Diarrheal pathogens trigger rapid evolution of the guanylate cyclase-C signaling axis in bats.

The pathogenesis of infectious diarrheal diseases is largely attributed to enterotoxins that cause dehydration by disrupting intestinal water absorption. We investigated patterns of genetic variation in mammalian guanylate cyclase-C (GC-C), an intestinal receptor targeted by bacterially encoded heat-stable enterotoxins (STa), to determine how host species adapt in response to diarrheal infections. Our phylogenetic and functional analysis of GC-C supports long-standing evolutionary conflict with diarrheal bacteria in primates and bats, with highly variable susceptibility to STa across species. In bats, we further show that GC-C diversification has sparked compensatory mutations in the endogenous uroguanylin ligand, suggesting an unusual scenario of pathogen-driven evolution of an entire signaling axis. Together, these findings suggest that conflicts with diarrheal pathogens have had far-reaching impacts on the evolution of mammalian gut physiology.

CBS-derived H2S facilitates host colonization of Vibrio cholerae by promoting the iron-dependent catalase activity of KatB.

Sensing and resisting oxidative stress is critical for Vibrio cholerae to survive in either the aquatic environment or the gastrointestinal tract. Previous studies mainly focused on the mechanisms of oxidative stress response regulation that rely on enzymatic antioxidant systems, while functions of non-enzymatic antioxidants are rarely discussed in V. cholerae. For the first time, we investigated the role of hydrogen sulfide (H2S), the simplest thiol compound, in protecting V. cholerae against oxidative stress. We found that degradation of L-cysteine by putative cystathionine ß-synthase (CBS) is the major source of endogenous H2S in V. cholerae. Our results indicate that intracellular H2S level has a positive correlation with cbs expression, while the enhanced H2S production can render V. cholerae cells less susceptible to H2O2 in vitro. Using proteome analysis and real-time qPCR assay, we found that cbs expression could stimulate the expression of several enzymatic antioxidants, including reactive oxygen species (ROS) detoxifying enzymes SodB, KatG and AhpC, the DNA protective protein DPS and the protein redox regulator Trx1. Assays of ROS detoxification capacities revealed that CBS-derived H2S could promote catalase activity at the post-translational level, especially for KatB, which serves as an important way that endogenous H2S participates in H2O2 detoxification. The enhancement of catalase activity by H2S is achieved through facilitating the uptake of iron. Adult mice experiments showed that cbs mutant has colonization defect, while either complementation of cbs or exogenous supplement of N-Acetyl-L-Cysteine restores its fitness in the host environment. Herein, we proposed that V. cholerae regulates CBS-dependent H2S production for better survival and proliferation under ROS stress.

Outer Membrane Vesicles of Vibrio cholerae Protect and Deliver Active Cholera Toxin to Host Cells via Porin-Dependent Uptake.

Outer membrane vesicles (OMVs) are an emerging research field due to their multifactorial composition and involvement in interspecies and intraspecies communication. Recent studies indicate that vesicle release by Gram-negative bacterial pathogens is increased during in vivo colonization, as exemplified by the facultative human pathogen Vibrio cholerae upon oral ingestion by the host. In this study, we investigate the fate of OMVs produced by the Gram-negative facultative pathogen V. cholerae. We show that vesicles produced by the clinically relevant El Tor biotype are readily taken up by human intestinal cell lines. We identify outer membrane porins of V. cholerae, i.e., OmpU and OmpT, as the required surface effectors on OMVs for cellular uptake, and we pinpoint the uptake mechanism as caveolin-mediated endocytosis. Furthermore, we show that OMVs derived from V. cholerae grown under virulence-inducing conditions act as potent vehicles for delivery of bioactive cholera toxin to intestinal epithelial cells. In contrast to free cholera toxin secreted via the type II secretion system, OMV-associated cholera toxin is protected from degradation by intestinal proteases. Taken together, these data show that OMV-associated cholera toxin can sustain longer periods in the intestinal tract and preserve toxin effects, as indicated by a prolonged increase of cAMP levels in the intestinal tissue. IMPORTANCE Cholera is still a massive global health burden because it causes large outbreaks with millions of infections and thousands of deaths every year. Several studies have contributed to the knowledge of this pathogen, although key parts are still missing. We aim to broaden our understanding of Vibrio cholerae infections, virulence, and toxicity by drawing attention to the involvement of OMVs in these core processes. Upon host entry, V. cholerae increases secretion of OMVs, which can carry the main virulence factor, cholera toxin, to distant host intestinal cells. We show that specific outer membrane porins on the vesicle surface mediate endocytosis of the vesicles into intestinal cells. With protection by the vesicles, cholera toxin activity endures even in the presence of intestinal proteases. It is tempting to hypothesize that the extended half-life of vesicle-associated cholera toxin allows it to target host cells distant from the primary colonization sites.

