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Modified tRNA anticodons are critical for proper mRNA translation during protein synthesis. It is generally thought that almost all bacterial tRNAsIleuse a modified cytidine—lysidine (L)—at the first position (34) of the anticodon to decipher the AUA codon as isoleucine (Ile). Here we report that tRNAsIlefrom plant organelles and a subset of bacteria contain a new cytidine derivative, designated 2-aminovaleramididine (ava2C). Like L34, ava2C34 governs both Ile-charging ability and AUA decoding. Cryo-electron microscopy structural analyses revealed molecular details of codon recognition by ava2C34 with a specific interaction between its terminal amide group and an mRNA residue 3′-adjacent to the AUA codon. These findings reveal the evolutionary variation of an essential tRNA modification and demonstrate the molecular basis of AUA decoding mediated by a unique tRNA modification.

The microbial cell wall is essential for maintenance of cell shape and resistance to external stressors1. The primary structural component of the cell wall is peptidoglycan, a glycopolymer with peptide crosslinks located outside of the cell membrane1. Peptidoglycan biosynthesis and structure are responsive to shifting environmental conditions such as pH and salinity2–6, but the mechanisms underlying such adaptations are incompletely understood. Precursors of peptidoglycan and other cell surface glycopolymers are synthesized in the cytoplasm and then delivered across the cell membrane bound to the recyclable lipid carrier undecaprenyl phosphate7(C55-P, also known as UndP). Here we identify the DUF368-containing and DedA transmembrane protein families as candidate C55-P translocases, filling a critical gap in knowledge of the proteins required for the biogenesis of microbial cell surface polymers. Gram-negative and Gram-positive bacteria lacking their cognate DUF368-containing protein exhibited alkaline-dependent cell wall and viability defects, along with increased cell surface C55-P levels. pH-dependent synthetic genetic interactions between DUF368-containing proteins and DedA family members suggest that C55-P transporter usage is dynamic and modulated by environmental inputs. C55-P transporter activity was required by the cholera pathogen for growth and cell shape maintenance in the intestine. We propose that conditional transporter reliance provides resilience in lipid carrier recycling, bolstering microbial fitness both inside and outside the host.

Filamentous bacteriophages play a critical role in biofilm formation and virulence in the opportunistic pathogen Pseudomonas aeruginosa. Here, studies of the filamentous Pf4 prophage life cycle within P. aeruginosabiofilms revealed that the prophage-encoded reverse transcriptase (RT) regulates phage genome dynamics. The RT and the non-coding RNA PhrD collaborate to edit the Pf4 phage genome to generate superinfective Pf4 variants capable of rapid propagation within biofilms by preserving genes essential for virion assembly and reconstituting a promoter for the phage excisionase gene. Mutant cells emerge in biofilms where intact Pf4 prophages are replaced by these reduced-genome phage variants, further enhancing virion production. The discovery of RT’s role in phage genome reduction expands understanding of RT functions and of the versatility of phage biology and its impact on microbial community dynamics within biofilms.

The microbial cell surface is a site of critical microbe–host interactions that often control infection outcomes. Defining the set of host proteins present at this interface has been challenging. Here we used a surface-biotinylation approach coupled to quantitative mass spectrometry to identify and quantify both bacterial and host proteins present on the surface of diarrheal fluid-derived Vibriocholeraein an infant rabbit model of cholera. The V. choleraesurface was coated with numerous host proteins, whose abundance were driven by the presence of cholera toxin, including the C-type lectin SP-D. Mice lacking SP-D had enhanced V. choleraeintestinal colonization, and SP-D production shaped both host and pathogen transcriptomes. Additional host proteins (AnxA1, LPO and ZAG) that bound V. choleraewere also found to recognize distinct taxa of the murine intestinal microbiota, suggesting that these host factors may play roles in intestinal homeostasis in addition to host defense.

The mucin Muc2 is a major constituent of the mucus layer that covers the intestinal epithelium and creates a barrier between epithelial cells and luminal commensal or pathogenic microorganisms. The Gram-positive foodborne pathogen Listeria monocytogenescan cause enteritis and also disseminate from the intestine to give rise to systemic disease. L. monocytogenescan bind to intestinal Muc2, but the influence of the Muc2 mucin barrier on L. monocytogenesintestinal colonization and systemic dissemination has not been explored.

Chemical modifications of the nucleosides that comprise transfer RNAs are diverse. However, the structure, location and extent of modifications have been systematically charted in very few organisms. Here, we describe an approach in which rapid prediction of modified sites through reverse transcription-derived signatures in high-throughput transfer RNA-sequencing (tRNA-seq) data is coupled with identification of tRNA modifications through RNA mass spectrometry. Comparative tRNA-seq enabled prediction of several Vibrio choleraemodifications that are absent from Escherichia coliand also revealed the effects of various environmental conditions on V. choleraetRNA modification. Through RNA mass spectrometric analyses, we showed that two of the V. cholerae-specific reverse transcription signatures reflected the presence of a new modification (acetylated acp3U (acacp3U)), while the other results from C-to-Ψ RNA editing, a process not described before. These findings demonstrate the utility of this approach for rapid surveillance of tRNA modification profiles and environmental control of tRNA modification.

Intelectin-1 (ITLN1) is a lectin secreted by intestinal epithelial cells (IECs) and upregulated in human ulcerative colitis (UC). We investigated how ITLN1 production is regulated in IECs and the biological effects of ITLN1 at the host–microbiota interface using mouse models. Our data show that ITLN1 upregulation in IECs from UC patients is a consequence of activating the unfolded protein response. Analysis of microbes coated by ITLN1 in vivo revealed a restricted subset of microorganisms, including the mucolytic bacterium Akkermansia muciniphila. Mice overexpressing intestinal ITLN1 exhibited decreased inner colonic mucus layer thickness and closer apposition of A. muciniphila to the epithelial cell surface, similar to alterations reported in UC. The changes in the inner mucus layer were microbiota and A. muciniphila dependent and associated with enhanced sensitivity to chemically induced and T cell–mediated colitis. We conclude that by determining the localization of a select group of bacteria to the mucus layer, ITLN1 modifies this critical barrier. Together, these findings may explain the impact of ITLN1 dysregulation on UC pathogenesis.

