Kegg Pathway: Cholera - Colonization

Proc Natl Acad Sci U S A. 2008 Jul 15; 105(28): 9769-74
Liu Z, Miyashiro T, Tsou A, Hsiao A, Goulian M, Zhu J

To successfully infect a host and cause the diarrheal disease Cholera, Vibrio Cholerae must penetrate the intestinal mucosal layer and express virulence genes. Previous studies have demonstrated that the transcriptional regulator HapR, which is part of the quorum sensing network in V. Cholerae, represses the expression of virulence genes. Here, we show that hapR expression is also modulated by the regulatory network that governs flagellar assembly. Specifically, FliA, which is the alternative sigma-factor (sigma(28)) that activates late-class flagellin genes in V. Cholerae, represses hapR expression. In addition, we show that mucin penetration by V. Cholerae is sufficient to break flagella and so cause the secretion of FlgM, the anti-sigma factor that inhibits FliA activity. During initial Colonization of host intestinal tissue, hapR expression is repressed because of low cell density. However, full repression of hapR expression does not occur in fliA mutants, which results in attenuated Colonization. Our results suggest that V. Cholerae uses flagellar machinery to sense particular intestinal signals before Colonization and enhance the expression of virulence genes by modulating the output of quorum sensing signaling.

Infect Immun. 2008 Aug; 76(8): 3595-605
Bina XR, Provenzano D, Nguyen N, Bina JE

Vibrio Cholerae is a gram-negative human intestinal pathogen that causes the diarrheal disease Cholera. Humans acquire Cholera by ingesting V. Cholerae-contaminated food or water. Upon ingestion, V. Cholerae encounters several barriers to Colonization, including bile acid toxicity and antimicrobial products of the innate immune system. In many gram-negative bacteria, resistance to the antimicrobial effects of these products is mediated by RND (resistance-nodulation-division) family efflux systems. In this study we tested the hypothesis that the V. Cholerae RND efflux systems are required for antimicrobial resistance and virulence. The six V. Cholerae genes encoding RND efflux pumps were deleted from the genome of the O1 El Tor strain N16961, resulting in the generation of 14 independent RND deletion mutants, including one RND-null strain. Determination of the antimicrobial susceptibilities of the mutants revealed that the RND efflux systems were responsible for resistance to multiple antimicrobial compounds, including bile acids, antimicrobial peptides, and antibiotics. VexB (VC0164) was found to be the RND efflux pump primarily responsible for the resistance of V. Cholerae to multiple antimicrobial compounds in vitro. In contrast, VexD (VC1757) and VexK (VC1673) encoded efflux pumps with detergent-specific substrate specificities that were redundant with VexB. A strain lacking VexB, VexD, and VexK was attenuated in the infant mouse model, and its virulence factor production was unaffected. In contrast, a V. Cholerae RND-null strain produced significantly less Cholera toxin and fewer toxin-coregulated pili than the wild type and was unable to colonize the infant mouse. The decreased virulence factor production in the RND-null strain was linked to reduced transcription of tcpP and toxT. Our findings show that the V. Cholerae RND efflux systems are required for antimicrobial resistance, optimal virulence factor production, and Colonization of the infant mouse.

Microb Pathog. 2008 Jul; 45(1): 7-11
Babiuk S, Asper DJ, Rogan D, Mutwiri GK, Potter AA

Type III secreted proteins from Escherichia coli O157:H7 are involved in the attachment of the organism to mammalian cells and have been shown to be effective vaccine components capable of reducing Colonization of cattle by the organism. In the current study, we used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against EspA and Tir were also monitored. Subcutaneous immunization of mice with type III secreted proteins induced significant EspA- and Tir-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant EspA- and Tir-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Only mice that were immunized intranasally with formulations containing mucosal adjuvants, either Cholera toxin or CpG-containing oligonucleotides, showed decreased E. coli O157:H7 shedding following experimental infection. Mice immunized subcutaneously with type III secreted proteins did not shed E. coli in feces. These results demonstrate the potential for the use of type III secreted proteins in mucosal vaccine formulations to prevent Colonization and shedding of E. coli O157:H7.

