ABSTRACT
Mast cells are now recognized as effective modulators of innate immunity. We recently reported that mast cells and secreted interleukin-4 (IL-4) effectively control intramacrophage replication of Francisella tularensis Live Vaccine Strain (LVS), and that mice deficient in mast cells or IL-4 receptor (IL-4R(-/-)) exhibit greater susceptibility to pulmonary challenge. In this study, we further evaluated the mechanism(s) by which mast cells/IL-4 control intramacrophage bacterial replication and host cell death, and found that IL-4R(-/-) mice exhibited significantly greater induction of active caspase-3 within lung macrophages than wild-type animals following intranasal challenge with either LVS or the human virulent type A strain SCHU S4. Treatment of LVS-infected bone-marrow-derived macrophages with a pancaspase inhibitor (zVAD) did not alter bacterial replication, but minimized active caspase-3 and other markers (Annexin V and propidium iodide) of cell death, whereas treatment with both rIL-4 and zVAD resulted in concomitant reduction of both parameters, suggesting that inhibition of bacterial replication by IL-4 was independent of caspase activation. Interestingly, IL-4-treated infected macrophages exhibited significantly increased ATP production and phagolysosomal acidification, as well as enhanced mannose receptor upregulation and increased internalization with acidification, which correlated with observations in mast cell-macrophage co-cultures, with resultant decreases in F. tularensis replication.
Subject(s)
Adenosine Triphosphate/biosynthesis , Francisella tularensis , Host-Pathogen Interactions , Interleukin-4/immunology , Mast Cells/immunology , Phagosomes/immunology , Tularemia/immunology , Animals , Caspase 3/metabolism , Cell Death/immunology , Cells, Cultured , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Francisella tularensis/growth & development , Francisella tularensis/immunology , Gene Expression Regulation , Lectins, C-Type/metabolism , Macrophages, Alveolar/enzymology , Macrophages, Alveolar/immunology , Macrophages, Alveolar/pathology , Mannose Receptor , Mannose-Binding Lectins/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Oligopeptides/pharmacology , Organelles/chemistry , Organelles/microbiology , Phagosomes/chemistry , Receptors, Cell Surface/metabolism , Receptors, Interleukin-4/genetics , Receptors, Interleukin-4/immunology , Signal Transduction/immunologyABSTRACT
Salmonella strains that lack or overproduce DNA adenine methylase (Dam) elicit a protective immune response to different Salmonella species. To generate vaccines against other bacterial pathogens, the dam genes of Yersinia pseudotuberculosis and Vibrio cholerae were disrupted but found to be essential for viability. Overproduction of Dam significantly attenuated the virulence of these two pathogens, leading to, in Yersinia, the ectopic secretion of virulence proteins (Yersinia outer proteins) and a fully protective immune response in vaccinated hosts. Dysregulation of Dam activity may provide a means for the development of vaccines against varied bacterial pathogens.
Subject(s)
Bacterial Vaccines/therapeutic use , Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics , Vibrio cholerae/pathogenicity , Yersinia pseudotuberculosis/pathogenicity , Animals , Bacterial Outer Membrane Proteins/metabolism , Bacterial Vaccines/genetics , Cholera/prevention & control , Cholera Vaccines/genetics , Cholera Vaccines/therapeutic use , Gene Expression Regulation, Bacterial , Mice , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Site-Specific DNA-Methyltransferase (Adenine-Specific)/biosynthesis , Vibrio cholerae/physiology , Yersinia pseudotuberculosis/physiology , Yersinia pseudotuberculosis Infections/prevention & controlABSTRACT
Vibrio cholerae causes the diarrheal disease cholera primarily because it expresses a colonization factor (toxin-coregulated pilus; TCP) and a potent toxin (cholera toxin; CT) within the human intestine. While the true environmental signals that induce CT and TCP expression within the intestine remain unknown, much progress has been made identifying the regulatory factors that modulate their expression. Transcriptional regulation of the genes encoding TCP and CT involves a cascade consisting of a number of regulatory factors located on recently acquired mobile genetic elements as well as others residing within the ancestral Vibrio genome. In vivo studies have revealed interesting differences between the regulation of TCP and CT expression in the laboratory and within the intestine.