ABSTRACTGram-negative bacteria are notoriously resistant to a variety of high-molecular-weight antibiotics due to the limited permeability of their outer membrane (OM). The basis of OM barrier function and the genetic factors required for its maintenance remain incompletely understood. Here, we employed transposon insertion sequencing to identify genes required for Vibrio choleraeresistance to vancomycin and bacitracin, antibiotics that are thought to be too large to efficiently penetrate the OM. The screen yielded several genes whose protein products are predicted to participate in processes important for OM barrier functions and for biofilm formation. In addition, we identified a novel factor, designated vigA(for vancomycin inhibits growth), that has not previously been characterized or linked to outer membrane function. The vigAopen reading frame (ORF) codes for an inner membrane protein, and in its absence, cells became highly sensitive to glycopeptide antibiotics (vancomycin and ramoplanin) and bacitracin but not to other large antibiotics or detergents. In contrast to wild-type (WT) cells, the vigAmutant was stained with fluorescent vancomycin. These observations suggest that VigA specifically prevents the periplasmic accumulation of certain large antibiotics without exerting a general role in the maintenance of OM integrity. We also observed marked interspecies variability in the susceptibilities of Gram-negative pathogens to glycopeptides and bacitracin. Collectively, our findings suggest that the OM barrier is not absolute but rather depends on specific OM-antibiotic interactions.

Activity-based protein profiling (ABPP) is a chemoproteomic tool for detecting active enzymes in complex biological systems. We used ABPP to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human choleric stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, and genetic disruption of all four proteases increased the abundance of intelectin, an intestinal lectin, and its binding to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting that it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Our work demonstrates the power of activity-based proteomics to reveal host-pathogen enzymatic dialog in an animal model of infection.

Transposon insertion sequencing (TIS) is a powerful approach that can be extensively applied to the genome-wide definition of loci that are required for bacterial growth under diverse conditions. However, experimental design choices and stochastic biological processes can heavily influence the results of TIS experiments and affect downstream statistical analysis. In this Opinion article, we discuss TIS experimental parameters and how these factors relate to the benefits and limitations of the various statistical frameworks that can be applied to the computational analysis of TIS data.

Vibrio choleraeis the agent of cholera, a potentially lethal diarrheal disease that remains a significant threat to populations in developing nations. The infant rabbit model of cholera is the only non‐surgical small animal model system that closely mimics human cholera. Following orogastric inoculation, V. choleraecolonizes the intestines of infant rabbits, and the animals develop severe cholera‐like diarrhea. In this unit, we provide a detailed description of the preparation of the V. choleraeinoculum, the inoculation process and the collection and processing of tissue samples. This infection model is useful for studies of V. choleraefactors and mechanisms that promote its intestinal colonization and enterotoxicity, as well as the host response to infection. The infant rabbit model of cholera enables investigations that will further our understanding of the pathophysiology of cholera and provides a platform for testing new therapeutics. © 2015 by John Wiley & Sons, Inc.

ABSTRACTDiverse environmental stimuli and a complex network of regulatory factors are known to modulate expression of Vibrio cholerae's principal virulence factors. However, there is relatively little known about how metabolic factors impinge upon the pathogen's well-characterized cascade of transcription factors that induce expression of cholera toxin and the toxin-coregulated pilus (TCP). Here, we used a transposon insertion site (TIS) sequencing-based strategy to identify new factors required for expression of tcpA, which encodes the major subunit of TCP, the organism's chief intestinal colonization factor. Besides identifying most of the genes known to modulate tcpAexpression, the screen yielded ptsIand ptsH, which encode the enzyme I (EI) and Hpr components of the V. choleraephosphoenolpyruvate phosphotransferase system (PTS). In addition to reduced expression of TcpA, strains lacking EI, Hpr, or the associated EIIAGlcprotein produced less cholera toxin (CT) and had a diminished capacity to colonize the infant mouse intestine. The PTS modulates virulence gene expression by regulating expression of tcpPHand aphAB, which themselves control expression of toxT, the central activator of virulence gene expression. One mechanism by which PTS promotes virulence gene expression appears to be by modulating the amounts of intracellular cyclic AMP (cAMP). Our findings reveal that the V. choleraePTS is an additional modulator of the ToxT regulon and demonstrate the potency of loss-of-function TIS sequencing screens for defining regulatory networks.

ABSTRACTThe Cpx two-component regulatory system has been shown in Escherichia colito alleviate stress caused by misfolded cell envelope proteins. The Vibrio choleraeCpx system was previously found to respond to cues distinct from those in the E. colisystem, suggesting that this system fulfills a different physiological role in the cholera pathogen. Here, we used microarrays to identify genes that were regulated by the V. choleraeCpx system. Our observations suggest that the activation of the V. choleraeCpx system does not induce expression of genes involved in the mitigation of stress generated by misfolded cell envelope proteins but promotes expression of genes involved in antimicrobial resistance. In particular, activation of the Cpx system induced expression of the genes encoding the VexAB and VexGH resistance-nodulation-division (RND) efflux systems and their cognate outer membrane pore protein TolC. The promoters for these loci contained putative CpxR consensus binding sites, and ectopic cpxRexpression activated transcription from the promoters for the RND efflux systems. CpxR was not required for intrinsic antimicrobial resistance, but CpxR activation enhanced resistance to antimicrobial substrates of VexAB and VexGH. Mutations that inactivated VexAB or VexGH efflux activity resulted in the activation of the Cpx response, suggesting that vexABand vexGHand the cpxP-cpxRAsystem are reciprocally regulated. We speculate that the reciprocal regulation of the V. choleraeRND efflux systems and the Cpx two-component system is mediated by the intracellular accumulation of an endogenously produced metabolic by-product that is normally extruded from the cell by the RND efflux systems.

ABSTRACTWe investigated the roles of the Vibrio choleraehigh-molecular-weight bifunctional penicillin binding proteins, PBP1a and PBP1b, in the fitness of this enteric pathogen. Using a screen for synthetic lethality, we found that the V. choleraePBP1a and PBP1b proteins, like their Escherichia colihomologues, are each essential in the absence of the other and in the absence of the other's putative activator, the outer membrane lipoproteins LpoA and LpoB, respectively. Comparative analyses of V. choleraemutants suggest that PBP1a/LpoA of V. choleraeplay a more prominent role in generating and/or maintaining the pathogen's cell wall than PBP1b/LpoB. V. choleraelacking PBP1b or LpoB exhibited wild-type growth under all conditions tested. In contrast, V. choleraelacking PBP1a or LpoA exhibited growth deficiencies in minimal medium, in the presence of deoxycholate and bile, and in competition assays with wild-type cells both in vitroand in the infant mouse small intestine. PBP1a pathway mutants are particularly impaired in stationary phase, which renders them sensitive to a product(s) present in supernatants from stationary-phase wild-type cells. The marked competitive defect of the PBP1a pathway mutants in vivowas largely absent when exponential-phase cells rather than stationary-phase cells were used to inoculate suckling mice. Thus, at least for V. choleraePBP1a pathway mutants, the growth phase of the inoculum is a key modulator of infectivity.

