cleavage sequence of MtpA. The cleavage sequence of MtpA can be located between the amino-terminal domain and carboxy-terminal domain of MtpA. Optionally, the first agent blocks cleavage of the carboxy-terminal domain of MtpA from the amino-terminal domain of MtpA. By blocking cleavage, it is meant that the agent prevents cleavage of the MtpA polypeptide between the carboxy- and amino-terminal domains. Without intending to be limited by theory, the first agent can, for example, bind the cleavage sequence and prevent access to the cleavage sequence by the protease responsible for cleavage of the polypeptide. Alternatively, the first agent can target the protease responsible for cleavage and prevent that protease from cleaving the MtpA polypeptide.
The first agent can, for example, be selected from the group consisting of a nucleic acid molecule, a polypeptide, a small molecule, or a peptidomimetic. The nucleic acid molecule can, for example, be selected from the group consisting of an antisense sequence, a short inhibitory RNA (siRNA) sequence, or a microRNA (miRNA) sequence. The polypeptide can, for example, be an antibody or a fragment thereof.
The second agent can, for example, be selected from the group consisting of ampicillin, tetracycline, chloramphenicol, ethambutol, isoniazid, p-aminosalicylic acid, cycloserine, vancomycin, streptomycin, clarithomycin, erythromycin A, rifampicin, ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, and norfloxacin.
As used herein, a nucleic acid molecule can be a short-interfering RNA (siRNA) sequence or a micro-RNA (miRNA) sequence. A 21-25 nucleotide siRNA or miRNA sequence can, for example, be produced from an expression vector by transcription of a short-hairpin RNA (shRNA) sequence, a 60-80 nucleotide precursor sequence, which is subsequently processed by the cellular RNAi machinery to produce either a siRNA or miRNA sequence. Alternatively, a 21-25 nucleotide siRNA or miRNA sequence can, for example, be synthesized chemically. Chemical synthesis of siRNA or miRNA sequences is commercially available from such corporations as Dharmacon, Inc. (Lafayette, Colo.), Qiagen (Valencia, Calif.), and Ambion (Austin, Tex.). A siRNA sequence preferably binds a unique sequence within the target mRNA with exact complementarity and results in the degradation of the target mRNA molecule. A siRNA sequence can bind anywhere within the target mRNA molecule. A miRNA sequence preferably binds a unique sequence within the target mRNA with exact or less than exact complementarity and results in the translational repression of the target mRNA molecule. A miRNA sequence can bind anywhere within the target mRNA sequence, but preferably binds within the 3′ untranslated region of the target mRNA molecule. Methods of delivering siRNA or miRNA molecules are known in the art. See, e.g., Oh and Park, Adv. Drug. Deliv. Rev. 61(10):850-62 (2009); Gondi and Rao, J. Cell Physiol. 220(2):285-91 (2009); and Whitehead et al., Nat. Rev. Drug. Discov. 8(2):129-38 (2009).
As used herein, a nucleic acid molecule can be an antisense nucleic acid sequence. Antisense nucleic acid sequences can, for example, be transcribed from an expression vector to produce an RNA which is complementary to at least a unique portion of the target mRNA and/or the endogenous gene which encodes the target. Hybridization of an antisense nucleic acid under specific cellular conditions results in inhibition of target protein expression by inhibiting transcription and/or translation.
The term antibody is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. The term can also refer to a human antibody and/or a humanized antibody. Examples of techniques for human monoclonal antibody production include those described by Cole et al. (Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol. 147(1):86-95 (1991)). Human antibodies (and fragments thereof) can also be produced using phage display libraries (Hoogenboom et al., J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)). The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551-5 (1993); Jakobovits et al., Nature 362:255-8 (1993); Bruggermann et al., Year in Immunol. 7:33 (1993)).
Also provided are methods of eliciting in a subject an immune response to an antigen. The methods comprise administering to the subject a modified Mycobacterium, wherein the modified Mycobacterium comprises any of the chimeric porin polypeptides disclosed herein. Optionally, the modified Mycobacterium comprises any of the chimeric porin polypeptides disclosed herein. Optionally, the modified Mycobacterium is selected from an attenuated Mycobacterium tuberculosis or Mycobacterium Bovis BCG. By attenuated, it is meant that the strain is modified to be less virulent than an unmodified strain. Strategies for using modified Mycobacterium are known in the art, see, e.g., Sambandamurthy and Jacobs, Microbes Infect. 7:955-61 (2005) and Zhang et al., Scand. J. Immunol. 72:349-57 (2010).
Also provided are methods of detecting an analyte in a conductive liquid medium. The method comprises applying an electric field to the Mycobacterium tuberculsos porin (Mtp) oligomers disclosed herein. The Mtp oligomers can, for example, have a vestibule and a constriction zone that define a tunnel, wherein the Mtp oligomer is positioned between a first conductive liquid medium and second conductive liquid medium. Optionally, the first or second conductive liquid medium comprises an analyte.
The method of detecting an analyte can, for example, comprise measuring an ion current as the analyte interacts with the tunnel to provide a current pattern. The appearance of a blockade in the current pattern indicates the presence of an analyte. Optionally, the methods comprise identifying the analyte, wherein identifying the analyte comprises comparing the current pattern to a known current pattern obtained using a known analyte under the same conditions.