The periplasmic domains of Vibriocholerae ToxR and ToxS are forming a strong heterodimeric complex independent on the redox state of ToxR cysteines.

The transmembrane protein ToxR plays a key role in the virulence expression system of Vibrio cholerae. The activity of ToxR is dependent on its periplasmic sensor domain (ToxRp) and on the inner membrane protein ToxS. Herein, we present the Nuclear Magnetic Resonance NMR solution structure of the sensory ToxRp containing an intramolecular disulfide bond. The presented structural and dynamic experiments with reduced and oxidized ToxRp propose an explanation for the increased proteolytic sensitivity of reduced ToxR. Additionally, for the first time, we could identify the formation of a strong heterodimer complex between the periplasmic domains of ToxR and ToxS in solution. NMR interaction studies reveal that binding of ToxS is not dependent on the redox state of ToxR cysteines, and formed complexes are structurally similar. By monitoring the proteolytic cleavage of ToxRp with NMR, we additionally provide a direct evidence of ToxS protective function. Taken together our results suggest that ToxR activity is regulated by its stability which is, on the one hand, dependent on the redox states of its cysteines, influencing the stability of its fold, and on the other hand, on its interaction with ToxS, which binds independent on the cysteines and acts as a protection against proteases.

A comprehensive review of therapeutic approaches available for the treatment of cholera.

OBJECTIVES: The oral rehydration solution is the most efficient method to treat cholera; however, it does not interfere in the action mechanism of the main virulence factor produced by Vibrio cholerae, the cholera toxin (CT), and this disease still stands out as a problem for human health worldwide. This review aimed to describe therapeutic alternatives available in the literature, especially those related to the search for molecules acting upon the physiopathology of cholera. KEY FINDINGS: New molecules have offered a protection effect against diarrhoea induced by CT or even by infection from V. cholerae. The receptor regulator cystic fibrosis channel transmembrane (CFTR), monosialoganglioside (GM1), enkephalinase, AMP-activated protein kinase (AMPK), inhibitors of expression of virulence factors and activators of ADP-ribosylarginine hydrolase are the main therapeutic targets studied. Many of these molecules or extracts still present unclear action mechanisms. CONCLUSIONS: Knowing therapeutic alternatives and their molecular mechanisms for the treatment of cholera could guide us to develop a new drug that could be used in combination with the rehydration solution.

Host stimuli and operator binding sites controlling protein interactions between virulence master regulator ToxR and ToxS in Vibrio cholerae.

Protein-protein interactions (PPIs) are key mechanisms in the maintenance of biological regulatory networks. Herein, we characterize PPIs within ToxR and its co-activator, ToxS, to understand the mechanisms of ToxR transcription factor activation. ToxR is a key transcription activator that is supported by ToxS for virulence gene regulation in Vibrio cholerae. ToxR comprises a cytoplasmic DNA-binding domain that is linked by a transmembrane domain to a periplasmic signal receiver domain containing two cysteine residues. ToxR-ToxR and ToxR-ToxS PPIs were detected using an adenylate-cyclase-based bacterial two-hybrid system approach in Escherichia coli. We found that the ToxR-ToxR PPIs are significantly increased in response to ToxR operators, the co-activator ToxS and bile salts. We suggest that ToxS and bile salts promote the interaction between ToxR molecules that ultimately results in dimerization. Upon binding of operators, ToxR-ToxR PPIs are found at the highest frequency. Moreover, disulfide-bond-dependent interaction in the periplasm results in homodimer formation that is promoted by DNA binding. The formation of these homodimers and the associated transcriptional activity of ToxR were strongly dependent on the oxidoreductases DsbA/DsbC. These findings show that protein and non-protein partners, that either transiently or stably interact with ToxR, fine-tune ToxR PPIs, and its associated transcriptional activity in changing environments.