Subject(s)
Cholera Toxin/genetics , Fimbriae Proteins , Vibrio cholerae/genetics , Vibrio cholerae/pathogenicity , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Cholera/physiopathology , Fimbriae, Bacterial/genetics , Gene Expression Regulation, Bacterial , Humans , Transcription Factors/genetics , Transcriptional Activation , Vibrio cholerae/growth & development , VirulenceABSTRACT
ToxR, the transmembrane regulatory protein required for expression of virulence factors in the human diarrheal pathogen Vibrio cholerae, directly activates and represses the transcription of two outer membrane porins, OmpU and OmpT, respectively. In an attempt to dissect the role of the OmpU and OmpT porins in viability and virulence factor expression, in-frame chromosomal deletions were constructed in the coding sequences of ompU and ompT of V. cholerae. Two separate deletions were introduced into ompU; the first (small) deletion, Delta ompU1, removed the coding sequence for 84 internal amino acids (aa), while the second (large) deletion, Delta ompU2, removed the coding sequence for the entire amino-terminal 274 aa. The Delta ompU1 strain had a growth defect that could not be complemented by episomal expression of full-length ompU. In contrast, a strain with Delta ompU2 displayed wild-type growth kinetics in rich media, suggesting that this is the true phenotype of a strain lacking OmpU and that the truncated OmpU protein, rather than the absence of OmpU, may be the cause for the Delta ompU1 phenotype. A large deletion removing the coding sequence for the entire N-terminal 273 aa of OmpT (Delta ompT) was also constructed in wild-type as well as Delta toxR and Delta ompU2 strains, and these strains displayed wild-type growth kinetics in rich media. However, the Delta ompU2 strain was deficient for growth in deoxycholate compared to wild-type, Delta ompT, and Delta ompU2 Delta ompT strains, reinforcing a positive role for the OmpU porin and a negative role for the OmpT porin in V. cholerae resistance to anionic detergents. The Delta ompU2, Delta ompT, and Delta ompU2 Delta ompT strains exhibited wild-type levels of in vitro virulence factor expression and resistance to polymyxin B and serum and in vivo colonization levels similar to a wild-type strain in the infant mouse intestine. Our results demonstrate that (i) OmpU and OmpT are not essential proteins, as was previously thought; (ii) these porins contribute to V. cholerae resistance to anionic detergents; and (iii) OmpU and OmpT are not essential for virulence factor expression in vitro or intestinal colonization in vivo.
Subject(s)
Adhesins, Bacterial/genetics , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins , DNA-Binding Proteins/genetics , Porins/genetics , Transcription Factors/genetics , Vibrio cholerae/genetics , Adhesins, Bacterial/metabolism , Animals , Animals, Suckling , Bacterial Outer Membrane Proteins/metabolism , Blotting, Western , DNA-Binding Proteins/metabolism , Detergents/pharmacology , Intestine, Small/microbiology , Mice , Mice, Inbred Strains , Mutagenesis, Site-Directed , Phenotype , Porins/metabolism , Sequence Deletion , Transcription Factors/metabolism , Vibrio cholerae/growth & development , Vibrio cholerae/pathogenicity , VirulenceABSTRACT
The human pathogen Vibrio cholerae is a highly motile organism by virtue of a polar flagellum. Flagellar transcriptional regulatory factors have been demonstrated to contribute to V. cholerae virulence, but the role these factors play in the transcription hierarchy controlling flagellar synthesis has been unclear. The flagellar genes revealed by the V. cholerae genome sequence are located in three large clusters, with the exception of the motor genes, which are found in three additional locations. It had previously been demonstrated that the alternative sigma factor sigma54 and the sigma54-dependent activators FlrA and FlrC are necessary for flagellar synthesis. The V. cholerae genome sequence revealed the presence of a fliA gene, which is predicted to encode the alternative flagellar sigma factor sigma28. A V. cholerae DeltafliA mutant strain is non-motile, and synthesizes a truncated flagellum. Vibrio cholerae FliA complements both V. cholerae and Salmonella typhimurium fliA mutants for motility, consistent with its function as an alternative flagellar sigma factor. Analysis of lacZ transcriptional fusions of the V. cholerae flagellar promoters in both V. cholerae and S. typhimurium identified sigma28-, sigma54-, FlrA- and FlrC-dependent promoters, as well as promoters that were independent of all these factors. Our results support a model of V. cholerae flagellar gene transcription as a novel hierarchy composed of four classes of genes. Class I is composed solely of the gene encoding the sigma54-dependent activator FlrA, which along with the sigma54-holoenzyme form of RNA polymerase activates expression of Class II genes. These genes include structural components of the MS ring, switch and export apparatus, as well as the genes encoding both FliA and FlrC. FlrC, along with sigma54-holoenzyme, activates expression of Class III genes, which include basal body, hook and filament genes. Finally, sigma28-holoenzyme activates expression of Class IV genes, which include additional filament genes as well as motor genes. Thus, this novel V. cholerae flagellar hierarchy has incorporated elements from both the sigma54-dependent Caulobacter crescentus polar flagellar hierarchy and the sigma28-dependent S. typhimurium peritrichous flagellar hierarchy.