The analyte can, for example, be selected from the group consisting of a nucleotide, a nucleic acid, an amino acid, a polypeptide, a protein, a polymer, a drug, an ion, a pollutant, a nanoscopic object, or a biological warfare agent. Optionally, the analyte is a polymer comprising more than one subunit. A polymer can, for example, comprise a polypeptide, a protein, or a nucleic acid.
The Mtp oligomers can, for example, be used to distinguish a first subunit of a polymer from at least a second subunit of the polymer. Distinguishing may comprise measuring the ion current produced as the first and second subunits separately translocate through the tunnel to produce a first and second current patter. The first and second current patters can, for example, differ from each other. Optionally, the Mtp oligomers can sequence the polymer. Sequencing the polymer can comprise measuring the ion current or optical signals as each unit of the polymer is separately translocated through the tunnel to provide a current pattern that is associated with each subunit of the polymer. Each current patter is compared to a known current patter of a known subunit under the same conditions, such that the polymer is sequenced.
The Mtp oligomers can, for example, also be used to determine the concentration, size, molecular weight, shape, or orientation of an analyte, or any combination thereof.
Also provided are methods of inducing necrotic cell death in a subject. Also provided are methods of treating or preventing excessive eye blinking, muscle pain disorders, hyperhidrosis, or cervical dystonia in a subject. The methods comprise administering to the subject an isolated polypeptide comprising the carboxy-terminal domain of MtpA. Optionally, the carboxy-terminal domain of MtpA comprises amino acids 650-834 of SEQ ID NO:1.
Also provided are methods of reducing wrinkles in a subject. The methods comprise administering into one or more muscles of the subject an isolated polypeptide comprising the carboxy-terminal domain of MtpA, wherein the contraction of the muscles is associated with wrinkles (e.g., facial muscles to reduce facial wrinkles). Optionally, the carboxy-terminal domain of MtpA comprises amino acids 650-834 of SEQ ID NO:1.
Further provided are methods of screening for an agent that modulates the activity of a Mtp. The methods comprise contacting a cell with the agent to be tested, wherein the cell comprises the Mtp, and determining the activity of the Mtp. An increase or decrease in the activity of the Mtp as compared to a control indicates that the agent modulates the activity of the Mtp. As used herein, a control can be a cell that is not contacted with the agent to be tested. Optionally, the Mtp is MtpA. Optionally, the Mtp comprises the amino-terminal domain of MtpA. The amino terminal domain of MtpA can, for example, comprise amino acids 1-443 of SEQ ID NO:1.
The activity of the Mtp can be selected from the group consisting of uptake of an antibiotic, output of antibiotic, and uptake of one or more nutrients or carbon sources. The antibiotic can be selected from the group consisting of ampicillin, tetracycline, chloramphenicol, ethambutol, isoniazid, p-aminosalicylic acid, cycloserine, vancomycin, streptomycin, clarithomycin, erythromycin A, rifampicin, ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, and norfloxacin. The carbon source can, for example, be glycerol.
Further provided are methods of screening for an agent that neutralizes a toxin produced by Mycobacterium tuberculosis. The methods comprise contacting the cell with the agent to be tested, contacting the cell with the toxin, and detecting the level of cell death in the presence of the agent. A decrease in cell death as compared to a control indicates the agent neutralizes the toxin. As used herein, a control can be a cell that is not contacted with the agent to be tested. Optionally, the toxin comprises the carboxy-terminal domain of MtpA. The carboxy-terminal domain of MtpA can, for example, comprise amino acids 650-834 of SEQ ID NO:1.
Provided herein are compositions containing the provided polypeptides, nucleic acid molecules, small molecules, and/or peptidomimetics and a pharmaceutically acceptable carrier described herein. The herein provided compositions are suitable for administration in vitro or in vivo. By pharmaceutically acceptable carrier is meant a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained. The carrier is selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject.
Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. The pH of the solution is generally about 5 to about 8 or from about 7 to 7.5. Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptides. Matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. Certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Carriers are those suitable for administration of the agent, e.g., the polypeptide, nucleic acid molecule, small molecule, and/or peptidomimetic, to humans or other subjects.
The compositions are administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. The compositions are administered via any of several routes of administration, including topically, orally, parenterally, intravenously, intra-articularly, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, intrahepatically, intracranially, nebulization/inhalation, or by installation via bronchoscopy. Optionally, the composition is administered by oral inhalation, nasal inhalation, or intranasal mucosal administration. Administration of the compositions by inhalant can be through the nose or mouth via delivery by spraying or droplet mechanism, for example, in the form of an aerosol.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives are optionally present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners and the like are optionally necessary or desirable.
Compositions for oral administration include powders or granules, suspension or solutions in water or non-aqueous media, capsules, sachets, or tables. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders are optionally desirable.
Optionally, the nucleic acid molecule or polypeptide is administered by a vector comprising the nucleic acid molecule or a nucleic acid sequence encoding the polypeptide. There are a number of compositions and methods which can be used to deliver the nucleic acid molecules and/or polypeptides to cells, either in vitro or in vivo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based deliver systems. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein.
As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without degradation and include a promoter yielding expression of the nucleic acid molecule and/or polypeptide in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general are described by Coffin et al., Retorviruses, Cold Spring Harbor Laboratory Press (1997), which is incorporated by reference herein for the vectors and methods of making them. The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virol. 61:1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83 (1986); Haj-Ahmad et al., J. Virol. 57:267-74 (1986); Davidson et al., J. Virol. 61:1226-39 (1987); Zhang et al., BioTechniques 15:868-72 (1993)). The benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infections viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.