Repositioning of a mucolytic drug to a selective antibacterial against Vibrio cholerae.

Drug repositioning, the approach to explore existing drugs for use in new therapeutic indications, has emerged as an alternative drug development strategy. In this study, we found that a mucolytic drug, N-acetylcysteine (NAC) showed antibacterial activity against Vibrio cholerae. NAC can provide acid stress that selectively inhibited the growth of V. cholerae among other bacterial pathogens. To address the antibacterial mechanism of NAC against V. cholerae, six acr (acetylcys-teine-resistant) mutants were isolated from 3,118 random transposon insertion clones. The transposon insertion sites of the six mutants were mapped at the five genes. All these mutants did not display NAC resistance under acidic conditions, despite their resistance to NAC under alkaline conditions, indicating that the NAC resistance directed by the acr mutations was independent of the unusual pH-sensitivity of V. cholerae. Furthermore, all these mutants displayed attenuated virulence and reduced biofilm formation, suggesting that the acr genes are required for pathogenesis of V. cholerae. This study validates the relevance of drug repositioning for antibacterials with new modes of action and will provide an insight into a novel antibacterial therapy for V. cholerae infections to minimize side effects and resistance emergence.

Anti-virulence activity of polyphenolic fraction isolated from Kombucha against Vibrio cholerae.

The use of traditional foods and beverages or their bioactive compounds as anti-virulence agents is a new alternative method to overcome the increased global emergence of antimicrobial resistance in enteric pathogens. In the present study, we investigated the anti-virulence activity of a polyphenolic fraction previously isolated from Kombucha, a 14-day fermented beverage of sugared black tea, against Vibrio cholerae O1. The isolated fraction was mainly composed of the polyphenols catechin and isorhamnetin. The fraction, the individual polyphenols and the combination of the individual polyphenols significantly inhibited bacterial swarming motility and expression of flagellar regulatory genes motY and flaC, even at sub-inhibitory concentrations. The polyphenolic compounds also decreased bacterial protease secretion and mucin penetration in vitro. In vivo study revealed that the polyphenolic fraction significantly inhibited V. cholerae induced fluid accumulation in the rabbit ileal loop model and intestinal colonization in suckling mice model. Therefore, the anti-virulence activity of the Kombucha polyphenolic fraction involved inhibition of motility and protease secretion of V. cholerae, thus preventing bacterial penetration through the mucin layer as well as fluid accumulation and bacterial colonization in the intestinal epithelial cells. The overall results implied that Kombucha might be considered as a potential alternative source of anti-virulence polyphenols against V. cholerae. To the best of our knowledge, this is the first report on the anti-virulence activity of Kombucha, mostly attributed to its polyphenolic content.

Large conformation shifts of Vibrio cholerae VqmA dimer in the absence of target DNA provide insight into DNA-binding mechanisms of LuxR-type receptors.

Quorum sensing regulates the biofilm formation and expression of virulence factors in Vibrio cholerae, an obligate human pathogen that continues to imperil human health. Cytoplasmic transcription factor VqmA is a LuxR-type receptor ubiquitous in the Vibrio genus and one vibriophage VP882 and plays an important role in V. cholerae pathogenicity. Here we presented the X-ray crystal structure of V. cholerae VqmA-DPO complex and compared it with the previously determined VqmA-DPO-DNA complex. To our knowledge, this is the first report on the crystal structures of the same LuxR-type receptor with two conformations of binding to DNA and not binding to DNA. Based on the results of structural analysis and biochemical assays, we revealed the secondary structure of the linker region between two function domains changed significantly, and DNA binding domains were covalently linked by a disulfide bond formed by the highly conserved Cys134. Besides, the distance between two DBD monomers became longer than that in DNA-binding conformation, and two α8 helixes underwent a large conformation shift. The results of the structure-function analyses presented here improve our understanding of the complex mechanisms in the conformational changes of LuxR-type receptors caused by DNA binding.