Vibrio parahaemolyticustype III secretion system 2 (T3SS2) is essential for the organism’s virulence, but the effectors required for intestinal colonization and induction of diarrhea by this pathogen have not been identified. Here, we identify a type III secretion system (T3SS2)-secreted effector, VopZ, that is essential for V. parahaemolyticuspathogenicity. VopZ plays distinct, genetically separable roles in enabling intestinal colonization and diarrheagenesis. Truncation of VopZ prevents V. parahaemolyticuscolonization, whereas deletion of VopZ amino acids 38–62 abrogates V. parahaemolyticus-induced diarrhea and intestinal pathology but does not impair colonization. VopZ inhibits activation of the kinase TAK1 and thereby prevents the activation of MAPK and NF-κB signaling pathways, which lie downstream. In contrast, the VopZ internal deletion mutant cannot counter the activation of pathways regulated by TAK1. Collectively, our findings suggest that VopZ’s inhibition of TAK1 is critical for V. parahaemolyticusto induce diarrhea and intestinal pathology.

ABSTRACTThe pathogenesis of the diarrheal disease caused by Vibrio parahaemolyticus, a leading cause of seafood-associated enteritis worldwide, is dependent upon a type III secretion system, T3SS2. This apparatus enables the pathogen to inject bacterial proteins (effectors) into the cytosol of host cells and thereby modulate host processes. T3SS effector proteins transit into the host cell via a membrane pore (translocon) typically formed by 3 bacterial proteins. We have identified the third translocon protein for T3SS2: VopW, which was previously classified as an effector protein for a homologous T3SS in V. cholerae. VopW is a hydrophilic translocon protein; like other such proteins, it is not inserted into the host cell membrane but is required for insertion of the two hydrophobic translocators, VopB2 and VopD2, that constitute the membrane channel. VopW is not required for secretion of T3SS2 effectors into the bacterial culture medium; however, it is essential for transfer of these proteins into the host cell cytoplasm. Consequently, deletion of vopWabrogates the virulence of V. parahaemolyticusin several animal models of diarrheal disease. Unlike previously described hydrophilic translocators, VopW is itself translocated into the host cell cytoplasm, raising the possibility that it functions as both a translocator and an effector.

Escherichia coli O157:H7 causes food and waterborne enteric infections that can result in hemorrhagic colitis and life-threatening hemolytic uremic syndrome. Intimate adherence of the bacteria to intestinal epithelial cells is mediated by intimin, but E. coli O157:H7 also possess several other putative adhesins, including curli and two operons that encode long polar fimbriae (Lpf). To assess the importance of Lpf for intestinal colonization, we performed competition experiments between E. coli O157:H7 and an isogenic ΔlpfA1 ΔlpfA2 double mutant in the infant rabbit model. The mutant was outcompeted in the ileum, cecum, and midcolon, suggesting that Lpf contributes to intestinal colonization. In contrast, the ΔlpfA1 ΔlpfA2 mutant showed increased adherence to colonic epithelial cells in vitro. Transmission electron microscopy revealed curli-like structures on the surface of the ΔlpfA1 ΔlpfA2 mutant, and the presence of curli was confirmed by Congo red binding, immunogold-labeling electron microscopy, immunoblotting, and quantitative real-time reverse transcription-PCR (qRT-PCR) measuring csgA expression. However, deletion of csgA, which encodes the major curli subunit, does not appear to affect intestinal colonization. In addition to suggesting that Lpf can contribute to EHEC intestinal colonization, our observations indicate that the regulatory pathways governing the expression of Lpf and curli are interdependent.

Escherichia coliO157:H7 causes food and waterborne enteric infections that can result in hemorrhagic colitis and life-threatening hemolytic uremic syndrome. Intimate adherence of the bacteria to intestinal epithelial cells is mediated by intimin, but E. coliO157:H7 also possess several other putative adhesins, including curli and two operons that encode long polar fimbriae (Lpf). To assess the importance of Lpf for intestinal colonization, we performed competition experiments between E. coliO157:H7 and an isogenic ?lpfA1?lpfA2double mutant in the infant rabbit model. The mutant was outcompeted in the ileum, cecum, and midcolon, suggesting that Lpf contributes to intestinal colonization. In contrast, the ?lpfA1?lpfA2mutant showed increased adherence to colonic epithelial cells in vitro. Transmission electron microscopy revealed curli-like structures on the surface of the ?lpfA1?lpfA2mutant, and the presence of curli was confirmed by Congo red binding, immunogold-labeling electron microscopy, immunoblotting, and quantitative real-time reverse transcription-PCR (qRT-PCR) measuring csgAexpression. However, deletion of csgA, which encodes the major curli subunit, does not appear to affect intestinal colonization. In addition to suggesting that Lpf can contribute to EHEC intestinal colonization, our observations indicate that the regulatory pathways governing the expression of Lpf and curli are interdependent.

ABSTRACTAvR2-V10.3 is an engineered R-type pyocin that specifically kills Escherichia coliO157, an enteric pathogen that is a major cause of food-borne diarrheal disease. New therapeutics to counteract E. coliO157 are needed, as currently available antibiotics can exacerbate the consequences of infection. We show here that orogastric administration of AvR2-V10.3 can prevent or ameliorate E. coliO157:H7-induced diarrhea and intestinal inflammation in an infant rabbit model of infection when the compound is administered either in a postexposure prophylactic regimen or after the onset of symptoms. Notably, administration of AvR2-V10.3 also reduces bacterial carriage and fecal shedding of this pathogen. Our findings support the further development of pathogen-specific R-type pyocins as a way to treat enteric infections.

AvR2-V10.3 is an engineered R-type pyocin that specifically kills Escherichia coli O157, an enteric pathogen that is a major cause of food-borne diarrheal disease. New therapeutics to counteract E. coli O157 are needed, as currently available antibiotics can exacerbate the consequences of infection. We show here that orogastric administration of AvR2-V10.3 can prevent or ameliorate E. coli O157:H7-induced diarrhea and intestinal inflammation in an infant rabbit model of infection when the compound is administered either in a postexposure prophylactic regimen or after the onset of symptoms. Notably, administration of AvR2-V10.3 also reduces bacterial carriage and fecal shedding of this pathogen. Our findings support the further development of pathogen-specific R-type pyocins as a way to treat enteric infections.