The provided polypeptides and/or nucleic acid molecules can be delivered via virus like particles. Virus like particles (VLPs) consist of viral protein(s) derived from the structural proteins of a virus. Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).
The provided polypeptides can be delivered by subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).
The provided polypeptides can be delivered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO 2006/110728.
Non-viral based delivery methods can include expression vectors comprising nucleic acid molecules and nucleic acid sequences encoding polypeptides, wherein the nucleic acids are operably linked to an expression control sequence. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clonetech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.). Vectors typically contain one or more regulatory regions. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters, e.g. β-actin promoter or EF1α promoter, or from hybrid or chimeric promoters (e.g., CMV promoter fused to the β-actin promoter). Of course, promoters from the host cell or related species are also useful herein.
Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5′ or 3′ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 base pairs (bp) in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
The promoter can be an inducible promoter (e.g. chemically or physically regulated promoter). A chemically regulated promoter can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal. Optionally, the inducible promoter is an acetamide-inducible promoter. A physically regulated promoter can, for example, be regulated by environmental factors, such as temperature and light. The promoter can be a cell type specific promoter (e.g. neuronal-specific, renal-specific, cardio-specific, liver-specific, or muscle-specific). A cell-type specific promoter is only expressed in the cell-type in which it is intended to be expressed. The promoter can be a promoter that is expressed independent of cell type. Examples of promoters that can be expressed independent of cell type include the cytomegalovirus (CMV) promoter, the Raus sarcoma virus (RSV) promoter, the adenoviral E1A promoter, and the EF-1α promoter. Optionally, the promoter is a psmyc promoter.
Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5′ or 3′ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 base pairs in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
The vectors also can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers. A marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Examples of marker genes include the E. coli lacZ gene, which encodes β galactosidase, green fluorescent protein (GFP), and luciferase. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, blasticidin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak, New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxy- or amino-terminus.
As used throughout, subject can be a vertebrate, more specifically a mammal (e.g., a human). The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, patient or subject may be used interchangeably and can refer to a subject with a disease or disorder (e.g., a Mycobcaterium tuberculosis (Mtb) infection or at risk for the same). The term patient or subject includes human and veterinary subjects.
A subject at risk of developing a disease or disorder (e.g., a Mtb infection) includes a subject with a known exposure or a potential exposure to Mycobacterium tuberculosis (e.g., due to employment at a prison or medical care facility) or due to the prevalence of Mycobacterium tuberculosis at a specific location (e.g., a prison or hospital), or show early signs or symptoms of the disease or disorder. A subject currently with a disease or disorder has one or more than one symptom of the disease or disorder and may have been diagnosed with the disease or disorder.
The methods and agents as described herein are useful for both prophylactic and therapeutic treatment. For prophylactic use, a therapeutically effective amount of the agents described herein are administered to a subject prior to onset (e.g., before obvious signs of Mtb infection) or during early onset (e.g., upon initial signs and symptoms of Mtb infection). Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of Mtb infection. Prophylactic administration can be used, for example, in the preventative treatment of subjects with a predisposition to Mtb infection (e.g., hospital workers). Therapeutic treatment involves administering to a subject a therapeutically effective amount of the agents described herein after diagnosis of an Mtb infection.
According to the methods taught herein, the subject is administered an effective amount of the agent. The terms effective amount and effective dosage are used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiologic response. Effective amounts and schedules for administering the agent may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex, type of disease, the extent of the disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
As used herein the terms treatment, treat, or treating refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
As used herein, the terms prevent, preventing, and prevention of a disease or disorder refers to an action, for example, administration of a therapeutic agent, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or exacerbation of one or more symptoms of the disease or disorder. As used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level. Such terms can include but do not necessarily include complete elimination.
Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to a number of molecules including the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.
Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.
Chemicals and Enzymes.
Hygromycin B was purchased from Calbiochem (San Diego, Calif.). All other chemicals were purchased from Merck (Whitehouse Station, N.J.) or Sigma (St. Louis, Mo.) at the highest purity available. Enzymes for DNA restriction and modification were from New England Biolabs (Ipswich, Mass.) and Invitrogen (Carlsbad, Calif.). Oligonucleotides were obtained from IDT (Coralville, Iowa).
Bacterial Strains, Media and Growth Conditions.
E. coli DH5α was used for cloning experiments and was routinely grown in Luria-Bertani broth at 37° C. M. smegmatis strains were grown at 37° C. in Middlebrook 7H9 medium (Difco) supplemented with 0.2% glycerol and 0.01% Tyloxapol or on Middlebrook 7H10 plates supplemented with 0.5% glycerol. M. bovis BCG (Strain Institute Pasteur) was grown in Middlebrook 7H9 medium (Difco) supplemented with 0.2% glycerol, 0.01% Tyloxapol and 10% OADC (Remel; Lenexa, Kans.) or on Middlebrook 7H10 plates supplemented with 0.5% glycerol and 10% OADC (Remel). Antibiotics were used when required at the following concentrations: hygromycin (200 μg/ml for E. coli, 50 μg/ml for mycobacteria), kanamycin (30 μg/ml). Hartmans-de Bont (HdB) medium (Smeulders et al., J. Bacteriol. 181:270-83 (1999)) supplemented with 0.01% Tyloxapol and appropriated carbon source (1% Tween-80 or 0.1% glycerol) was used as a minimal medium for growth experiments.