Posttranslational Regulation of IL-23 Production Distinguishes the Innate Immune Responses to Live Toxigenic versus Heat-Inactivated Vibrio cholerae.

Vibrio cholerae infection provides long-lasting protective immunity, while oral, inactivated cholera vaccines (OCV) result in more-limited protection. To identify characteristics of the innate immune response that may distinguish natural V. cholerae infection from OCV, we stimulated differentiated, macrophage-like THP-1 cells with live versus heat-inactivated V. cholerae with and without endogenous or exogenous cholera holotoxin (CT). Interleukin 23A gene (IL23A) expression was higher in cells exposed to live V. cholerae than in cells exposed to inactivated organisms (mean change, 38-fold; 95% confidence interval [95% CI], 4.0 to 42; P < 0.01). IL-23 secretion was also higher in cells exposed to live V. cholerae than in cells exposed to inactivated V. cholerae (mean change, 5.6-fold; 95% CI, 4.4 to 11; P < 0.001). This increase in IL-23 secretion was more marked than for other key innate immune cytokines (e.g., IL-1ß and IL-6) and dependent on exposure to the combination of both live V. cholerae and CT. While IL-23 secretion was reduced following stimulation with either heat-inactivated wild-type V. cholerae or a live isogenic ctxAB mutant of V. cholerae, the addition of exogenous CT restored IL-23 secretion in combination with the live isogenic ctxAB mutant V. cholerae, but not when it was paired with stimulation by heat-inactivated V. cholerae The posttranslational regulation of IL-23 under these conditions was dependent on the activity of the cysteine protease cathepsin B. In humans, IL-23 promotes the differentiation of Th17 cells to T follicular helper cells, which maintain and support long-term memory B cell generation after infection. Based on these findings, the stimulation of IL-23 production may be a determinant of protective immunity following V. cholerae infection.IMPORTANCE An episode of cholera provides better protection against reinfection than oral cholera vaccines, and the reasons for this are still under study. To better understand this, we compared the immune responses of human cells exposed to live Vibrio cholerae with those of cells exposed to heat-killed V. cholerae (similar to the contents of oral cholera vaccines). We also compared the effects of active cholera toxin and the inactive cholera toxin B subunit (which is included in some cholera vaccines). One key immune signaling molecule, IL-23, was uniquely produced in response to the combination of live bacteria and active cholera holotoxin. Stimulation with V. cholerae that did not produce the active toxin or was killed did not produce an IL-23 response. The stimulation of IL-23 production by cholera toxin-producing V. cholerae may be important in conferring long-term immunity after cholera.

Cepas chilenas de origen clínico de Vibrio cholerae no-O1, no-O139 portan los genes vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF del sistema de secreción T3SS2 presentes en una isla de patogenicidad / Chilean strains of clinical origin of non-O1, non-O139 Vibrio cholerae carry the genes vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF from secretion system T3SS2 present in an island of pathogenicity

Resumen Introducción. Los factores de virulencia de las cepas de Vibrio cholerae no-O1, no-O139 no son claramente conocidos. La cepa de origen septicémico NN1 Vibrio cholerae no-O1, no-O139 fue secuenciada previamente mediante la plataforma Illumina, detectándose en su genoma un fragmento de la isla de patogenicidad VPaI-7 de V. parahaemolyticus. Objetivo: detectar los genes de virulencia vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF en cepas chilenas clínicas de V. cholerae no-O1, no-O139. Material y Métodos: Un total de 9 cepas chilenas de origen clínico de Vibrio cholerae no-O1, no-O139 aisladas entre 2006-2012 fueron analizadas mediante ensayos de reacción de polimerasa en cadena (RPC, en inglés PCR) convencional para los genes de secreción tipo III codificados en dicha isla: vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF. Adicionalmente se determinó la presencia de los genes de virulencia hylA y rtxA. Además, se realizaron ensayos de repetitive element palindromic PCR (REP-PCR) y Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Resultados: la mayoría (6/9) de las cepas chilenas de V. cholerae no-O1, no-O139 contiene todos los genes de secreción tipo III vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF, codificados en una isla de patogenicidad. Además, el total de las cepas (9/9) contiene los genes de virulencia hylA y rtxA. Conclusión: Estos resultados sugieren fuertemente la posibilidad que dichas cepas posean un potencial de virulencia importante en seres humanos.