ABSTRACTIn addition to causing diarrhea, Escherichia coliO157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. Differences in HUS frequency among E. coliO157:H7 outbreaks have been noted, but our understanding of bacterial factors that promote HUS is incomplete. In 2006, in an outbreak of E. coliO157:H7 caused by consumption of contaminated spinach, there was a notably high frequency of HUS. We sequenced the genome of the strain responsible (TW14359) with the goal of identifying candidate genetic factors that contribute to an enhanced ability to cause HUS. The TW14359 genome contains 70 kb of DNA segments not present in either of the two reference O157:H7 genomes. We identified seven putative virulence determinants, including two putative type III secretion system effector proteins, candidate genes that could result in increased pathogenicity or, alternatively, adaptation to plants, and an intact anaerobic nitric oxide reductase gene, norV. We surveyed 100 O157:H7 isolates for the presence of these putative virulence determinants. A norVdeletion was found in over one-half of the strains surveyed and correlated strikingly with the absence of stx1. The other putative virulence factors were found in 8 to 35% of the O157:H7 isolates surveyed, and their presence also correlated with the presence of norVand the absence of stx1, indicating that the presence of norVmay serve as a marker of a greater propensity for HUS, similar to the correlation between the absence of stx1and a propensity for HUS.

In addition to causing diarrhea, Escherichia coli O157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. Differences in HUS frequency among E. coli O157:H7 outbreaks have been noted, but our understanding of bacterial factors that promote HUS is incomplete. In 2006, in an outbreak of E. coli O157:H7 caused by consumption of contaminated spinach, there was a notably high frequency of HUS. We sequenced the genome of the strain responsible (TW14359) with the goal of identifying candidate genetic factors that contribute to an enhanced ability to cause HUS. The TW14359 genome contains 70 kb of DNA segments not present in either of the two reference O157:H7 genomes. We identified seven putative virulence determinants, including two putative type III secretion system effector proteins, candidate genes that could result in increased pathogenicity or, alternatively, adaptation to plants, and an intact anaerobic nitric oxide reductase gene, norV. We surveyed 100 O157:H7 isolates for the presence of these putative virulence determinants. A norV deletion was found in over one-half of the strains surveyed and correlated strikingly with the absence of stx1. The other putative virulence factors were found in 8 to 35% of the O157:H7 isolates surveyed, and their presence also correlated with the presence of norV and the absence of stx1, indicating that the presence of norV may serve as a marker of a greater propensity for HUS, similar to the correlation between the absence of stx1and a propensity for HUS.

ABSTRACTAdherence of pathogenic Escherichia colistrains to intestinal epithelia is essential for infection. For enterohemorrhagic E. coli(EHEC) serotype O157:H7, we have previously demonstrated that multiple factors govern this pathogen's adherence to HeLa cells (A. G. Torres and J. B. Kaper, Infect. Immun. 71:4985-4995, 2003). One of these factors is CadA, a lysine decarboxylase, and this protein has been proposed to negatively regulate virulence in several enteric pathogens. In the case of EHEC strains, CadA modulates expression of the intimin, an outer membrane adhesin involved in pathogenesis. Here, we inactivated cadAin O157:H7 strain 86-24 to investigate the role of this gene in EHEC adhesion to tissue-cultured monolayers, global gene expression patterns, and colonization of the infant rabbit intestine. The cadAmutant did not possess lysine decarboxylation activity and was hyperadherent to tissue-cultured cells. Adherence of the cadAmutant was nearly twofold greater than that of the wild type, and the adherence phenotype was independent of pH, lysine, or cadaverine in the media. Additionally, complementation of the cadAdefect reduced adherence back to wild-type levels, and it was found that the mutation affected the expression of the intimin protein. Disruption of the eaegene (intimin-encoding gene) in the cadAmutant significantly reduced its adherence to tissue-cultured cells. However, adherence of the cadA eaedouble mutant was greater than that of an 86-24 eaemutant, suggesting that the enhanced adherence of the cadAmutant is not entirely attributable to enhanced expression of intimin in this background. Gene array analysis revealed that the cadAmutation significantly altered EHEC gene expression patterns; expression of 1,332 genes was downregulated and that of 132 genes was upregulated in the mutant compared to the wild-type strain. Interestingly, the gene expression variation shows an EHEC-biased gene alteration including intergenic regions. Two putative adhesins, flagella and F9 fimbria, were upregulated in the cadAmutant, suggestive of their association with adherence in the absence of the Cad regulatory mechanism. In the infant rabbit model, the cadAmutant outcompeted the wild-type strain in the ileum but not in the cecum or mid-colon, raising the possibility that CadA negatively regulates EHEC pathogenicity in a tissue-specific fashion.

Adherence of pathogenic Escherichia coli strains to intestinal epithelia is essential for infection. For enterohemorrhagic E. coli (EHEC) serotype O157:H7, we have previously demonstrated that multiple factors govern this pathogen's adherence to HeLa cells (A. G. Torres and J. B. Kaper, Infect. Immun. 71:4985-4995, 2003). One of these factors is CadA, a lysine decarboxylase, and this protein has been proposed to negatively regulate virulence in several enteric pathogens. In the case of EHEC strains, CadA modulates expression of the intimin, an outer membrane adhesin involved in pathogenesis. Here, we inactivated cadA in O157:H7 strain 86-24 to investigate the role of this gene in EHEC adhesion to tissue-cultured monolayers, global gene expression patterns, and colonization of the infant rabbit intestine. The cadA mutant did not possess lysine decarboxylation activity and was hyperadherent to tissue-cultured cells. Adherence of the cadA mutant was nearly twofold greater than that of the wild type, and the adherence phenotype was independent of pH, lysine, or cadaverine in the media. Additionally, complementation of the cadA defect reduced adherence back to wild-type levels, and it was found that the mutation affected the expression of the intimin protein. Disruption of the eae gene (intimin-encoding gene) in the cadA mutant significantly reduced its adherence to tissue-cultured cells. However, adherence of the cadA eae double mutant was greater than that of an 86-24 eae mutant, suggesting that the enhanced adherence of the cadA mutant is not entirely attributable to enhanced expression of intimin in this background. Gene array analysis revealed that the cadA mutation significantly altered EHEC gene expression patterns; expression of 1,332 genes was downregulated and that of 132 genes was upregulated in the mutant compared to the wild-type strain. Interestingly, the gene expression variation shows an EHEC-biased gene alteration including intergenic regions. Two putative adhesins, flagella and F9 fimbria, were upregulated in the cadA mutant, suggestive of their association with adherence in the absence of the Cad regulatory mechanism. In the infant rabbit model, the cadA mutant outcompeted the wild-type strain in the ileum but not in the cecum or mid-colon, raising the possibility that CadA negatively regulates EHEC pathogenicity in a tissue-specific fashion.