Construction of Plasmids.
Plasmids used are listed in Table 2; oligonucleotides used are listed in Table 3. To construct a new integrative vector without a bla resistance cassette and with low copy number in E. coli, plasmid pML113 (Wolschendorf et al., J. Bacteriol. 189:2435-42 (2007)) was digested with SspI and ClaI followed by T4 Polymerase treatment to remove sticky ends. The pBR322 origin of replication was cloned in from pET21a(+) (Novagen; Madison, Wis.), digested with FspI and DraI. In order to be able to use PacI restriction site for further applications, the obtained plasmid was digested with PacI, followed by blunt-end reaction with T4 polymerase and re-ligation resulting in construction of pML2008. MspA expression cassette under control of pimyc was cloned from pMN013 (Stephan et al., Antimicrob. Agents Chemother. (2004)) by digestion with ClaI followed by fill-in reaction with T4 Polymerase and digestion with SpeI. The obtained fragment was cloned into pML2008 digested with SpeI and PmeI resulting in pML2009. PCR amplification of rv3903c (also referred to as Mycobacterium tuberculosis porin (MtpA)) was done from genomic DNA of H37Rv (obtained from Colorado State University as part of the National Institutes of Health (NIAID) Contract HHSN266200400091C entitled “Tuberculosis Vaccine Testing and Research Materials”) using the oligonucleotides CN1537 and CN1397, which introduced the PacI restriction site at the 5′-end and HindIII restriction site at the 3′-end. The obtained PCR fragment and pML2009 were digested with PacI and HindIII and ligated resulting in pML2010. To obtain complementation vector pML2046 for the N-terminal domain of MtpA (amino acids (aa) 1-444), PCR amplification was done using CN1537 and CN1729, followed by digestion with PacI and HindIII and ligation into pML2009 digested with the same enzymes. M. smegmatis expression vector pML2061 was obtained by digestion of pML2046 and pMN016 (Stephan et al., Mol. Microbiol. 58:714-30 (2005)) with PmeI and HindIII. To obtain a tagged version of MtpA, two step PCR amplification was employed: (1) the His-tag was added by using CN1393 (introduction of NdeI site on 5′ end) and CN1824; (2) the HA-tag was added by using CN1393 and CN1825 (introduction of HindIII site on 3′ end and EcoRV site between MtpA and tags). The obtained PCR fragment was digested with NdeI and HindIII and ligated into pNIT-1::gfp (Pandey et al., Tuberculosis 89:12-6 (2009)) digested with the same enzymes resulting in pML2024. To exchange the resistance cassette, pML2024 was digested with HpaI and XbaI and ligated into pMS2 (Kaps et al., Gene 278:115-24 (2001)) digested with EcoRV and XbaI to obtain pML2031. The nitrile-inducible vector pML2040 for the N-terminal domain of MtpA (aa 1-444) was obtained by PCR amplification of MtpA using CN1393 and CN1720 followed by NdeI and EcoRV digestion and ligation into pML2031 digested with the same enzymes. To construct the E. coli expression vector for the N-terminal domain of MtpA (aa 49-444), the gene was codon optimized for efficient expression in E. coli and synthesized by GenScript (Piscataway, N.J.). Domain 1 (without signal sequence), was amplified by PCR using two step protocol: (i) 5′-end Strep-tag was introduced with CN1806 and 3′-end HA-tag was introduced with CN1809, (ii) 5′-end NdeI site was introduced with CN1753 and 3′-end His-tag and HindIII was introduced with CN1781. The obtained PCR fragment was digested with NdeI and HindIII and cloned into pET-28b(+) digested with the same enzymes.
GGCGTAGTCCGGCACGTCGTACGGGTACGGCGTGGTCGGTT
Subcellular Fractionation of M. smegmatis.
Experiments were carried out as described previously with some modifications (Song et al., Tuberculosis 88:526-44 (2008)). Briefly, M. smegmatis mc2155 containing nitrile-inducible full-length MtpA (pML2024) or the N-terminal domain (pML2040) were grown until an OD600 of 0.2 was reached. 5 μM isovalernitrile was added to induce protein expression. After 48 hours of growth, cultures were harvested by centrifugation and washed twice with PBS (140 mM NaCl, 2 mM KCl, 10 mM K2HPO4/KH2PO4 pH 7.4) containing 1 mM PMSF and 1 mM EDTA. The cells were resuspended and lysed by sonication (10 minutes, 12 Watt output power). Unbroken cells were removed by low speed centrifugation (4,000×g for 15 minutes). The resulting supernatant was ultra-centrifuged at 100,000×g for 1 hour to separate cytosolic proteins (SN-100.1) from membrane fraction (P-100.1). P-100.1 was washed extensively to remove all cytosolic and membrane-attached proteins. All samples were mixed with protein loading buffer (160 mM Tris-Cl pH 7.0, 12% SDS, 32% glycerol, 0.4% Bromophenol blue), boiled for 10 minutes and loaded on the 10% SDS-PAGE gel. The protein gel was blotted overnight at 50 mA in transfer buffer (25 mM Tris base, 192 mM Glycine, 0.1% SDS, 20% methanol) onto a polyvinylidene difluoride (PVDF) membrane (Amersham; Piscataway, N.J.). Tagged versions of full-length Rv3903c and the N-terminal domain were detected with anti-HA-HRP antibody (Sigma), MspA was detected with rabbit antiserum against MspA (pAK #813) (Kartmann et al., J. Bacteriol. 181:6543-6 (1999)) using ECL plus kit (Pierce; Rockford, Ill.). LabWorks (UVP; Upland, Calif.) chemoluminescence imaging system and the software were used to visualize and quantify the luminescence.