Backgound: The virulence factors of the Vibrio cholerae non-O1, non-O139 strains are not clearly known. The strain of septicemic origin NN1 Vibrio cholerae non-O1, non-O139 was sequenced previously by the Illumina platform. A fragment of the pathogenicity island VPaI-7 of V. parahaemolyticus was detected in its genome. Aim: To detect the virulence genes vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF in Chilean strains of V. cholerae non-O1, non-O139. Methods: A total of 9 Chilean strains of clinical origin of Vibrio cholerae non-O1, non-O139 isolated between 2006-2012 were analyzed by conventional PCR assays for type III secretion genes encoded on that island: vcsN2, vcsC2, vcsV2, vspD, toxR2 and vopF. Additionally, the presence of the virulence genes hylA and rtxA was determined. In addition, REP-PCR and ERIC-PCR assays were performed. Results: most (6/9) Chilean V. cholerae non-O1, non-O139 strains contain the type III secretion genes vcsN2, vcsC2, vcsV2, vspD, toxR2 and vopF, encoded in an island of pathogenicity. In addition, all (9/9) the strains contain the virulence genes hylA and rtxA. Conclusion: These results strongly suggest the possibility that those strains possess an important virulence potential in humans.

Bacteriemia por Vibrio cholerae no-O1/no-O139 que porta una región homóloga a la isla de patogenicidad VpaI-7 / Bacteremia caused by Vibrio cholerae non-O1/non-O139 carrying a region homologous to pathogenicity island VpaI-7

Resumen Presentamos un caso de bacteriemia por Vibrio cholerae no-O1/ no-O139 en una mujer de 81 años con un cuadro de dolor abdominal, fiebre, vómitos, diarrea, coluria e ictericia, mientras visitaba una zona rural sin acceso a agua potable. La identificación se realizó por la técnica de espectrometría de masa MALDI-TOF, confirmándose una cepa no toxigénica no-O1/no-139. La caracterización molecular del aislado demostró la ausencia del gen de la toxina del cólera (CTX), y pilus TCP; sin embargo, presentó cinco de los seis genes de virulencia presentes en la isla de patogenicidad homóloga denominada VPaI-7 del V. parahaemolyticus (vcs N2+, vcs C2+, vcs V2+,toxR-, vspD+, T vopF+). Además, el aislado presentó los genes de virulencia hylA y rtxA. Este es el primer caso reportado en Chile de una cepa clínica de V. cholerae no-O1, no-O139 aislada de hemocultivos portador de un segmento homólogo de la isla de patogenicidad denominada VPaI-7 de V. parahaemolyticus, el cual codifica para un sistema de secreción tipo III (TTSS), que probablemente contribuye a su virulencia.

We report a case of V. cholerae non-O1 / non-O139 bacteremia in an 81-year-old woman with abdominal pain, fever, vomiting, liquid stools, choluria and jaundice, while visiting a rural area without access to potable water. The identification was made by the MALDI-TOF mass spectrometry technique and subsequently the non-toxigenic non-O1 / non-139 strain was confirmed in the national reference laboratory. The molecular characterization demonstrated the absence of the cholera toxin gene (CTX), and the TCP pilus, however, presented 5 of 6 virulence genes present in an island of homologous pathogenicity named VPaI-7 of V. parahaemolyticus (vcs N2 +, vcs C2 +, vcs V2 +, toxR-, vspD +, T vopF +) and in addition it was positive for hylAy rtxA virulence genes recognized outside the island. This is the first case reported in Chile of a clinical strain of V. cholerae non-O1, non-O139 isolated from blood culture that carries in its genome a homologous segment of the pathogenicity island named VPaI-7 of V. parahaemolyticus, which codifies for a type III secretion system (TTSS) that probably contributes to his virulence.