J Biotechnol. 2008 May 20; 135(1): 22-7
Sharma MK, Jani D, Thungapathra M, Gautam JK, Meena LS, Singh Y, Ghosh A, Tyagi AK, Sharma AK

In earlier study from our group, Cholera toxin B subunit had been expressed in tomato for developing a plant-based vaccine against Cholera. In the present investigation, gene for accessory Colonization factor (acf) subunit A, earlier reported to be essential for efficient Colonization in the intestine, has been expressed in Escherichia coli as well as tomato plants. Gene encoding for a chimeric protein having a fusion of Cholera toxin B subunit and accessory Colonization factor A was also expressed in tomato to generate more potent combinatorial antigen. CaMV35S promoter with a duplicated enhancer sequence was used for expression of these genes in tomato. Integration of transgenes into tomato genome was confirmed by PCR and Southern hybridization. Expression of the genes was confirmed at transcript and protein levels. Accessory Colonization factor A and Cholera toxin B subunit fused to this protein accumulated up to 0.25% and 0.08% of total soluble protein, respectively, in the fruits of transgenic plants. Whereas protein purified from E. coli, in combination with Cholera toxin B subunit can be used for development of conventional subunit vaccine, tomato fruits expressing these proteins can be used together with tomato plants expressing Cholera toxin B subunit for development of oral vaccine against Cholera.

Infect Immun. 2008 Jun; 76(6): 2678-84
Trzciński K, Thompson CM, Srivastava A, Basset A, Malley R, Lipsitch M

CD4(+) T-cell-dependent acquired immunity confers antibody-independent protection against pneumococcal Colonization. Since this mechanism is poorly understood for extracellular bacteria, we assessed the antigen specificity of the induction and recall of this immune response by using BALB/c DO11.10Rag(-/-) mice, which lack mature B and T cells except for CD4(+) T cells specific for the OVA(323-339) peptide derived from ovalbumin. Serotype 6B Streptococcus pneumoniae strain 603S and unencapsulated strain Rx1Delta lytA were modified to express OVA(323-339) as a fusion protein with surface protein A (PspA) (strains 603OVA(1) and Rx1Delta lytAOVA(1)) or with PspA, neuraminidase A, and pneumolysin (Rx1Delta lytAOVA(3)). Whole-cell vaccines (WCV) were made of ethanol-killed cells of Rx1Delta lytA plus Cholera toxin (CT) adjuvant, of Rx1Delta lytAOVA(1) + CT (WCV-OVA(1)), and of Rx1Delta lytAOVA(3) + CT (WCV-OVA(3)). Mice intranasally immunized with WCV-OVA(1), but not with WCV or CT alone, were protected against intranasal challenge with 603OVA(1). There was no protection against strain 603S in mice immunized with WCV-OVA(1). These results indicate antigen specificity of both immune induction and the recall response. Effector action was not restricted to antigen-bearing bacteria since Colonization by 603S was reduced in animals immunized with vaccines made of OVA-expressing strains when ovalbumin or killed Rx1Delta lytAOVA(3) antigen was administered around the time of challenge. CD4(+) T-cell-mediated protection against pneumococcal Colonization can be induced in an antigen-specific fashion and requires specific antigen for effective bacterial clearance, but this activity may extend beyond antigen-expressing bacteria. These results are consistent with the recruitment and/or activation of phagocytic or other nonspecific effectors by antigen-specific CD4(+) T cells.