Subject(s)
Bacterial Proteins/genetics , DNA-Binding Proteins , DNA-Directed RNA Polymerases/genetics , Flagella/genetics , Sigma Factor/genetics , Transcription, Genetic , Vibrio cholerae/genetics , Bacterial Proteins/classification , Chemotactic Factors/genetics , Chemotaxis/genetics , Flagellin , Gene Expression Regulation, Bacterial , Gene Order , Mutation , Promoter Regions, Genetic , RNA Polymerase Sigma 54 , Salmonella typhimurium/geneticsABSTRACT
Throughout most of history, epidemic and pandemic cholera was caused by Vibrio cholerae of the serogroup O1. In 1992, however, a V. cholerae strain of the serogroup O139 emerged as a new agent of epidemic cholera. Interestingly, V. cholerae O139 forms biofilms on abiotic surfaces more rapidly than V. cholerae O1 biotype El Tor, perhaps because regulation of exopolysaccharide synthesis in V. cholerae O139 differs from that in O1 El Tor. Here, we show that all flagellar mutants of V. cholerae O139 have a rugose colony morphology that is dependent on the vps genes. This suggests that the absence of the flagellar structure constitutes a signal to increase exopolysaccharide synthesis. Furthermore, although exopolysaccharide production is required for the development of a three-dimensional biofilm, inappropriate exopolysaccharide production leads to inefficient colonization of the infant mouse intestinal epithelium by flagellar mutants. Thus, precise regulation of exopolysaccharide synthesis is an important factor in the survival of V. cholerae O139 in both aquatic environments and the mammalian intestine.
Subject(s)
Biofilms/growth & development , Flagella , Gene Expression Regulation, Bacterial , Vibrio cholerae/growth & development , Vibrio cholerae/pathogenicity , Animals , Cholera/microbiology , Cholera/physiopathology , Flagella/metabolism , Humans , Mice , Microscopy, Confocal , Mutation , Polysaccharides, Bacterial/genetics , Polysaccharides, Bacterial/metabolism , Vibrio cholerae/genetics , Virulence/geneticsABSTRACT
Recently we described the isolation of spontaneous bacteriophage K139-resistant Vibrio cholerae O1 El Tor mutants. In this study, we identified phage-resistant isolates with intact O antigen but altered core oligosaccharide which were also affected in galactose catabolism; this strains have mutations in the galU gene. We inactivated another gal gene, galE, and the mutant was also found to be defective in the catabolism of exogenous galactose but synthesized an apparently normal lipopolysaccharide (LPS). Both gal mutants as well as a rough LPS (R-LPS) mutant were investigated for the ability to colonize the mouse small intestine. The galU and R-LPS mutants, but not the galE mutant, were defective in colonization, a phenotype also associated with O-antigen-negative mutants. By investigating several parameters in vitro, we could show that galU and R-LPS mutants were more sensitive to short-chain organic acids, cationic antimicrobial peptides, the complement system, and bile salts as well as other hydrophobic agents, indicating that their outer membrane no longer provides an effective barrier function. O-antigen-negative strains were found to be sensitive to complement and cationic peptides, but they displayed significant resistance to bile salts and short-chain organic acids. Furthermore, we found that galU and galE are essential for the formation of a biofilm in a spontaneous phage-resistant rugose variant, suggesting that the synthesis of UDP-galactose via UDP-glucose is necessary for biosynthesis of the exopolysaccharide. In addition, we provide evidence that the production of exopolysaccharide limits the access of phage K139 to its receptor, the O antigen. In conclusion, our results indicate involvement of galU in V. cholerae virulence, correlated with the observed change in LPS structure, and a role for galU and galE in environmental survival of V. cholerae.