Proteinase Accessibility Assay.
The assays were done as described previously (Song et al., Tuberculosis 88:526-44 (2008)). PE-PGRS-33HA (pMV61015.1) (Cascioferro et al., Mol. Microbiol. 66:1536-47 (2007)) and PhoAHA (pML970) (Song et al., Tuberculosis 88:526-44 (2008)) were used as controls for proteinase accessibility assay. Strains were grown as described above. Proteinase K treatment was done at 4° C. for 30 minutes.
Purification of the N-Terminal Domain of MtpA.
Nitrile induction of the N-terminal domain of MtpA (aa 1-444) was done in M. smegmatis mc2155 carrying plasmid pML2040 as described above. The cells were harvested, washed twice with PBS buffer, and disrupted at 4° C. using a Sonicator 3000 ultrasonic liquid processor (Misonix, Inc.; Farmingdale, N.Y.) (15 minutes, 12 W output power). The pellet was obtained by low speed centrifugation at 4,000×g and was treated with 1 mg/ml of lysozyme and 0.01 mg/ml of DNaseI for 2 hours at 37° C. The sonication step was performed again as described above. The pellet obtained by low speed centrifugation (4,000×g) contains the majority of the N-terminal domain of MtpA and therefore was used for further purification steps. The proteins were solubilized by incubation with 0.8% sodium dodecyl sulphate (SDS) overnight at 37° C. Non-soluble material was removed by centrifugation at 16,000×g for 10 minutes at room temperature. Extracted proteins were then loaded onto a 1.7 ml POROS 20MC Ni-NTA column (BioLogic duoflow, BioRad HPLC system) (Bio-Rad; Hercules, Calif.) and eluted off using an imidazole gradient from 0 M to 0.5 M. Purification was done according to the manufacturer's protocol. Anion-exchange purification was done next using 1.7 ml POROS 20 HZ anion-exchange column as described elsewhere (Siroy et al., J. Biol. Chem. 283:17827-37 (2008)). Elution was done using salt concentration gradient ranging from 10 mM to 2 M NaCl. To perform gel purification, protein samples were incubated at 37° C. for 1 hour and loaded on 8% SDS-PAGE gel. To visualize proteins, reverse staining by imidazole-zinc salts was performed (Fernandez-Patron et al., Biotechniques 12; 564-73 (1992)) and subsequently confirmed using Western blot analysis. Protein bands of interest were excised from the gel, and eluted using D-Tubes (Novagen). Electroelution was done using cathode buffer (0.1 M Tris-HCl pH 8.25, 0.1 M Tricine, 0.1% SDS) overnight at 50 mA (4° C.). The obtained protein was dialyzed overnight at 4° C. using D-Tube Dialyzer kit (EMD Chemicals; Gibbstown, N.J.) (according to manufacturer's protocol) against PBS containing 0.5% n-octylpolyethylene oxide (OPOE, Alexis Biochemicals; Farmingdale, N.Y.). The channel forming activity of the purified protein was analyzed using the lipid bilayer method. Protein concentrations were determined using BCA kit (Pierce). In addition, a calibration curve with known amounts of bovine serum albumin (BSA) was used to estimate protein concentrations in Coomassie Blue-stained SDS-PAGE gels using the imagine analysis software (LabWorks 4.6, UVP). For protein expression and purification of the N-terminal domain of MtpA (aa 49-444) from E. coli, BL21 carrying pML2069 plasmid was used. Using a previously published protocol (Song et al., Tuberculosis 88:526-44 (2008)), the N-terminal domain was found to be localized both in soluble fractions and in inclusion bodies. Soluble fractions were pooled together and Ni-affinity purification was performed as described above. Strep-tag batch purification was done using Strep-Tactin Sepharose (IBA; Gottingen, Germany) according to the manufacturer' instruction. 0.5% OPOE was added to the elution buffer. Gel elution, dialyzes and protein concentration determination were done as described above.
Channel Activity Analysis.
The single-channel conductance of the N-terminal domain of MtpA was analyzed as described previously (Siroy et al., J. Biol. Chem. 283:17827-37 (2008)). As a negative control for contaminations or intrinsic ability of buffer to form channels in bilayer, a gel peace of approximately the same size which does not contain any detectable proteins was cut from the SDS-PAGE gel and treated the same way as described above for the N-terminal domain of MtpA. At least 3 different membranes were recorded (at 10 and 100 mV) using this sample before addition of the protein.
Glycerol Uptake Experiments.