Infect Immun. 2008 Apr; 76(4): 1617-27
Tamayo R, Schild S, Pratt JT, Camilli A

In Vibrio Cholerae, the second messenger cyclic di-GMP (c-di-GMP) positively regulates biofilm formation and negatively regulates virulence and is proposed to play an important role in the transition from persistence in the environment to survival in the host. Herein we describe a characterization of the infection-induced gene cdpA, which encodes both GGDEF and EAL domains, which are known to mediate diguanylate cyclase and c-di-GMP phosphodiesterase (PDE) activities, respectively. CdpA is shown to possess PDE activity, and this activity is regulated by its inactive degenerate GGDEF domain. CdpA inhibits biofilm formation but has no effect on Colonization of the infant mouse small intestine. Consistent with these observations, cdpA is expressed during in vitro growth in a biofilm but is not expressed in vivo until the late stage of infection, after Colonization has occurred. To test for a role of c-di-GMP in the early stages of infection, we artificially increased c-di-GMP and observed reduced Colonization. This was attributed to a significant reduction in toxT transcription during infection. Cumulatively, these results support a model of the V. Cholerae life cycle in which c-di-GMP must be down-regulated early after entering the small intestine and maintained at a low level to allow virulence gene expression, Colonization, and motility at appropriate stages of infection.

Mol Microbiol. 2008 Feb; 67(4): 849-60
Hsiao A, Toscano K, Zhu J

The pathogen Vibrio Cholerae modulates the expression of many genes in order to transition from its environmental reservoir to its niche in the human host. Among these are genes encoding two related Type IV pili, the mannose-sensitive haemagglutinin (MSHA) pilus, which aids V. Cholerae persistence in aquatic environments but causes clearance of bacteria by host immune defences, and the toxin co-regulated pilus (TCP) required for Colonization. These antagonistic effects are resolved transcriptionally by the regulator ToxT, which represses msh genes while activating tcp genes during infection. We show that these two pili systems are also intertwined post-transcriptionally through the ToxT-regulated pre-pilin peptidase TcpJ. We found that the major MSHA pilin, MshA, was degraded in V. Cholerae in a TcpJ-dependent fashion. In a heterologous Escherichia coli system, TcpJ can recognize both MshA and its cognate substrate, the TCP subunit TcpA, but that processing by TcpJ causes the degradation of MshA. Through site-directed mutagenesis and chimeric pilin analysis, we show that this process targets a combination of MshA N-terminal motifs and depends on the proteolytic activity of TcpJ. Moreover, overexpression of tcpJ partially restored the ability of bacteria unable to transcriptionally downregulate msh genes to colonize infant mice. These findings describe co-ordinated proteolysis as a regulatory mechanism in V. Cholerae and illustrate this organism's adaptability in the face of dramatic environmental changes.

Cell Host Microbe. 2007 Apr 19; 1(2): 95-107
Tam VC, Serruto D, Dziejman M, Brieher W, Mekalanos JJ

We have previously characterized a non-O1, non-O139 Vibrio Cholerae strain, AM-19226, that lacks the known virulence factors but contains components of a type III secretion system (T3SS). In this study, we demonstrated that the T3SS is functional and is required for intestinal Colonization in the infant mouse model. We also identified VopF, which is conserved among T3SS-positive V. Cholerae strains, as an effector containing both formin homology 1-like (FH1-like) and WASP homology 2 (WH2) domains. Translocation of VopF by V. Cholerae or expression by transfection altered the actin cytoskeletal organization of the eukaryotic host cells. In vitro domain analysis indicated that both FH1-like and WH2 domains are required for actin nucleation and polymerization activity. These data correlate with in vivo data, suggesting that VopF-mediated alteration of actin polymerization homeostasis is required for efficient intestinal Colonization by T3SS+V. Cholerae in the infant mouse model.

Mol Microbiol. 2007 Dec; 66(6): 1331-41
Shakhnovich EA, Sturtevant D, Mekalanos JJ