Cholix toxin, an eukaryotic elongation factor 2 ADP-ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes.

Vibrio cholerae produced-Cholix toxin (Cholix) is a cytotoxin that ADP-ribosylates eukaryotic elongation factor 2, inhibiting protein synthesis, and inducing apoptosis. Here, we identified prohibitin (PHB) 1 and 2 as novel Cholix-interacting membrane proteins in immortalised human hepatocytes and HepG2 cells by Cholix immunoprecipitation assays. The expression level of PHB1 was decreased by Cholix after a 12hr incubation. Cholix-induced poly (ADP-ribose) polymerase (PARP) cleavage was significantly enhanced in PHB (PHB1 or PHB2) knockdown cells. In contrast, transiently overexpressed PHB in hepatocytes attenuated Cholix-induced Bax/Bak conformational changes and PARP cleavage. In addition, Cholix-induced reactive oxygen species production and accumulation of fragmented mitochondria were enhanced in PHB-knockdown cells. Furthermore, Cholix induced activation of Rho-associated coiled coil-containing protein kinase 1 (ROCK1), which was enhanced in PHB-knockdown cells, followed by actin filament depolymerisation and accumulation of tubulin in the blebbing cells. Inhibition of ROCK1 by siRNA or its inhibitor suppressed Cholix-induced PARP cleavage and reactive oxygen species generation. Our findings identify PHB as a new protein that interacts with Cholix and is involved in Cholix-induced mitochondrial dysfunction and cytoskeletal rearrangement by ROCK1 activation during apoptosis.

Antibiotic Korormicin A Kills Bacteria by Producing Reactive Oxygen Species.

Korormicin is an antibiotic produced by some pseudoalteromonads which selectively kills Gram-negative bacteria that express the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR.) We show that although korormicin is an inhibitor of Na+-NQR, the antibiotic action is not a direct result of inhibiting enzyme activity. Instead, perturbation of electron transfer inside the enzyme promotes a reaction between O2 and one or more redox cofactors in the enzyme (likely the flavin adenine dinucleotide [FAD] and 2Fe-2S center), leading to the production of reactive oxygen species (ROS). All Pseudoalteromonas contain the nqr operon in their genomes, including Pseudoalteromonas strain J010, which produces korormicin. We present activity data indicating that this strain expresses an active Na+-NQR and that this enzyme is not susceptible to korormicin inhibition. On the basis of our DNA sequence data, we show that the Na+-NQR of Pseudoalteromonas J010 carries an amino acid substitution (NqrB-G141A; Vibrio cholerae numbering) that in other Na+-NQRs confers resistance against korormicin. This is likely the reason that a functional Na+-NQR is able to exist in a bacterium that produces a compound that typically inhibits this enzyme and causes cell death. Korormicin is an effective antibiotic against such pathogens as Vibrio cholerae, Aliivibrio fischeri, and Pseudomonas aeruginosa but has no effect on Bacteroides fragilis and Bacteroides thetaiotaomicron, microorganisms that are important members of the human intestinal microflora.IMPORTANCE As multidrug antibiotic resistance in pathogenic bacteria continues to rise, there is a critical need for novel antimicrobial agents. An essential requirement for a useful antibiotic is that it selectively targets bacteria without significant effects on the eukaryotic hosts. Korormicin is an excellent candidate in this respect because it targets a unique respiratory enzyme found only in prokaryotes, the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR). Korormicin is synthesized by some species of the marine bacterium Pseudoalteromonas and is a potent and specific inhibitor of Na+-NQR, an enzyme that is essential for the survival and proliferation of many Gram-negative human pathogens, including Vibrio cholerae and Pseudomonas aeruginosa, among others. Here, we identified how korormicin selectively kills these bacteria. The binding of korormicin to Na+-NQR promotes the formation of reactive oxygen species generated by the reaction of the FAD and the 2Fe-2S center cofactors with O2.