Glycerol uptake experiments were carried out as described previously (Danilchanka et al., Antimicrob. Agents Chemother. 52:3127-34 (2008b)) with some modifications. The cells were harvested at an OD600 of 0.5-1.0 by centrifugation, washed twice in the uptake buffer (50 mM Tris pH 6.9, 15 mM KCl, 10 mM (NH4)2SO4, 1 mM MgSO4, 0.02% Tyloxapol), and resuspended in the same buffer (mean dry weight is 0.5-1 mg/ml). The cells were pre-incubated at 37° C. for 5 minutes (M. smegmatis) or 15 minutes (M. bovis BCG) before addition of [14C] glycerol (specific activity: 146 mCi/mmol). The final concentration of radioactively-labeled glycerol was 1.5 μM for M. smegmatis and 3 μM for M. bovis BCG. The experiment was performed in triplicate at least two independent times.
Determination of Antibiotic Resistance.
Determination of the minimal inhibitory concentration (MIC) was done using the microplate Alamar Blue assay (MABA) (Franzblau et al., J. Clin. Microbiol. 36:362-6 (1998)) with some modifications (Danilchanka et al., Antimicrob. Agents Chemother. 52:2503-11 (2008); Danilchanka et al., Antimicrob. Agents Chemother. 52:3127-34 (2008b)). The final drug concentrations were as follows: for isoniazid and ethambutol, 0.03125-1 μg/ml; for streptomycin and rifampicin, 0.04 to 1.28 μg/ml; for levofloxacin, ofloxacin and clarithromycin, 0.0625-2 μg/ml; for p-aminosalicylic acid and moxifloxacin, 0.25-8 μg/ml; for ciprofloxacin and norfloxacin, 0.5-16 μg/ml; for chloramphenicol and cycloserine, 1 to 32 μg/ml; for vancomycin, 2.5 to 80 μg/ml; for erythromycin A and tetracycline, 8 to 256 μg/ml; for ampicillin, 30 to 960 μg/ml. The MABA was performed in triplicate at least two independent times.
Sensitivity to Nitric Oxide.
M. bovis BCG strains were grown in 7H9/OADC/0.01% Tyloxapol to an OD600 of 2. The cultures were pelleted down, washed and re-suspended in 1/10 of original volume. 0.1-ml aliquots of the suspension were added to 0.9 ml of fresh media containing 0 mM, 5 mM, 25 mM and 100 mM sodium nitroferricyanide (III) dehydrate (SNP, Sigma). Each suspension was incubated at 37° C. (200 rpm) for 0, 1, 3 or 7 days. Serial dilutions of the cultures were plated on 7H10/OADC/Hyg plates and incubated at 37° C. for 3 weeks, after which colony forming units (CFU) were counted. The data were recalculated as the percent survival ([number of CFU exposed at day X/number of CFU unexposed at day X]×100%). Each strain was tested in triplicate; assay was repeated three independent times.
Macrophage Experiments.
Human acute monocytotic leukemia cells (THP-1) were maintained as described previously (Jordao et al., Cell Microbiol. 10:529-48 (2008)). Differentiation of THP-1 monocytes into macrophages was done overnight with 50 mM phorbol 12-myreistate 13-acetate (PMA). Human monocyte-derived macrophages (HMDM) were obtained from a buffy coat preparation kindly provided by Instituto Português do Sangue, and prepared as previously described (Jordao et al., Cell Microbiol. 10:529-48 (2008)). The THP-1 cells were infected with a multiplicity of infection (MOI) of 20 and HMDMs were infected with MOI between 1 and 3. In each experiment, after 3 hours of infection, the cells were washed 3 times with PBS to remove non-internalized bacteria. At different time points after infection (3 hours, 1, 3, 5 and 7 days), the cells were washed with PBS and lysed with 1% Igepal (Sigma) solution in water. Serial dilutions were performed in water and plated on Middlebrook 7H10 medium supplemented with 10% OADC. Colony forming units (CFU) were counted upon 2 weeks of incubation at 37° C. When required, HMDM were treated with human IFN-γ (200 IU) overnight before infection.
Analysis of Secondary Structure of Rv3903c.
Analysis was done as described previously (Siroy et al., J. Biol. Chem. 283:17827-37 (2008)).
Statistical Analysis.
Data were presented as mean±standard deviation; p values were calculated using Student t test, and a P value of <0.05 was considered to be significant.