Virstatin is a previously described small molecule inhibitor of Vibrio Cholerae virulence. We have demonstrated that the molecule inhibits the activity of the transcriptional activator ToxT, thereby preventing elaboration of the toxin co-regulated pilus (TCP) and Cholera toxin in vitro and in vivo in O1 strains of V. Cholerae. While strains of the O1 and O139 serogroups are the cause of most epidemic and endemic Cholera currently seen globally, sporadic disease caused by strains of non-O1/non-O139 serogroups suggests that understanding the pathogenic mechanisms of these unusual strains is relevant for disease. Although some non-O1/non-O139 strains have acquired the pathogenicity island that encodes the TCP, the role that this essential Colonization factor of O1/O139 strains plays in the virulence of non-O1/non-O139 strains has not been determined. In this study, we utilize virstatin in a 'chemical genetic approach' to examine the role of ToxT, and thus by inference TCP, in the Colonization of a panel of predominantly non-O1/non-O139 tcp+ strains. We identified nine strains whose Colonization was resistant to virstatin inhibition in the infant mouse model. These strains presumably colonize by a TCP-independent mechanism or contain a naturally occurring virstatin-resistant ToxT. Four strains contained the typical toxT gene found in O1/O139 strains (toxT(EPI)) isolated from Cholera epidemics. Interruption of toxT in one of these strains did not affect Colonization of the infant mouse small intestine. The remaining five strains were found to contain a sequence divergent toxT gene that has been previously designated toxT(ENV) because of its occurrence in isolates of V. Cholerae from the environment. We show that ToxT(ENV) is resistant to virstatin in two separate heterologous systems and is necessary for efficient Colonization of the infant mouse small intestine. These results support the new concept that chemical genetic probes for the in vivo function or expression of virulence genes can be used to identify strains that express alternative virulence factors or novel regulatory systems that are functional in vivo.

J Bacteriol. 2008 Jan; 190(1): 231-9
Morris DC, Peng F, Barker JR, Klose KE

Vibrio Cholerae, the causative agent of Cholera, has a sheathed, polar flagellum, and motility has been linked to virulence. An operon with two genes, flgO and flgP (VC2207 and VC2206), is positively regulated by FlrC, the activator of class III flagellar genes. Deletion of flgP results in a nonmotile phenotype, demonstrating the requirement of this gene for V. Cholerae motility. V. Cholerae delta flgP cells synthesize fragile and defective flagella but transcribe flagellar genes similar to the wild-type strain. PhoA fusion analysis indicated that the putative lipoprotein FlgP is localized external to the cytoplasm, and fractionation demonstrated that it was localized to the outer membrane. Mutagenesis of the site of lipidation of FlgP (C18G) prevented [3H]palmitate incorporation and outer membrane localization. Interestingly, FlgP with the mutation C18G [FlgP(C18G)] could complement the delta flgP mutant for motility, and the cells synthesized wild-type flagella. The delta flgP mutant strain was defective for intestinal Colonization (approximately 20-fold), but FlgP(C18G) was unable to complement this defect, demonstrating that lipidation of FlgP is essential for its role in intestinal Colonization but not flagellar synthesis. FlgP thus represents a novel V. Cholerae intestinal Colonization factor that is regulated by the flagellar transcription hierarchy.

Plant Cell Rep. 2008 Feb; 27(2): 307-18
Sharma MK, Singh NK, Jani D, Sisodia R, Thungapathra M, Gautam JK, Meena LS, Singh Y, Ghosh A, Tyagi AK, Sharma AK

For protection against Cholera, it is important to develop efficient vaccine capable of inducing anti-toxin as well as anti-colonizing immunity against Vibrio Cholerae infections. Earlier, expression of Cholera toxin B subunit (CTB) in tomato was reported by us. In the present investigation, toxin co-regulated pilus subunit A (TCPA), earlier reported to be an antigen capable of providing anti-Colonization immunity, has been expressed in tomato. Further, to generate more potent combinatorial antigens, nucleotides encoding P4 or P6 epitope of TCPA were fused to Cholera toxin B subunit gene (ctxB) and expressed in tomato. Presence of transgenes in the tomato genome was confirmed by PCR and expression of genes was confirmed at transcript and protein level. TCPA, chimeric CTB-P4 and CTB-P6 proteins were also expressed in E. coli. TCPA protein expressed in E. coli was purified to generate anti-TCPA antibodies in rabbit. Immunoblot and G(M1)-ELISA verified the synthesis and assembly of pentameric chimeric proteins in fruit tissue of transgenic tomato plants. The chimeric protein CTB-P4 and CTB-P6 accumulated up to 0.17 and 0.096% of total soluble protein (TSP), respectively, in tomato fruits. Whereas expression of TCPA, CTB-P4 and CTB-P6 in E. coli can be utilized for development of conventional vaccine, expression of these antigens which can provide both anti-toxin as well as anti-Colonization immunity, has been demonstrated in plants, in a form which is potentially capable of inducing immune response against Cholera infection.