Human evolutionary loss of epithelial Neu5Gc expression and species-specific susceptibility to cholera.

While infectious agents have typical host preferences, the noninvasive enteric bacterium Vibrio cholerae is remarkable for its ability to survive in many environments, yet cause diarrheal disease (cholera) only in humans. One key V. cholerae virulence factor is its neuraminidase (VcN), which releases host intestinal epithelial sialic acids as a nutrition source and simultaneously remodels intestinal polysialylated gangliosides into monosialoganglioside GM1. GM1 is the optimal binding target for the B subunit of a second virulence factor, the AB5 cholera toxin (Ctx). This coordinated process delivers the CtxA subunit into host epithelia, triggering fluid loss via cAMP-mediated activation of anion secretion and inhibition of electroneutral NaCl absorption. We hypothesized that human-specific and human-universal evolutionary loss of the sialic acid N-glycolylneuraminic acid (Neu5Gc) and the consequent excess of N-acetylneuraminic acid (Neu5Ac) contributes to specificity at one or more steps in pathogenesis. Indeed, VcN was less efficient in releasing Neu5Gc than Neu5Ac. We show enhanced binding of Ctx to sections of small intestine and isolated polysialogangliosides from human-like Neu5Gc-deficient Cmah-/- mice compared to wild-type, suggesting that Neu5Gc impeded generation of the GM1 target. Human epithelial cells artificially expressing Neu5Gc were also less susceptible to Ctx binding and CtxA intoxication following VcN treatment. Finally, we found increased fluid secretion into loops of Cmah-/- mouse small intestine injected with Ctx, indicating an additional direct effect on ion transport. Thus, V. cholerae evolved into a human-specific pathogen partly by adapting to the human evolutionary loss of Neu5Gc, optimizing multiple steps in cholera pathogenesis.

AC6 is the major adenylate cyclase forming a diarrheagenic protein complex with cystic fibrosis transmembrane conductance regulator in cholera.

The World Health Organization(WHO) has reported a worldwide surge in cases of cholera caused by the intestinal pathogen Vibrio cholerae, and, combined, such surges have claimed several million lives, mostly in early childhood. Elevated cAMP production in intestinal epithelial cells challenged with cholera toxin (CTX) results in diarrhea due to chloride transport by a cAMP-activated channel, the cystic fibrosis transmembrane conductance regulator (CFTR). However, the identity of the main cAMP-producing proteins that regulate CFTR in the intestine and may be relevant for secretory diarrhea is unclear. Here, using RNA-Seq to identify the predominant AC isoform in mouse and human cells and extensive biochemical analyses for further characterization, we found that the cAMP-generating enzyme adenylate cyclase 6 (AC6) physically and functionally associates with CFTR at the apical surface of intestinal epithelial cells. We generated epithelium-specific AC6 knockout mice and demonstrated that CFTR-dependent fluid secretion is nearly abolished in AC6 knockout mice upon CTX challenge in ligated ileal loops. Furthermore, loss of AC6 function dramatically impaired CTX-induced CFTR activation in human and mouse intestinal spheroids. Our finding that the CFTR-AC6 protein complex is the key mediator of CTX-associated diarrhea may facilitate development of antidiarrheal agents to manage cholera symptoms and improve outcomes.

Neutralization of cholera toxin with nanoparticle decoys for treatment of cholera.

Diarrheal diseases are a major cause of morbidity and mortality worldwide. In many cases, antibiotic therapy is either ineffective or not recommended due to concerns about emergence of resistance. The pathogenesis of several of the most prevalent infections, including cholera and enteroxigenic Escherichia coli, is dominated by enterotoxins produced by lumen-dwelling pathogens before clearance by intestinal defenses. Toxins gain access to the host through critical host receptors, making these receptors attractive targets for alternative antimicrobial strategies that do not rely on conventional antibiotics. Here, we developed a new nanotechnology strategy as a countermeasure against cholera, one of the most important and prevalent toxin-mediated enteric infections. The key host receptor for cholera toxin, monosialotetrahexosylganglioside (GM1), was coated onto the surface of polymeric nanoparticles. The resulting GM1-polymer hybrid nanoparticles were shown to function as toxin decoys by selectively and stably binding cholera toxin, and neutralizing its actions on epithelial cells in vitro and in vivo. Furthermore, the GM1-coated nanoparticle decoys attenuated epithelial 3',5'-cyclic adenosine monophosphate production and fluid responses to infection with live Vibrio cholera in cell culture and a murine infection model. Together, these studies illustrate that the new nanotechnology-based platform can be employed as a non-traditional antimicrobial strategy for the management of enteric infections with enterotoxin-producing pathogens.