In a previous attempt to identify outer membrane proteins of Mtb involved in the uptake of small hydrophilic compounds, a transposon library of M. bovis BCG was constructed and screened for mutants resistant to ampicillin (Danilchanka et al., Antimicrob. Agents Chemother. 52:2503-11 (2008)). There were no potential outer membrane proteins in this library identified based on criteria previously established for prediction of mycobacterial outer membrane proteins (Song et al., Tuberculosis 88:526-44 (2008)). However, OmpATb is an outer membrane protein of Mtb which does not have a classical Sec-signal sequence (Alahari et al., J. Bacteriol. 189:6351-8 (2007)) as observed for porins of gram-negative bacteria (de Keyzer et al., Cell Mol. Life. Sci. 60:2034-52 (2003)). Therefore, the ampicillin-resistant mutants in the library were re-analyzed for hypothetical proteins that have the properties of known porins with the exception of a signal sequence (Song et al., Tuberculosis 88:526-44 (2008)). One of the mutants in the screen, ML1012, contains an insertion in bcg3960c that encodes for a hypothetical alanine-proline-rich protein (
Of note, extended signal sequences with a remarkably conserved N-terminal extended signal peptide region have only been found in Gram-negative bacterial proteins from the classes β- and γ-Proteobacteria, and exclusively in proteins secreted via the Type V secretion pathway, such as autotransporters, two-partner secretion system, and trimeric autotransporters (Desvaux et al., FEMS Microbiol. Lett. 264:22-30 (2006)). However, proteins of the Type V secretion system have not been characterized in mycobacteria yet. Based on available sequence information, only genomes of slow-growing mycobacteria such as Mtb, M. bovis and M. bovis BCG have full-length homologues of the MtpA protein. BLAST analysis (
To examine whether MtpA is a surface associated protein, the full-length protein and its N-terminal domain (aa 1-444) (domain 1) tagged with HA and His-tags at the C-termini, under control of a nitrile-inducible promoter was cloned and expressed them in M. smegmatis mc2155. Both proteins were tested to determine if they were localized with membranes using subcellular fractionation. The cell envelope fraction (P100) and the fraction containing water-soluble cytoplasmic and periplasmic proteins (SN100) were separated by ultracentrifugation of whole cell lysates (100,000×g) (Song et al., Tuberculosis 88:526-44 (2008)). Both the N-terminal domain and the full-length MtpA were associated with the membrane fraction P100 and not with the soluble fraction SN100 (FIG. 2A). Similar fractionation profiles have been found previously for other known mycobacterial outer membrane proteins such as MspA, OmpATb, and Rv1698 (Song et al., Tuberculosis 88:526-44 (2008)). Because the P100 fraction contains inner and outer membrane proteins and membrane-associated proteins, the surface accessibility of MtpA was determined To this end, accessibility of MtpA to proteinase K was tested (Song et al., Tuberculosis 88:526-44 (2008)), which is based on the inability of proteinase K to penetrate the mycobacterial cell envelope. Therefore, only proteins with extracellular loops are digested in whole cells. As controls for the assay, the surface protein PE_PGRS33HA was overexpressed (Cascioferro et al., Mol. Microbiol. 66:1536-47 (2007)), which was significantly digested in the presence of proteinase K, and a periplasmic protein PhoAHA (Siroy et al., J. Biol. Chem. 283:17827-37 (2008)), which was unaffected by the proteinase K treatment (
Mutations in porins of gram-negative bacteria and M. smegmatis can result in decreased growth rates due to decreased uptake of small or hydrophilic solutes such as glucose and glycerol when used as sole carbon sources (Liu and Ferenci, J. Bacteriol. 180:3917-22 (1998); Stephan et al., Mol. Microbiol. 58:714-30 (2005)). To determine if uptake of nutrients in slow-growing mycobacteria is dependent on MtpA, the growth of wild-type M. bovis BCG was analyzed in comparison to the ΔMtpA mutant. No difference in growth was observed when oleic acid was used as a sole carbon source, suggesting that the utilization of hydrophobic solutes as nutrients in the ΔMtpA mutant is the same as in the wild-type strain of M. bovis BCG and the ΔMtpA mutant does not have an intrinsic growth defect. Using rich Middlebrook 7H9 medium containing glycerol, glucose and oleic acid as carbon sources, a slight growth defect was observed for the ΔMtpA mutant. To identify the compound utilization of which was impaired in this medium in the ΔMtpA mutant, minimal Hartmans-de Bond (HdB) media was used with various carbon sources. Growth of the ΔMtpA mutant was only minimal when 0.1% glycerol was added as the sole carbon source (
The bioinformatic analysis (
Based on the observation that both the N-terminal domain of MtpA and MspA complements the growth defect of the ΔMtpA mutant, this domain was speculated to have porin activity. Porins are water-filled channel proteins that enable diffusion of small molecules and ions that are not able to diffuse through lipid membranes directly (Schulz, Curr. Opin. Struct. Biol. 6:485-90 (1996)). Passage of ions through porins can be measured electrophysiologically using a planar lipid bilayer assay. This method detects the incorporation of channel proteins into planar lipid membranes, which corresponds in discrete increases in the membrane current measured over time (Benz et al., Biochim. Biophys. Acta 511:305-19 (1978)). To this end, the N-terminal domain (aa 1-444) of MtpA was expressed under the control of a nitrile-inducible promoter in M. smegmatis. The N-terminal domain of MtpA was extracted from the pellet fraction with 0.8% SDS at 37° C. and purified on Ni+-NTA affinity column followed by anion-exchange purification. To exclude the possibility that the measured channel activity originates from other protein contaminants not detected using Coomassie staining, the protein band corresponding to the N-terminal domain of MtpA was excised from SDS-PAGE gel and purified using electroelution (