Subject(s)
Bacterial Proteins/physiology , Biofilms , Escherichia coli Proteins , Lipopolysaccharides/chemistry , UDPglucose 4-Epimerase/physiology , UTP-Glucose-1-Phosphate Uridylyltransferase , Vibrio cholerae/genetics , Animals , Bacterial Proteins/genetics , Bile/physiology , Fimbriae, Bacterial/physiology , Galactose/metabolism , Mice , Mutation , O Antigens/physiology , Open Reading Frames , UDPglucose 4-Epimerase/genetics , Vibrio cholerae/immunology , Vibrio cholerae/physiology , VirulenceABSTRACT
Bacteriophage K139 was recently characterized as a temperate phage of O1 Vibrio cholerae. In this study we have determined the phage adsorption site on the bacterial cell surface. Phage-binding studies with purified lipopolysaccharide (LPS) of different O1 serotypes and biotypes revealed that the O1 antigen serves as the phage receptor. In addition, phage-resistant O1 El Tor strains were screened by using a virulent isolate of phage K139. Analysis of the LPS of such spontaneous phage-resistant mutants revealed that most of them synthesize incomplete LPS molecules, composed of either defective O1 antigen or core oligosaccharide. By applying phage-binding studies, it was possible to distinguish between receptor mutants and mutations which probably caused abortion of later steps of phage infection. Furthermore, we investigated the genetic nature of O1-negative strains by Southern hybridization with probes specific for the O antigen biosynthesis cluster (rfb region). Two of the investigated O1 antigen-negative mutants revealed insertions of element IS1004 into the rfb gene cluster. Treating one wbeW::IS1004 serum-sensitive mutant with normal human serum, we found that several survivors showed precise excision of IS1004, restoring O antigen biosynthesis and serum resistance. Investigation of clinical isolates by screening for phage resistance and performing LPS analysis of nonlysogenic strains led to the identification of a strain with decreased O1 antigen presentation. This strain had a significant reduction in its ability to colonize the mouse small intestine.
Subject(s)
Bacteriophages/physiology , DNA Transposable Elements , Enterotoxins/physiology , Receptors, Virus , Vibrio cholerae/physiology , Adsorption , Animals , Bacteriophages/drug effects , Base Sequence , Cell Membrane/ultrastructure , Cell Membrane/virology , Chromosomes, Bacterial/genetics , Enterotoxins/analysis , Humans , Kinetics , Lipopolysaccharides/pharmacology , Mice , Molecular Sequence Data , Serotyping , Vibrio cholerae/drug effects , Vibrio cholerae/genetics , Vibrio cholerae/virologyABSTRACT
The transmembrane transcriptional activators ToxR and TcpP modulate expression of Vibrio cholerae virulence factors by exerting control over toxT, which encodes the cytoplasmic transcriptional activator of the ctx, tcp, and acf virulence genes. However, ToxR, independently of TcpP and ToxT, activates and represses transcription of the genes encoding two outer-membrane porins, OmpU and OmpT. To determine the role of ToxR-dependent porin regulation in V. cholerae pathogenesis, the ToxR-activated ompU promoter was used to drive ompT transcription in a strain lacking OmpU. Likewise, the ToxR-repressed ompT promoter was used to drive ompU transcription in a strain lacking both ToxR and OmpT. This strategy allowed the generation of a toxR(+) strain that expresses OmpT in place of OmpU, and a toxR(-) strain that expresses OmpU in place of OmpT. Growth rates in the presence of bile salts and other anionic detergents were retarded for the toxR(+) V. cholerae expressing OmpT in place of OmpU, but increased in toxR(-) V. cholerae expressing OmpU in place of OmpT. Additionally, the toxR(+) V. cholerae expressing OmpT in place of OmpU expressed less cholera toxin and toxin-coregulated pilus, and this effect was shown to be caused by reduced toxT transcription in this strain. Finally, the toxR(+) V. cholerae expressing OmpT in place of OmpU was approximately 100-fold reduced in its ability to colonize the infant-mouse intestine. Our results indicate that ToxR-dependent modulation of the outer membrane porins OmpU and OmpT is critical for V. cholerae bile resistance, virulence factor expression, and intestinal colonization.
Subject(s)
Adhesins, Bacterial/genetics , Bacterial Outer Membrane Proteins/genetics , Bile/physiology , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Bacterial , Intestine, Small/microbiology , Porins/genetics , Transcription Factors/metabolism , Vibrio cholerae/physiology , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Kinetics , Mice , Transcription Factors/genetics , Transcription, Genetic , Vibrio cholerae/genetics , Vibrio cholerae/pathogenicity , VirulenceABSTRACT
The human pathogen Vibrio cholerae specifically expresses virulence factors within the host, including cholera toxin (CT) and the toxin co-regulated pilus (TCP), which allow it to colonize the intestine and cause disease. V. cholerae is a highly motile organism by virtue of a polar flagellum, and motility has been inferred to be an important aspect of virulence, yet the exact role of motility in pathogenesis has remained undefined. The two-component regulatory system FlrB/FlrC is required for polar flagellar synthesis; FlrC is a sigma54-dependent transcriptional activator. We demonstrate that the transcriptional activity of FlrC affects both motility and colonization of V. cholerae. In a purified in vitro reaction, FlrB transfers phosphate to the wild-type FlrC protein, but not to a mutant form in which the aspartate residue at amino acid position 54 has been changed to alanine (D54A), consistent with this being the site of phosphorylation of FlrC. The wild-type FlrC protein, but not the D54A protein, activates sigma54-dependent transcription in a heterologous system, demonstrating that phospho-FlrC is the transcriptionally active form. A V. cholerae strain containing a chromosomal flrCD54A allele did not synthesize a flagellum and had no detectable levels of transcription of the critical sigma54-dependent flagellin gene flaA. The V. cholerae flrCD54A mutant strain was also defective in its ability to colonize the infant mouse small intestine, approximately 50-fold worse than an isogenic wild-type strain. Another mutation of FlrC (methionine 114 to isoleucine; M114I) confers constitutive transcriptional activity in the absence of phosphorylation, but a V. cholerae flrCM114I mutant strain, although flagellated and motile, was also defective in its ability to colonize. The strains carrying D54A or M114I mutant FlrC proteins expressed normal levels of CT and TCP under in vitro inducing conditions. Our results show that FlrC 'locked' into either an inactive (D54A) or an active (M114I) state results in colonization defects, thereby demonstrating a requirement for modulation of FlrC activity during V. cholerae pathogenesis. Thus, the sigma54-dependent transcriptional activity of the flagellar regulatory protein FlrC contributes not only to motility, but also to colonization of V. cholerae.