Appl Environ Microbiol. 2007 Dec; 73(24): 7926-33
Rawlings TK, Ruiz GM, Colwell RR

The association of Vibrio Cholerae with zooplankton has been suggested as an important factor in transmission of human epidemic Cholera, and the ability to colonize zooplankton surfaces may play a role in the temporal variation and predominance of the two different serogroups (V. Cholerae O1 El Tor and O139) in the aquatic environment. To date, interactions between specific serogroups and species of plankton remain poorly understood. Laboratory microcosm experiments were carried out to compare quantitatively the Colonization of two copepod species, Acartia tonsa and Eurytemora affinis, by each of the epidemic serogroups. V. Cholerae O1 consistently achieved higher abundances than V. Cholerae O139 in colonizing adults of each copepod species as well as the multiple life stages of E. affinis. This difference in Colonization may be significant in the general predominance of V. Cholerae O1 in Cholera epidemics in rural Bangladesh where water supplies are taken directly from the environment.

Biochim Biophys Acta. 2007 Oct; 1774(10): 1331-8
Chatterjee T, Saha RP, Chakrabarti P

The transcription activator ToxR controls the expression of Cholera toxin, pilus Colonization factor and outer membrane protein in Vibrio Cholerae. It binds to the 5'-TTTTGAT-3' tandemly repeated DNA sequence in the Cholera toxin promoter region. ToxR is a membrane protein having distinct periplasmic and cytoplasmic domains. The two domains have been cloned, over-expressed and purified for structural studies. The cytoplasmic domain is more compact than the periplasmic domain. The periplasmic domain exists as dimer due to the presence of an interchain disulfide linkage, while the cytoplasmic domain is monomeric in solution implying the importance of the disulfide bond to homodimerize the native ToxR. By replacing one of the cysteines C293 with alanine, using site-directed mutagenesis, a C293A mutant was created at the periplasmic domain to elucidate the role of cysteine in dimerization of ToxR.

Microbiology. 2007 Sep; 153(Pt 9): 2964-75
Liang W, Pascual-Montano A, Silva AJ, Benitez JA

Vibrio Cholerae is the causative agent of Cholera, which continues to be a major public health concern in Asia, Africa and Latin America. The bacterium can persist outside the human host and alternates between planktonic and biofilm community lifestyles. Transition between the different lifestyles is mediated by multiple signal transduction pathways including quorum sensing. Expression of the Zn-metalloprotease haemagglutinin (HA)/protease is subject to a dual regulation which involves the quorum-sensing regulator HapR and the cAMP receptor protein. In a previous study, we observed that a mutant defective in the cAMP-receptor protein (CRP) expressed lower levels of HapR. To further investigate the role of CRP in modulating HapR and other signal transduction pathways, we performed global gene expression profiling of a Deltacrp mutant of El Tor biotype V. Cholerae. Here we show that CRP is required for the biosynthesis of Cholera autoinducer 1 (CAI-1) and affects the expression of multiple HapR-regulated genes. As expected, the Deltacrp mutant produced more Cholera toxin and enhanced biofilm. Expression of flagellar genes, reported to be affected in DeltahapR mutants, was diminished in the Deltacrp mutant. However, an epistasis analysis indicated that cAMP-CRP affects motility by a mechanism independent of HapR. Inactivation of crp inhibited the expression of multiple genes reported to be strongly induced in vivo and to affect the ability of V. Cholerae to colonize the small intestine and cause disease. These genes included ompU, ompT and ompW encoding outer-membrane proteins, the alternative sigma factor sigma(E) required for intestinal Colonization, and genes involved in anaerobic energy metabolism. Our results indicate that CRP plays a crucial role in the V. Cholerae life cycle by affecting quorum sensing and multiple genes required for survival of V. Cholerae in the human host and the environment.