To eliminate the possibility that the observed channel activity of the N-terminal domain of MtpA results from a co-purified contaminant protein of M. smegmatis, expression of the protein was carried out in E. coli. A truncated N-terminal domain of MtpA (aa 49-444) was codon optimized and cloned into T7-expression vector, containing an N-terminal Strep-tag, and C-terminal HA- and His-tags. The N-terminal domain of MtpA was purified by Ni2+- and Strep-tag affinity chromatography and gel elution (
Previously, it has been shown that the uptake of small hydrophilic molecules in M. smegmatis is mediated by Msp-like porins (Stahl et al., Mol. Microbiol. 40:451-64 (2001); Stephan et al., Mol. Microbiol. 58:714-30 (2005)). Furthermore, a reduction in the number of porins results in a decreased growth rate (Stephan et al., Mol. Microbiol. 58:714-30 (2005)). Therefore, if the N-terminal domain of MtpA has a porin function, an increase in the rate of growth and nutrient uptake would be observed in a M. smegmatis porin mutant upon expression of the N-terminal domain of MtpA. When an untagged N-terminal domain of MtpA (aa 1-444) was expressed in ML16, a triple porin mutant of M. smegmatis (ΔmspA ΔmspB ΔmspD) (Stephan et al., Mol. Microbiol. 58:714-30 (2005)), a statistically significant increase in an accumulation of [14C] glycerol was observed for this strain compared to ML16 (
Deletion of porins in M. smegmatis results in improved survival in macrophages due to reduced uptake of bactericidal components present in the phagosome (Fabrino et al., Microbes Infect. 11:868-75 (2009); Purdy et al., Mol. Microbiol. 73:844-57 (2009)). To define the role of MtpA for survival of Mtb in macrophages, human acute monocytic leukemia cells (THP-1) were infected with wild-type M. bovis BCG, the ΔMtpA mutant, or the mutant complemented with either full-length MtpA or mspA. As reported previously, THP-1 cells retained a constant low level of live wild-type M. bovis BCG bacteria over seven days of infection (
The production of reactive nitrogen and oxygen intermediates plays a role in control of tuberculosis infection (MacMicking et al., Annu. Rev. Immunol. 15:323-50 (1997)). Previously it has been shown that the absence of msp porin genes confers resistance of M. smegmatis to nitric oxide (Fabrino et al., Microbes Infect. 11:868-75 (2009)). To investigate if MtpA is important for the resistance of slow-growing mycobacteria to nitric oxide, wild-type M. bovis BCG, the ΔMtpA mutant, or the mutant complemented with either full-length MtpA or mspA were treated with the nitric oxide donor sodium nitroferricyanide (III) dehydrate (SNP). Treatment of all strains with SNP in liquid Middlebrook 7H9 medium reduced the number of colonies recovered in a dose- and time-dependent manner (
To determine if increased resistance of the ΔMtpA strain to nitric oxide has any advantages in vivo, infection of human monocyte-derived macrophages (HMDM) with wild-type M. bovis BCG was done with or without γ-interferon treatment. γ-Interferon is known to induce nitric oxide synthase activity in macrophages with a significant increase in nitric oxide levels after the first two days of infection with M. bovis BCG (Jordao et al., Cell Microbiol. 10:529-48 (2008)). Consistent with that, both wild-type and the ΔMtpA strains grew steadily over the course of infection in untreated HMDM, however, less bacteria were recovered for the mutant compared to the wild-type strain. This was especially pronounced on day 7 of infection (
Lack of porins in gram-negative bacteria confers only low level resistance to small, hydrophilic drugs (Nikaido, Microbiol. Mol. Biol. Rev. 67:593-656 (2003)), while loss of porins in M. smegmatis result in higher resistance levels (Danilchanka et al., Antimicrob. Agents Chemother. 52:3127-34 (2008)). Uptake pathways for drugs in slow-growing mycobacteria have not been identified thus far. However, it has been suggested that porins mediate uptake of β-lactam antibiotics, ethambutol, isoniazid and streptomycin (Stephan et al., Antimicrob. Agents Chemother. 48:4163-70 (2004)). To determine if drug resistance of Mtb is mediated by MtpA, a microplate Alamar Blue assay (MABA) was employed. The resistance of M. bovis BCG to several small, hydrophilic antibiotics (Table 4, group A) was increased drastically in the absence of MtpA. For example, a 32- and 16-fold increased minimal inhibitory concentration (MIC) was observed for ampicillin and chloramphenicol respectively in the ΔMtpA strain while the resistance to the anti-tuberculosis drugs ethambutol, isoniazid and p-aminosalicylic acid (PAS) was increased more than eight-fold. Surprisingly, MICs for large and hydrophobic antibiotics such as erythromycin and rifamicin were also significantly increased in the ΔMtpA mutant (Table 4, group C). The resistance of the ΔMtpA strain expressing MspA or MtpA to small or/and hydrophilic antibiotics was identical to that of the wild-type M. bovis BCG (Table 4, groups A, D) suggesting that MtpA makes M. bovis BCG more susceptible to these drugs and that MspA and MtpA function by a similar mechanism.
Attempts to express the MtpA CTD in vivo failed in M. smegmatis, E. coli, and Saccharomyces cerevisiae while in vitro (cell-free) expression allowed recovery of minute amounts of protein. To test whether the CTD of MtpA is toxic in mammalian cells, a pRK7-based vector (Graycar et al., Mol. Endocrinol. 3:1977-86 (1989)) containing the MtpA CTD fused with an HA-tag under control of the cytomegalovirus (CMV) promoter was transiently transfected into the human 293T cell line. Microscopy of cells 24 hours after transfection revealed that the majority of cells were dead (
This application claims benefit of U.S. Provisional Application No. 61/529,010, filed Aug. 30, 2011, which is hereby incorporated herein by reference in its entirety.
This invention was made with government funding under Grant No. RO1 AI63432 from the National Institutes of Health. The government has certain rights in this invention.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US2012/053091 | 8/30/2012 | WO | 00 | 5/30/2014 |
Number | Date | Country | |
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61529010 | Aug 2011 | US |