Subject(s)
Bacterial Proteins/metabolism , DNA-Binding Proteins , Flagella/metabolism , Vibrio cholerae/metabolism , Animals , Aspartic Acid/metabolism , Bacterial Adhesion/genetics , Bacterial Proteins/genetics , Cell Division , DNA-Directed RNA Polymerases/physiology , Flagellin/genetics , Gene Expression Regulation, Bacterial , Hemolysin Proteins/metabolism , Humans , Mice , Movement , Mutation , Phosphates/metabolism , Phosphorylation , Phosphotransferases/genetics , Phosphotransferases/metabolism , RNA Polymerase Sigma 54 , Sigma Factor/physiology , Transcription, Genetic , Vibrio cholerae/genetics , Vibrio cholerae/growth & developmentABSTRACT
The transmembrane regulatory protein ToxR is required for expression of virulence factors in the human diarrheal pathogen Vibrio cholerae, including cholera toxin (CT) and the toxin coregulated pilus (TCP). ToxR is necessary for transcription of the gene encoding a second regulatory protein, ToxT, which is the direct transcriptional activator of CT and TCP genes. However, ToxR, independent of ToxT, directly activates and represses transcription of the outer membrane porins OmpU and OmpT, respectively. The genes encoding TCP and CT (and including ToxT) lie on horizontally acquired genetic elements, while the toxR, ompU, and ompT genes are apparently in the ancestral Vibrio chromosome. The contribution of ToxR-dependent modulation of outer membrane porins to cholera pathogenesis has remained unknown. We demonstrate that ToxR mediates enhanced bile resistance in a ToxT-independent manner. In both classical and El Tor biotypes of V. cholerae, a toxR mutant strain has a reduced minimum bactericidal concentration (MBC) of bile, the bile component deoxycholate (DC), and the anionic detergent sodium dodecyl sulfate (SDS) compared to both wild-type and toxT mutant strains. Classical and El Tor toxR mutant strains also exhibit reduced growth rates at subinhibitory concentrations of DC and SDS. Growth of either V. cholerae biotype in subinhibitory concentrations of bile or DC induces increased ToxR-dependent production of a major 38-kDa outer membrane protein, which was confirmed to be OmpU by Western blot. Measurement of transcription of a ompUp-lacZ fusion in both biotypes reveals stimulation (about two- to threefold) of ToxR-dependent ompU transcription by the presence of bile or DC, suggesting that ToxR may respond to the presence of bile. The toxR mutant strains of three additional human intestinal pathogenic Vibrio species, V. mimicus, V. fluvialis, and V. parahaemolyticus, display lower MBCs of bile, DC, and SDS and have altered outer membrane protein profiles compared to the parental wild-type strains. Our results demonstrate a conserved role for ToxR in the modulation of outer membrane proteins and bile resistance of pathogenic Vibrio species and suggest that these ToxR-dependent outer membrane proteins may mediate enhanced resistance to bile. We speculate that ToxR-mediated bile resistance was an early step in the evolution of V. cholerae as an intestinal pathogen.