A Bacteriophage Capsid Protein Is an Inhibitor of a Conserved Transcription Terminator of Various Bacterial Pathogens.

Rho is a hexameric molecular motor that functions as a conserved transcription terminator in the majority of bacterial species and is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibits Escherichia coli Rho by obstructing its ATPase and translocase activities. In this study, we explored the anti-Rho activity of Psu for Rho proteins from different pathogens. Sequence alignment and homology modeling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens, including Mycobacterium smegmatis, Mycobacterium bovis, Mycobacterium tuberculosis, Xanthomonas campestris, Xanthomonas oryzae, Corynebacterium glutamicum, Vibrio cholerae, Salmonella enterica, and Pseudomonas syringae The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on poly(rC) as the substrate and were capable of releasing RNA from the E. coli transcription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins. In vivo pulldown assays revealed direct binding of Psu with many of these Rho proteins. In vivo expression of psu induced killing of M. smegmatis, M. bovis, X. campestris, and E. coli expressing S. enterica Rho indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the "universal" inhibitory function of the Psu protein against the Rho proteins from both Gram-negative and Gram-positive bacteria could be useful for designing peptides with antimicrobial functions and that these peptides could contribute to synergistic antibiotic treatment of the pathogens by compromising the Rho functions.IMPORTANCE Bacteriophage-derived protein factors modulating different bacterial processes could be converted into unique antimicrobial agents. Bacteriophage P4 capsid protein Psu is an inhibitor of the E. coli transcription terminator Rho. Here we show that apart from antagonizing E. coli Rho, Psu is able to inhibit Rho proteins from various phylogenetically unrelated Gram-negative and Gram-positive pathogens. Upon binding to these Rho proteins, Psu inhibited them by affecting their ATPase and RNA release functions. The expression of Psu in vivo kills various pathogens, such as Mycobacterium and Xanthomonas species. Hence, Psu could be useful for identifying peptide sequences with anti-Rho activities and might constitute part of synergistic antibiotic treatment against pathogens.

Vibrio cholerae OmpU induces IL-8 expression in human intestinal epithelial cells.

Although Vibrio cholerae colonizes the small intestine and induces acute inflammatory responses, less is known about the molecular mechanisms of V. cholerae-induced inflammatory responses in the intestine. We recently reported that OmpU, one of the most abundant outer membrane proteins of V. cholerae, plays an important role in the innate immunity of the whole bacteria. In this study, we evaluated the role of OmpU in induction of IL-8, a representative chemokine that recruits various inflammatory immune cells, in the human intestinal epithelial cell (IEC) line, HT-29. Recombinant OmpU (rOmpU) of V. cholerae induced IL-8 expression at the mRNA and protein levels in a dose- and time-dependent manner. Interestingly, IL-8 was secreted through both apical and basolateral sides of the polarized HT-29 cells upon apical exposure to rOmpU but not upon basolateral exposure. rOmpU-induced IL-8 expression was inhibited by interference of lipid raft formation with nystatin, but not by blocking the formation of clathrin-coated pits with chlorpromazine. In addition, rOmpU-induced IL-8 expression was mediated via ERK1/2 and p38 kinase pathways, but not via JNK signaling pathway. Finally, V. cholerae lacking ompU elicited decreased IL-8 expression and adherence to HT-29 cells compared to the parental strain. Collectively, these results suggest that V. cholerae OmpU might play an important role in intestinal inflammation by inducing IL-8 expression in human IECs.