Infect Immun. 2007 Oct; 75(10): 5043-51
Olivier V, Salzman NH, Satchell KJ

Cholera epidemics caused by Vibrio Cholerae El Tor O1 strains are typified by a large number of asymptomatic carriers who excrete vibrios but do not develop diarrhea. This carriage state was important for the spread of the seventh Cholera pandemic as the bacterium was mobilized geographically, allowing the global dispersion of this less virulent strain. Virulence factors associated with the development of the carriage state have not been previously identified. We have developed an animal model of Cholera in adult C57BL/6 mice wherein V. Cholerae colonizes the mucus layer and forms microcolonies in the crypts of the distal small bowel. Colonization occurred 1 to 3 h after oral inoculation and peaked at 10 to 12 h, when bacterial loads exceeded the inoculum by 10- to 200-fold, indicating bacterial growth within the small intestine. After a clearance phase, the number of bacteria within the small intestine, but not those in the cecum or colon, stabilized and persisted for at least 72 h. The ability of V. Cholerae to prevent clearance and establish this prolonged Colonization was associated with the accessory toxins hemolysin, the multifunctional autoprocessing RTX toxin, and hemagglutinin/protease and did not require Cholera toxin or toxin-coregulated pili. The defect in Colonization attributed to the loss of the accessory toxins may be extracellularly complemented by inoculation of the defective strain with an isogenic Colonization-proficient V. Cholerae strain. This work thus demonstrates that secreted accessory toxins modify the host environment to enable prolonged Colonization of the small intestine in the absence of overt disease symptoms and thereby contribute to disease dissemination via asymptomatic carriers.

Future Microbiol. 2007 Jun; 2: 335-44
Childers BM, Klose KE

Vibrio Cholerae is a gram-negative bacterium that is the causative agent of Cholera. This disease consists of enormous fluid loss through stools, which can be fatal. Cholera epidemics appear in explosive outbreaks that have occurred repeatedly throughout history. The virulence factors toxin coregulated pilus (TCP) and Cholera toxin (CT) are essential for Colonization of the host and enterotoxicity, respectively. These virulence factors are under the control of ToxT, an AraC/XylS family protein that activates transcription of the genes encoding TCP and CT. ToxT is under the control of a virulence regulatory cascade known as the ToxR regulon, which responds to environmental stimuli to ensure maximal virulence-factor induction within the human intestine. An understanding of this intricate signaling pathway is essential for the development of methods to treat and prevent this devastating disease.

Vaccine. 2007 Aug 14; 25(33): 6201-10
Zhang P, Lewis JP, Michalek SM, Katz J

The gingipains of Porphyromonas gingivalis have been implicated in the virulence of this bacterium, and antibodies to the hemagglutinin/adhesin domain (HArep) of the gingipains have been shown to protect against P. gingivalis Colonization. However, the cellular mechanisms involved in host responses to HArep have not been elucidated. The purpose of the present study was to determine the functional role of CD80 and CD86 in mediating systemic and mucosal immune responses to the recombinant HArep derived from the gingipain Kgp (Kgp-HArep) after intranasal (i.n.) immunization. We also investigated the effect of the mucosal adjuvants the B subunit of Cholera toxin (CTB) and monophosphoryl lipid A (MPL) on the functional role of the costimulatory molecules for the induction of systemic and mucosal responses to Kgp-HArep. The in vivo functional roles of CD80 and CD86 were assessed in C57BL/6 wild-type (wt), CD80(-/-), CD86(-/-) and CD80/CD86(-/-) mice following intranasal immunization with Kgp-HArep with or without adjuvant. Serum IgG and mucosal IgA antibody responses were induced following i.n. immunization of mice with Kgp-HArep, and were potentiated by CTB or MPL. A differential requirement of CD80and/or CD86 was observed for systemic IgG anti-Kgp-HArep responses following the primary and secondary immunization with antigen alone or antigen+adjuvant. Compared to wt and CD80(-/-) mice, CD86(-/-) mice had reduced serum IgG anti-Kgp-HArep responses following the second immunization with antigen alone or antigen+CTB, whereas similar levels of serum IgG anti-Kgp-HArep antibody activity were observed in wt, CD80(-/-) and CD86(-/-) mice immunized with antigen+MPL. Analysis of the serum IgG subclass responses revealed that CD80 influenced both Th1- and Th2-like IgG subclass responses, while CD86 preferentially influenced a Th2-associated IgG subclass response to Kgp-HArep. Mucosal IgA anti-Kgp-HArep responses in saliva and vaginal washes were diminished in CD86(-/-) mice. In vitro stimulation of murine bone marrow-derived dendritic cells with Kgp-HArep, CTB and MPL resulted in an up-regulation of CD80 and especially CD86 expression. Taken together, our results demonstrate that CD80 and CD86 can play distinct as well as redundant roles in mediating a systemic immune response and that CD86 plays a unique role in mediating a mucosal response to Kgp-HArep following immunization via the i.n. route alone or with adjuvant.