Subject(s)
Bacterial Proteins/physiology , Bile/physiology , DNA-Binding Proteins/physiology , Transcription Factors/physiology , Vibrio cholerae/pathogenicity , Vibrio/pathogenicity , Adhesins, Bacterial/genetics , Bacterial Outer Membrane Proteins/genetics , Humans , Sodium Dodecyl Sulfate/pharmacology , Transcription, Genetic , Vibrio/drug effects , VirulenceABSTRACT
The virulence regulatory protein ToxR of Vibrio cholerae is unique in that it contains a cytoplasmic DNA-binding-transcriptional activation domain, a transmembrane domain, and a periplasmic domain. Although ToxR and other transmembrane transcriptional activators have been discovered in other bacteria, little is known about their mechanism of activation. Utilizing degenerate oligonucleotides and PCR, we have amplified internal toxR gene sequences from seven Vibrio and Photobacterium species and subspecies, demonstrating that toxR is an ancestral gene of the family Vibrionaceae. Sequence alignment of all available ToxR amino acid sequences revealed a region between the transcriptional activation and transmembrane domains that displays wide divergence among Vibrio species. We hypothesize that this region merely tethers the transcriptional activation domain to the cytoplasmic membrane and thus can tolerate wide divergence and multiple insertions and deletions. The divergence in the tether region at the nucleotide level may provide a useful tool for the distinction of Vibrio and Photobacterium species.
Subject(s)
Bacterial Proteins , DNA-Binding Proteins/metabolism , Intracellular Membranes/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Vibrio/metabolism , Amino Acid Sequence , Cytoplasm/metabolism , DNA-Binding Proteins/chemistry , Molecular Sequence Data , Photobacterium/metabolism , Phylogeny , Sequence Alignment , Software , Transcription Factors/chemistryABSTRACT
We have previously shown that more prominent immune responses are induced to antigens expressed from multicopy plasmids in live attenuated vaccine vector strains of Vibrio cholerae than to antigens expressed from single-copy genes on the V. cholerae chromosome. Here, we report the construction of a DeltaglnA derivative of V. cholerae vaccine strain Peru2. This mutant strain, Peru2DeltaglnA, is unable to grow on medium that does not contain glutamine; this growth deficiency is complemented by pKEK71-NotI, a plasmid containing a complete copy of the Salmonella typhimurium glnA gene, or by pTIC5, a derivative of pKEK71-NotI containing a 1. 8-kbp fragment that directs expression of CtxB with a 12-amino-acid epitope of the serine-rich Entamoeba histolytica protein fused to the amino terminus. Strain Peru2DeltaglnA(pTIC5) produced 10-fold more SREHP-12-CtxB in supernatants than did ETR3, a Peru2-derivative strain containing the same fragment inserted on the chromosome. To assess immune responses to antigens expressed by this balanced lethal system in vivo, we inoculated germfree mice on days 0, 14, 28, and 42 with Peru2DeltaglnA, Peru2DeltaglnA(pKEK71-NotI), Peru2(pTIC5), Peru2DeltaglnA(pTIC5), or ETR3. All V. cholerae strains were recoverable from stool for 8 to 12 days after primary inoculation, including Peru2DeltaglnA; strains containing plasmids continued to harbor pKEK71-NotI or pTIC5 for 8 to 10 days after primary inoculation. Animals were sacrificed on day 56, and serum, stool and biliary samples were analyzed for immune responses. Vibriocidal antibody responses, reflective of in vivo colonization, were equivalent in all groups of animals. However, specific anti-CtxB immune responses in serum (P = 0.05) and bile (P = 0. 001) were significantly higher in animals that received Peru2DeltaglnA(pTIC5) than in those that received ETR3, confirming the advantage of higher-level antigen expression in vivo. The development of this balanced lethal system thus permits construction and maintenance of vaccine and vector strains of V. cholerae that express high levels of immunogenic antigens from plasmid vectors without the need for antibiotic selection pressure.
Subject(s)
Antigens, Bacterial/genetics , Glutamate-Ammonia Ligase/genetics , Glutamate-Ammonia Ligase/immunology , Plasmids/genetics , Vibrio cholerae/genetics , Vibrio cholerae/immunology , Administration, Oral , Animals , Antibodies, Bacterial/blood , Antigens, Protozoan/genetics , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/genetics , Bacterial Vaccines/immunology , Entamoeba histolytica/genetics , Entamoeba histolytica/immunology , Female , Gene Expression , Genes, Bacterial , Genetic Complementation Test , Genetic Vectors , Germ-Free Life , Glutamine/deficiency , Intestines/immunology , Intestines/microbiology , Mice , Mutation , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Salmonella typhimurium/genetics , Salmonella typhimurium/immunology , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunologyABSTRACT
Vibrio cholerae colonization of the suckling mouse intestine is a commonly used animal model for the human diarrheal disease cholera. This model has a number of advantages as well as disadvantages, and has been extremely useful in the identification and characterization of proven and putative virulence factors involved in human cholera.