ABSTRACTEnterohemorrhagic Escherichia coli(EHEC) is a noninvasive food-borne pathogen that colonizes the distal ileum and colon. Proteins encoded in the EHEC locus of enterocyte effacement (LEE) pathogenicity island are known to contribute to this pathogen's adherence to epithelial cells and intestinal colonization. The role of non-LEE-encoded proteins in these processes is not as clear. We found that the Z2053 gene (designated adfOhere), a gene located in a cryptic EHEC prophage, exhibits similarity to adherence and/or colonization factor genes found in several other enteric pathogens. An EHEC adfOmutant exhibited marked reductions in adherence to HeLa cells and in the secretion of several proteins into the supernatant. YodA, one of these secreted proteins, was found to be a substrate of the EHEC pO157-encoded type 2 secretion system (T2SS). Both the T2SS and YodA proved to be essential for EHEC adherence to cultured HeLa cell monolayers. Using an infant rabbit model of infection, we found that the adfOmutation did not affect colonization but that the colonization of an etpC(T2SS) mutant was reduced ∼5-fold. A strain deficient in YodA had a more severe colonization defect; however, this strain also exhibited a growth defect in vitro. Overall, our findings indicate that the pO157-encoded T2SS contributes to EHEC adherence and intestinal colonization and thus show that EHEC pathogenicity depends on type 2 secretion as well as type 3 secretion.

Enterohemorrhagic Escherichia coli (EHEC) is a noninvasive food-borne pathogen that colonizes the distal ileum and colon. Proteins encoded in the EHEC locus of enterocyte effacement (LEE) pathogenicity island are known to contribute to this pathogen's adherence to epithelial cells and intestinal colonization. The role of non-LEE-encoded proteins in these processes is not as clear. We found that the Z2053 gene (designated adfO here), a gene located in a cryptic EHEC prophage, exhibits similarity to adherence and/or colonization factor genes found in several other enteric pathogens. An EHEC adfO mutant exhibited marked reductions in adherence to HeLa cells and in the secretion of several proteins into the supernatant. YodA, one of these secreted proteins, was found to be a substrate of the EHEC pO157-encoded type 2 secretion system (T2SS). Both the T2SS and YodA proved to be essential for EHEC adherence to cultured HeLa cell monolayers. Using an infant rabbit model of infection, we found that the adfO mutation did not affect colonization but that the colonization of an etpC (T2SS) mutant was reduced ∼5-fold. A strain deficient in YodA had a more severe colonization defect; however, this strain also exhibited a growth defect in vitro. Overall, our findings indicate that the pO157-encoded T2SS contributes to EHEC adherence and intestinal colonization and thus show that EHEC pathogenicity depends on type 2 secretion as well as type 3 secretion.

Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal+strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal+parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal+revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.

ABSTRACTEnterohemorrhagic Escherichia coli(EHEC), especially E. coliO157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coliO157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coliO157:H7 galmutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the galmutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coliO157:H7. The galmutants could easily be reverted to a wild-type Gal+strain using P1 transduction. We found that the O157:H7 galETKM::aad-7deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal+parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal+revertant of this mutant outcompeted the galETKMdeletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC galmutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.

ABSTRACTCholera toxin, one of the main virulence factors of Vibrio cholerae, is encoded in the genome of CTXφ, a V. cholerae-specific lysogenic filamentous bacteriophage. Although the genes encoding cholera toxin, ctxAB, are known to have their own promoter, the toxin genes can also be transcribed from an upstream CTXφ promoter, PrstA. The V. choleraeSOS response to DNA damage induces the CTX prophage by stimulating gene expression initiating from PrstA. Here, we investigated whether ctxAmRNA levels increase along with the levels of the transcripts for the other CTXφ genes following stimulation of the V. choleraeSOS response. Treatment of V. choleraewith the SOS-inducing agent mitomycin C increased the level of ctxAmRNA approximately sevenfold, apparently by augmenting the activity of PrstA. However, using suckling mice as a model host, we found that intraintestinal ctxAtranscription does not depend on PrstA. In fact, the suckling mouse intestine does not appear to be a potent inducer of the V. choleraeSOS response. Furthermore, alleviation of LexA-mediated repression of the V. choleraeSOS regulon was not required for V. choleraegrowth in the suckling mouse intestine. Our observations suggest that pathogenicity of V. choleraedoes not depend on its SOS response.

Cholera toxin, one of the main virulence factors of Vibrio cholerae, is encoded in the genome of CTXφ, a V. cholerae-specific lysogenic filamentous bacteriophage. Although the genes encoding cholera toxin, ctxAB, are known to have their own promoter, the toxin genes can also be transcribed from an upstream CTXφ promoter, PrstA. The V. cholerae SOS response to DNA damage induces the CTX prophage by stimulating gene expression initiating from PrstA. Here, we investigated whether ctxA mRNA levels increase along with the levels of the transcripts for the other CTXφ genes following stimulation of the V. cholerae SOS response. Treatment of V. cholerae with the SOS-inducing agent mitomycin C increased the level of ctxA mRNA approximately sevenfold, apparently by augmenting the activity of PrstA. However, using suckling mice as a model host, we found that intraintestinal ctxA transcription does not depend on PrstA. In fact, the suckling mouse intestine does not appear to be a potent inducer of the V. cholerae SOS response. Furthermore, alleviation of LexA-mediated repression of the V. cholerae SOS regulon was not required for V. cholerae growth in the suckling mouse intestine. Our observations suggest that pathogenicity of V. cholerae does not depend on its SOS response.