J Clin Microbiol. 2007 Sep; 45(9): 3068-71
Ghosal A, Bhowmick R, Nandy RK, Ramamurthy T, Chatterjee NS

Colonization factor antigens (CFAs) of enterotoxigenic Escherichia coli (ETEC) have been classified into several groups based on their distinct antigenicity. We describe here a PCR-based method to detect common CFAs of ETEC, which were characterized using conventional serology. This PCR assay is simple and sensitive for the detection of expressed CFA genes.

FEMS Immunol Med Microbiol. 2007 Jul; 50(2): 213-9
Sutton P, Doidge C, Pinczower G, Wilson J, Harbour S, Swierczak A, Lee A

Several studies have explored the production and immunogenicity of HpaA as a potential protective antigen against Helicobacter pylori but little is known regarding its protective capabilities. We therefore evaluated the protective efficacy of recombinant HpaA (rHpaA) as a candidate vaccine antigen against H. pylori. To explore the impact of genetic diversity, inbred and outbred mice were prophylactically and therapeutically immunized with rHpaA adjuvanted with Cholera toxin (CT). Prophylactic immunization induced a reduction in bacterial Colonization in BALB/c and QS mice, but was ineffective in C57BL/6 mice, despite induction of antigen-specific antibodies. By contrast, therapeutic immunization was effective in all three strains of mice. Prophylactic immunization with CT-adjuvanted rHpaA was more effective when delivered via the nasal route than following intragastric delivery in BALB/c mice. However, HpaA-mediated protection was inferior to that induced by bacterial lysate. Hence, protective efficacy is inducible with vaccines containing HpaA, most relevantly shown in an outbred population of mice. The effectiveness of protection induced by HpaA antigen was influenced by host genetics and was less effective than lysate. HpaA therefore has potential for the development of effective immunization against H. pylori but this would probably entail the antigen to be one component of a multiantigenic vaccine.

Vaccine. 2007 May 30; 25(22): 4392-400
Sack DA, Shimko J, Torres O, Bourgeois AL, Francia DS, Gustafsson B, Kärnell A, Nyquist I, Svennerholm AM

We tested the efficacy of a killed oral vaccine for enterotoxigenic Escherichia coli (ETEC) diarrhoea to determine if two doses of vaccine with Colonization factor antigens (CF) and Cholera B subunit would protect against ETEC diarrhoea of travellers. Six hundred seventy-two healthy travellers going to Mexico or Guatemala were studied in a prospective, randomised, placebo-controlled trial. The primary outcome was a vaccine preventable outcome (VPO), defined as an episode of ETEC diarrhoea with an ETEC organism producing heat labile toxin (LT) or CF homologous with the vaccine, without other known causes. The vaccine was safe and stimulated anti-heat labile toxin antibodies. There was a significant decrease in more severe VPO episodes (PE=77%, p=0.039) as defined by symptoms that interfered with daily activities or more than five loose stools in a day, although the total number of VPO events did not differ significantly in the vaccine and placebo groups. We conclude that the new oral ETEC vaccine reduces the rate of more severe episodes of traveller's diarrhoea (TD) due to VPO-ETEC, but it did not reduce the overall rate of ETEC diarrhoea or of travellers' diarrhoea due to other causes.