Subject(s)
Cholera , Disease Models, Animal , Mice , Vibrio cholerae/physiology , Vibrio cholerae/pathogenicity , Animals , Animals, Newborn , Animals, Suckling , Cholera/microbiology , Cholera/mortality , Cholera/pathology , Disease Susceptibility , Humans , Intestines/microbiology , Intestines/pathology , Vibrio cholerae/genetics , VirulenceABSTRACT
The regulatory protein ToxT directly activates the transcription of virulence factors in Vibrio cholerae, including cholera toxin (CT) and the toxin-coregulated pilus (TCP). Specific environmental signals stimulate virulence factor expression by inducing the transcription of toxT. We demonstrate that transcriptional activation by the ToxT protein is also modulated by environmental signals. ToxT expressed from an inducible promoter activated high-level expression of CT and TCP in V. cholerae at 30 degrees C, but expression of CT and TCP was significantly decreased or abolished by the addition of 0.4% bile to the medium and/or an increase of the temperature to 37 degrees C. Also, expression of six ToxT-dependent TnphoA fusions was modulated by temperature and bile. Measurement of ToxT-dependent transcription of genes encoding CT and TCP by ctxAp- and tcpAp-luciferase fusions confirmed that negative regulation by 37 degrees C or bile occurs at the transcriptional level in V. cholerae. Interestingly, ToxT-dependent transcription of these same promoters in Salmonella typhimurium was relatively insensitive to regulation by temperature or bile. These data are consistent with ToxT transcriptional activity being modulated by environmental signals in V. cholerae and demonstrate an additional level of complexity governing the expression of virulence factors in this pathogen. We propose that negative regulation of ToxT-dependent transcription by environmental signals prevents the incorrect temporal and spatial expression of virulence factors during cholera pathogenesis.
Subject(s)
Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/metabolism , Cholera Toxin/genetics , Fimbriae Proteins , Gene Expression Regulation, Bacterial , Transcription Factors/metabolism , Transcription, Genetic , Vibrio cholerae/growth & development , Vibrio cholerae/genetics , Bile/microbiology , Cholera/physiopathology , Cloning, Molecular , Fimbriae, Bacterial/genetics , Humans , Promoter Regions, Genetic , Recombinant Fusion Proteins/biosynthesis , Salmonella typhimurium/genetics , Signal Transduction , Temperature , Transcriptional Activation , Vibrio cholerae/pathogenicityABSTRACT
In the pathogenic bacterium Vibrio cholerae, the alternate sigma factor sigma54 is required for expression of multiple sets of genes, including an unidentified gene(s) necessary for enhanced colonization within the host. To identify sigma54-dependent transcriptional activators involved in colonization, PCR was performed with V. cholerae chromosomal DNA and degenerate primers, revealing six novel and distinct coding sequences with homology to sigma54-dependent activators. One sequence had high homology to the luxO gene of V. harveyi, which in that organism is involved in quorum sensing. Phenotypes of V. cholerae strains containing mutations in each of the six putative sigma54-dependent activator genes identified one as a probable ntrC homologue. None of the mutant strains exhibited a defect in the ability to colonize infant mice, suggesting the presence of additional sigma54-dependent activators not identified by this technique.
Subject(s)
DNA-Binding Proteins , DNA-Directed RNA Polymerases/physiology , Sigma Factor/physiology , Trans-Activators/physiology , Vibrio cholerae/genetics , Vibrio cholerae/pathogenicity , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Bacterial/genetics , Glutamate-Ammonia Ligase/genetics , Mice , Molecular Sequence Data , Mutation , Polymerase Chain Reaction/methods , RNA Polymerase Sigma 54 , Sequence Homology, Amino Acid , Virulence/geneticsABSTRACT
Vibrio cholerae, the bacterium that causes cholera, has a pathogenic cycle consisting of a free-swimming phase outside its host, and a sessile virulent phase when colonizing the human small intestine. We have cloned the V. cholerae homologue of the rpoN gene (encoding sigma54) and determined its role in the cholera pathogenic cycle by constructing an rpoN null mutant. The V. cholerae rpoN mutant is non-motile; examination of this mutant by electron microscopy revealed that it lacks a flagellum. In addition to flagellar synthesis, sigma54 is involved in glutamine synthetase expression. Moreover, the rpoN mutant is defective for colonization in an infant mouse model of cholera. We present evidence that the colonization defect is distinct from the non-motile and Gln phenotypes of the rpoN mutant, implicating multiple and distinct roles of sigma54 during the V. cholerae pathogenic cycle. RNA polymerase containing sigma54 (sigma54-holoenzyme) has an absolute requirement for an activator protein to initiate transcription. We have identified three regulatory genes, flrABC (flagellar regulatory proteins ABC) that are additionally required for flagellar synthesis. The flrA and flrC gene products are sigma54-activators and form a flagellar transcription cascade. flrA and flrC mutants are also defective for colonization; this phenotype is probably independent of non-motility. An flrC constitutive mutation (M114-->I) was isolated that is independent of its cognate kinase FlrB. Expression of the constitutive FlrCM114-->I from the cholera toxin promoter resulted in a change in cell morphology, implicating involvement of FlrC in cell division. Thus, sigma54 holoenzyme, FlrA and FlrC transcribe genes for flagellar synthesis and possibly cell division during the free-swimming phase of the V. cholerae life cycle, and some as yet unidentified gene(s) that aid colonization within the host.