ABSTRACTThe genes encoding the enterohemorrhagic Escherichia coli(EHEC) type III secretion system (TTSS) and five effector proteins secreted by the TTSS are located on the locus of enterocyte effacement (LEE) pathogenicity island. Deletion of tir, which encodes one of these effector proteins, results in a profound reduction (∼10,000-fold) in EHEC colonization of the infant rabbit intestine, but the in vivo phenotypes of other LEE genes are unknown. Here, we constructed in-frame deletions in escN, the putative ATPase component of the TTSS, and the genes encoding the four other LEE-encoded effector proteins, EspH, Map, EspF, and EspG, to investigate the contributions of the TTSS and the translocated effector proteins to EHEC pathogenicity in infant rabbits. We found that the TTSS is required for EHEC colonization and attaching and effacing (A/E) lesion formation in the rabbit intestine. Deletion of escNreduced EHEC recovery from the rabbit intestine by ∼10,000-fold. Although EspH, Map, EspF, and EspG were not required for A/E lesion formation in the rabbit intestine or in HeLa cells, these effector proteins promote EHEC colonization. Colonization by the espHand espFmutants was reduced throughout the intestine. In contrast, colonization by the mapand espGmutants was reduced only in the small intestine, indicating that Map and EspG have organ-specific effects. EspF appears to down-regulate the host response to EHEC, since we observed increased accumulation of polymorphonuclear leukocytes in the colonic mucosa of rabbits infected with the EHEC espFmutant. Thus, all the known LEE-encoded effector proteins influence EHEC pathogenicity.

ABSTRACTBPI (bactericidal/permeability-increasing) is a potent antimicrobial protein that was recently reported to be expressed as a surface protein on human gastrointestinal tract epithelial cells. In this study, we investigated the resistance of Vibrio cholerae, a small-bowel pathogen that causes cholera, to a BPI-derived peptide, P2. Unlike in Escherichia coliand Salmonella entericaserovar Typhimurium, resistance to P2 in V. choleraewas not dependent on the BipA GTPase. Instead, we found that ToxR, the master regulator of V. choleraepathogenicity, controlled resistance to P2 by regulating the production of the outer membrane protein OmpU. Both toxRand ompUmutants were at least 100-fold more sensitive to P2 than were wild-type cells. OmpU also conferred resistance to polymyxin B sulfate, suggesting that this porin may impart resistance to cationic antibacterial proteins via a common mechanism. Studies of stationary-phase cells revealed that the ToxR-repressed porin OmpT may also contribute to P2 resistance. Finally, although the mechanism of porin-mediated resistance to antimicrobial peptides remains elusive, our data suggest that the BPI peptide sensitivity of OmpU-deficient V. choleraeis not attributable to a generally defective outer membrane.

Enterohemorrhagic Escherichia coli (EHEC) is a group of food-borne pathogens that can cause diarrhea, colitis, and the hemolytic uremic syndrome (HUS). The importance of several of the proposed EHEC virulence factors lacks experimental verification in animal models. The limitations of current animal models led us to reexamine the infant rabbit model for the study of EHEC pathogenicity. Here, we report that intragastric inoculation of a Shiga toxin 2 (Stx2)-producing E. coli O157:H7 clinical isolate into infant rabbits led to severe diarrhea and intestinal inflammation but no signs of HUS. We constructed a set of isogenic derivatives of this isolate with deletions in several putative virulence genes, including stx(2), eae, tir, and ehxA, to investigate the contribution of individual virulence factors to EHEC pathogenicity. stx(2) increased the severity and duration of EHEC-induced diarrhea. Furthermore, although stx(2) had no role in EHEC intestinal colonization nor was it required for EHEC-induced inflammation, stx(2) altered how the host responded to EHEC infection by promoting heterophilic infiltration of the colonic epithelium and lamina propria. Intragastric inoculation of purified Stx2 also induced inflammation and diarrhea in this model. Diarrhea and intestinal inflammation were also dependent on EHEC colonization, as EHEC derivatives with deletions in eae and tir did not colonize, form attaching and effacing lesions, or develop clinical signs of disease. Our studies indicate that infant rabbits are a useful model for investigation of the intestinal stage of EHEC pathogenesis and suggest that Shiga toxin may play a critical role in causing diarrhea and inflammation in patients infected with EHEC.

ABSTRACTEnterohemorrhagic Escherichia coli(EHEC) is a group of food-borne pathogens that can cause diarrhea, colitis, and the hemolytic uremic syndrome (HUS). The importance of several of the proposed EHEC virulence factors lacks experimental verification in animal models. The limitations of current animal models led us to reexamine the infant rabbit model for the study of EHEC pathogenicity. Here, we report that intragastric inoculation of a Shiga toxin 2 (Stx2)-producing E. coliO157:H7 clinical isolate into infant rabbits led to severe diarrhea and intestinal inflammation but no signs of HUS. We constructed a set of isogenic derivatives of this isolate with deletions in several putative virulence genes, including stx2, eae, tir, and ehxA, to investigate the contribution of individual virulence factors to EHEC pathogenicity. stx2increased the severity and duration of EHEC-induced diarrhea. Furthermore, although stx2had no role in EHEC intestinal colonization nor was it required for EHEC-induced inflammation, stx2altered how the host responded to EHEC infection by promoting heterophilic infiltration of the colonic epithelium and lamina propria. Intragastric inoculation of purified Stx2 also induced inflammation and diarrhea in this model. Diarrhea and intestinal inflammation were also dependent on EHEC colonization, as EHEC derivatives with deletions in eaeand tirdid not colonize, form attaching and effacing lesions, or develop clinical signs of disease. Our studies indicate that infant rabbits are a useful model for investigation of the intestinal stage of EHEC pathogenesis and suggest that Shiga toxin may play a critical role in causing diarrhea and inflammation in patients infected with EHEC.

ClC chloride channels are found in all three kingdoms of life though little is known about their functions in prokaryotes. Here we investigated the role of a Vibrio cholerae ClC channel in acid resistance and intestinal colonization. The putative V. cholerae ClC channel was found to confer mild resistance to acid when pH was adjusted with HCl, but not with other acids. Surprisingly, a ClC channel deletion mutant exhibited enhanced intestinal colonization in infant mice.

ABSTRACTClC chloride channels are found in all three kingdoms of life though little is known about their functions in prokaryotes. Here we investigated the role of a Vibrio choleraeClC channel in acid resistance and intestinal colonization. The putative V. choleraeClC channel was found to confer mild resistance to acid when pH was adjusted with HCl, but not with other acids. Surprisingly, a ClC channel deletion mutant exhibited enhanced intestinal colonization in infant mice.