Subject(s)
DNA-Binding Proteins , DNA-Directed RNA Polymerases/physiology , Sigma Factor/physiology , Vibrio cholerae/physiology , Vibrio cholerae/pathogenicity , Animals , Base Sequence , Cell Division/genetics , DNA-Directed RNA Polymerases/genetics , Flagella/metabolism , Flagellin/genetics , Gene Expression , Gene Expression Regulation, Bacterial , Genes, Bacterial , Genes, Regulator , Glutamate-Ammonia Ligase/genetics , Humans , Mice , Microscopy, Electron , Molecular Sequence Data , Movement , Plasmids/genetics , Promoter Regions, Genetic , RNA Polymerase Sigma 54 , Salmonella typhimurium/genetics , Sigma Factor/genetics , Transcriptional Activation , Vibrio cholerae/genetics , Vibrio cholerae/ultrastructure , VirulenceABSTRACT
Vibrio cholerae, the causative agent of the human diarrheal disease cholera, is a motile bacterium with a single polar flagellum. Motility has been implicated as a virulence determinant in some animal models of cholera, but the relationship between motility and virulence has not yet been clearly defined. We have begun to define the regulatory circuitry controlling motility. We have identified five V. cholerae flagellin genes, arranged in two chromosomal loci, flaAC and flaEDB; all five genes have their own promoters. The predicted gene products have a high degree of homology to each other. A strain containing a single mutation in flaA is nonmotile and lacks a flagellum, while strains containing multiple mutations in the other four flagellin genes, including a flaCEDB strain, remain motile. Measurement of fla promoter-lacZ fusions reveals that all five flagellin promoters are transcribed at high levels in both wild-type and flaA strains. Measurement of the flagellin promoter-lacZ fusions in Salmonella typhimurium indicates that the promoter for flaA is transcribed by the sigma54 holoenzyme form of RNA polymerase while the flaE, flaD, and flaB promoters are transcribed by the sigma28 holoenzyme. These results reveal that the V. cholerae flagellum is a complex structure with multiple flagellin subunits including FlaA, which is essential for flagellar synthesis and is differentially regulated from the other flagellins.
Subject(s)
DNA-Binding Proteins , Flagellin/genetics , Gene Expression Regulation, Bacterial , Vibrio cholerae/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , DNA, Bacterial , DNA-Directed RNA Polymerases/metabolism , Molecular Sequence Data , Promoter Regions, Genetic , RNA Polymerase Sigma 54 , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Sigma Factor/metabolism , Transcription, Genetic , Vibrio cholerae/physiologyABSTRACT
In Salmonella typhimurium, transcription of the glnA gene (encoding glutamine synthetase) is under the control of the nitrogen-regulatory (ntr) system comprising the alternate sigma factor sigma54 (NtrA) and the two-component sensor-transcriptional activator pair NtrB and NtrC. The glnA, ntrB, and ntrC genes form an operon. We measured the virulence of S. typhimurium strains with nitrogen-regulatory mutations after intraperitoneal (i.p.) or oral inoculations of BALB/c mice. Strains with single mutations in glnA, ntrA, ntrB, or ntrC had i.p. 50% lethal doses (LD50s) of <10 bacteria, similar to the wild-type strain. However, a strain with a delta(glnA-ntrC) operon deletion had an i.p. LD50 of >10(5) bacteria, as did delta glnA ntrA and delta glnA ntrC strains, suggesting that glnA strains require an ntr-transcribed gene for full virulence. High-level transcription of the glutamine transport operon (glnHPQ) is dependent upon both ntrA and ntrC, as determined by glnHp-lacZ fusion measurements. Moreover, delta glnA glnH and delta glnA glnQ strains are attenuated, similar to delta glnA ntrA and delta glnA ntrC strains. These results reveal that access of S. typhimurium to host glutamine depends on the ntr system, which apparently is required for the transcription of the glutamine transport genes. The delta(glnA-ntrC) strain exhibited a reduced ability to survive within the macrophage cell line J774, identifying a potential host environment with low levels of glutamine. Finally, the delta(glnA-ntrC) strain, when inoculated at doses as low as 10 organisms, provided mice with protective immunity against challenge by the wild-type strain, demonstrating its potential use as a live vaccine.