ABSTRACTMany recent Asian clinical Vibrio choleraeE1 Tor O1 and O139 isolates are resistant to the antibiotics sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm), and streptomycin (Sm). The corresponding resistance genes are located on large conjugative elements (SXT constins) that are integrated into prfCon the V. choleraechromosome. We determined the DNA sequences of the antibiotic resistance genes in the SXT constin in MO10, an O139 isolate. In SXTMO10, these genes are clustered within a composite transposon-like structure found near the element's 5′ end. The genes conferring resistance to Cm (floR), Su (sulII), and Sm (strAand strB) correspond to previously described genes, whereas the gene conferring resistance to Tm, designated dfr18, is novel. In some other O139 isolates the antibiotic resistance gene cluster was found to be deleted from the SXT-related constin. The El Tor O1 SXT constin, SXTET, does not contain the same resistance genes as SXTMO10. In this constin, the Tm resistance determinant was located nearly 70 kbp away from the other resistance genes and found in a novel type of integron that constitutes a fourth class of resistance integrons. These studies indicate that there is considerable flux in the antibiotic resistance genes found in the SXT family of constins and point to a model for the evolution of these related mobile elements.

ABSTRACTThe Shiga toxins (Stx) are critical virulence factors forEscherichia coliO157:H7 and other serotypes of enterohemorrhagic E. coli(EHEC). These potent toxins are encoded in the genomes of temperate lambdoid bacteriophages. We recently demonstrated that induction of the resident Stx2-encoding prophage in an O157:H7 clinical isolate is required for toxin production by this strain. Since several factors produced by human cells, including hydrogen peroxide (H2O2), are capable of inducing lambdoid prophages, we hypothesized that such molecules might also induce toxin production by EHEC. Here, we studied whether H2O2and also human neutrophils, an important endogenous source of H2O2, induced Stx2 expression by an EHEC clinical isolate. Both H2O2and neutrophils were found to augment Stx2 production, raising the possibility that these agents may lead to prophage induction in vivo and thereby contribute to EHEC pathogenesis.

ABSTRACTIn toxigenic Vibrio cholerae, the cholera enterotoxin (CT) is encoded by CTXΦ, a lysogenic bacteriophage. The propagation of this filamentous phage can result in the origination of new toxigenic strains. To understand the nature of possible environmental factors associated with the propagation of CTXΦ, we examined the effects of temperature, pH, salinity, and exposure to direct sunlight on the induction of the CTX prophage and studied the transmission of the phage to potential recipient strains. Exposure of cultures of CTXΦ lysogens to direct sunlight resulted in ∼10,000-fold increases in phage titers. Variation in temperature, pH, or salinity of the culture did not have a substantial effect on the induction of the prophage, but these factors influenced the stability of CTXΦ particles. Exposure of mixed cultures of CTXΦ lysogens and potential recipient strains to sunlight significantly increased both the in vitro and in vivo (in rabbit ileal loops) transduction of the recipient strains by CTXΦ. Included in these transduction experiments were two environmental nontoxigenic (CTXΦ−) strains of V. choleraeO139. These two O139 strains were transduced at high efficiency by CTXΦ, and the phage genome integrated into the O139 host chromosome. The resulting CTXΦ lysogens produced biologically active CT both in vitro and in rabbit ileal loops. This finding suggests a possible mechanism explaining the origination of toxigenic V. choleraeO139 strains from nontoxigenic progenitors. This study indicates that sunlight is a significant inducer of the CTX prophage and suggests that sunlight-induced transmission of CTXΦ may constitute part of a natural mechanism for the origination of new toxigenic strains of V. cholerae.

ABSTRACTCryptosporidium parvumis a significant cause of diarrheal disease worldwide. The specific molecules that mediateC. parvum-host cell interactions and the molecular mechanisms involved in the pathogenesis of cryptosporidiosis are unknown. In this study we have shown that gp40, a mucin-like glycoprotein, is localized to the surface and apical region of invasive stages of the parasite and is shed from its surface. gp40-specific antibodies neutralize infection in vitro, and native gp40 binds specifically to host cells, implicating this glycoprotein in C. parvumattachment to and invasion of host cells. We have cloned and sequenced a gene designated Cpgp40/15that encodes gp40 as well as gp15, an antigenically distinct, surface glycoprotein also implicated in C. parvum-host cell interactions. Analysis of the deduced amino acid sequence of the 981-bp Cpgp40/15revealed the presence of an N-terminal signal peptide, a polyserine domain, multiple predicted O-glycosylation sites, a single potential N-glycosylation site, and a hydrophobic region at the C terminus, a finding consistent with what is required for the addition of a GPI anchor. There is a single copy ofCpgp40/15in the C. parvumgenome, and this gene does not contain introns. Our data indicate that the twoCpgp40/15-encoded proteins, gp40 and gp15, are products of proteolytic cleavage of a 49-kDa precursor protein which is expressed in intracellular stages of the parasite. The surface localization of gp40 and gp15 and their involvement in the host-parasite interaction suggest that either or both of these glycoproteins may serve as effective targets for specific preventive or therapeutic measures for cryptosporidiosis.

ABSTRACTVibrio mimicusdiffers from Vibrio choleraein a number of genotypic and phenotypic traits but like V. choleraecan give rise to diarrheal disease. We examined clinical isolates of V. mimicusfor the presence of CTXΦ, the lysogenic filamentous bacteriophage that carries the cholera toxin genes in epidemic V. choleraestrains. Four V. mimicusisolates were found to contain complete copies of CTXΦ. Southern blot analyses revealed that V. mimicusstrain PT5 contains two CTX prophages integrated at different sites within theV. mimicusgenome whereas V. mimicusstrains PT48, 523-80, and 9583 each contain tandemly arranged copies of CTXΦ. We detected the replicative form of CTXΦ, pCTX, in all four of theseV. mimicusisolates. The CTX prophage in strain PT5 was found to produce infectious CTXΦ particles. The nucleotide sequences of CTXΦ genes orfUand zotfrom V. mimicusstrain PT5 and V. choleraestrain N16961 were identical, indicating contemporary horizontal transfer of CTXΦ between these two species. The receptor for CTXΦ, the toxin-coregulated pilus, which is encoded by another lysogenic filamentous bacteriophage, VPIΦ, was also present in the CTXΦ-positive V. mimicusisolates. The nucleotide sequences of VPIΦ genes aldAand toxTfromV. mimicusstrain PT5 and V. choleraeN16961 were identical, suggesting recent horizontal transfer of this phage between V. mimicusand V. cholerae. In V. mimicus, the vibrio pathogenicity island prophage was integrated in the same chromosomal attachment site as in V. cholerae. These results suggest that V. mimicusmay be a significant reservoir for both CTXΦ and VPIΦ and may play an important role in the emergence of new toxigenic V. choleraeisolates.

ABSTRACTWe produced isogenic Escherichia coliK-12 lysogens of seven different Shiga toxin 2 (Stx2)-encoding bacteriophages derived from clinical Shiga toxin-producing E. coli(STEC) isolates of serotypes O157:H7, O145, O111, and O83 to assess the variability among these phages and determine if there were phage-related differences in toxin production. Phage genomic restriction fragment length polymorphisms (RFLP) and superinfection resistance studies revealed significant differences among these phages and allowed the seven phages to be placed into five distinct groups. Experiments revealed striking differences in spontaneous phage and toxin production that were correlated with the groupings derived from the RFLP and resistance studies. These results suggest that the genotype of the Stx2 prophage can influence the level of phage release and toxin expression by host strains and thus may be relevant to STEC pathogenesis.

ABSTRACTHorizontal transfer of genes encoding virulence factors has played a central role in the evolution of many pathogenic bacteria. The unexpected discovery that the genes encoding cholera toxin (ctxAB), the main cause of the profuse secretory diarrhea characteristic of cholera, are encoded on a novel filamentous phage named CTXΦ, has resulted in a renewed interest in the potential mechanisms of transfer of virulence genes among Vibrio cholerae. We describe here an alternative mechanism of cholera toxin gene transfer into nontoxigenic V. choleraeisolates, including strains that lack both the CTXΦ receptor, the toxin coregulated pilus (TCP), and attRS, the chromosomal attachment site for CTXΦ integration. A temperature-sensitive mutant of the V. choleraegeneralized transducing bacteriophage CP-T1 (CP-T1ts) was used to transfer a genetically marked derivative of the CTX prophage into four nontoxigenicV. choleraestrains, including two V. choleraevaccine strains. We demonstrate that CTXΦ transduced by CP-T1ts can replicate and integrate into these nontoxigenic V. choleraestrains with high efficiency. In fact, CP-T1ts transduces the CTX prophage preferentially when compared with other chromosomal markers. These results reveal a potential mechanism by which CTXΦ+V. choleraestrains that lack the TCP receptor may have arisen. Finally, these findings indicate an additional pathway for reversion of live-attenuated V. choleraevaccine strains.

ABSTRACTThe suckling mouse has been used as a model to identifyVibrio choleraeintestinal colonization factors for over two decades, yet little is known about the location of recoverable organisms along the gastrointestinal (GI) tract following intragastric inoculation. In the present study, we determined the population dynamics of wild-type and avirulent mutant derivatives of both classical and El Tor biotype strains throughout the entire suckling mouse GI tract at various times after intragastric inoculation. Wild-type strains preferentially colonized the middle small bowel with a sharp demarcation between more proximal segments which had manyfold-fewer recoverable cells. Surprisingly, large and stable populations of viable cells were also recovered from the cecum and large bowel. Strains lacking toxin-coregulated pili (TCP−) were cleared from the small bowel; however, an El Tor TCP−strain colonized the cecum and large bowel almost as well as the wild-type strain. Strains lacking lipopolysaccharide O antigen (OA−) were efficiently cleared from the small bowel at early times but then showed net growth for the remainder of the infections. Moreover, large populations of the OA−strains were maintained in the large bowel. These results show that for the El Tor biotype neither TCP nor OA is required for colonization of the suckling mouse large bowel. Finally, similar percent recoveries of wild-type, TCP−, and OA−strains from the small bowel at an early time after infection suggest that TCP and OA are not required for strains of either biotype to resist bactericidal mechanisms in the suckling mouse GI tract.

Abstract: The morphological and physical characteristics of the capsule of Vibrio cholerae O139 were examined. An electron microscopic study using the freeze-substitution technique showed that all of the V. cholerae strains of the O139 serogroup examined have a very thin fibrous layer on the outside of the outer membrane. In contrast, the mutants of strain O139, strain MO10T4 (which lacks capsule synthesis), and strain Bengal-2R1 (which fails to synthesize both the capsule and the O-antigen of lipopolysaccharide) were all found to have lost the surface layer. In addition, the capsule layer could also not be observed on the surface of V. cholerae strain O1. To determine the biological characteristics of the capsule of strains of the O139 serogroup, we investigated the serum killing activity and bacterial phagocytosis by polymorphonuclear leukocytes. The O139 strains were more resistant to the serum killing activity than were the V. cholerae O1 strain and the O139 mutant strains, thus suggesting that the existence of the capsule gave a serum-resistant character to the O139 strains. The surface character of the O139 strains had the same hydrophobic character as did that of the O139 mutant strains and the O1 strain. In addition, all the V. cholerae O1 and O139 strains examined, including the mutant strains, were effectively ingested by the human polymorphonuclear leukocytes. The number of ingested bacteria was not significantly different among the strains, and the ingestion of the acapsular O139 mutants thus showed that the capsule does not play an antiphagocytic role. These data suggest that the capsule of V. cholerae O139 has a physiological function different from that of the ordinal hydrophilic capsule that is found in invasive bacteria such as Klebsiella pneumoniae.

ABSTRACTPathogenic strains of Vibrio choleraeare lysogens of the filamentous phage CTXφ, which carries the genes for cholera toxin (ctxAB). We found that the titers of infective CTXφ in culture supernatants of El Tor CTXφ lysogens increased rapidly during exponential growth but dropped to undetectable levels late in stationary-phase growth. When CTXφ transducing particles were mixed with stationary-phase culture supernatants of El Tor strains, CTXφ infectivity was destroyed. Our data indicate that this growth phase-regulated factor, designated CDF (CTXφ-destroying factor), is the secreted hemagglutinin/protease (HA/P) of V. cholerae. A strain containing a disrupted hapgene, which encodes HA/P of V. cholerae, did not produce CDF activity in culture supernatants. Introduction of the HA/P-expressing plasmid pCH2 restored CDF activity. Also, CDF activity in culture supernatants of a variety of pathogenic V. choleraeisolates varied widely but correlated with the levels of secreted HA/P, as measured by immunoblotting with anti-HA/P antibody. CDF was purified from V. choleraeculture supernatants and shown to contain a 45-kDa polypeptide which bound anti-HA/P antibodies and which comigrated with HA/P in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The production of high levels of secreted HA/P by certain V. choleraestrains may be a factor in preventing CTXφ reinfection in natural environments and in the human host.

A monoclonal antibody to I-Agene products of the immune response gene complex attenuates both humoral and cellular responses to acetylcholine receptor and appears to suppress clinical manifestations of experimental autoimmune myasthenia gravis. This demonstrates that use of antibodies against immune response gene products that are associated with susceptibility to disease may be feasible for therapy in autoimmune conditions such as myasthenia gravis.