Tailored multivalent peptide targeting the B-subunit pentamer of cholera toxin inhibits its intestinal toxicity by inducing aberrant transport of the toxin in cells.

Cholera toxin (Ctx) is a major virulence factor produced by Vibrio cholerae that can cause gastrointestinal diseases, including severe watery diarrhea and dehydration, in humans. Ctx binds to target cells through multivalent interactions between its B-subunit pentamer and the receptor ganglioside GM1 present on the cell surface. Here, we identified a series of tetravalent peptides that specifically bind to the receptor-binding region of the B-subunit pentamer using affinity-based screening of multivalent random-peptide libraries. These tetravalent peptides efficiently inhibited not only the cell-elongation phenotype but also the elevated cAMP levels, both of which are induced by Ctx treatment in CHO cells or a human colon carcinoma cell line (Caco-2 cells), respectively. Importantly, one of these peptides, NRR-tet, which was highly efficient in these two activities, markedly inhibited fluid accumulation in the mouse ileum caused by the direct injection of Ctx. In consistent, NRR-tet reduced the extensive Ctx-induced damage of the intestinal villi. After NRR-tet bound to Ctx, the complex was incorporated into the cultured epithelial cells and accumulated in the recycling endosome, affecting the retrograde transport of Ctx from the endosome to the Golgi, which is an essential process for Ctx to exert its toxicity in cells. Thus, NRR-tet may be a novel type of therapeutic agent against cholera, which induces the aberrant transport of Ctx in the intestinal epithelial cells, detoxifying the toxin.

Calcium-sensing receptor activator cinacalcet for treatment of cyclic nucleotide-mediated secretory diarrheas.

Cyclic nucleotide elevation in intestinal epithelial cells is the key pathology causing intestinal fluid loss in secretory diarrheas such as cholera. Current secretory diarrhea treatment is primarily supportive, and oral rehydration solution is the mainstay of cholera treatment. There is an unmet need for safe, simple and effective diarrhea treatments. By promoting cAMP hydrolysis, extracellular calcium-sensing receptor (CaSR) is a regulator of intestinal fluid transport. We studied the antidiarrheal mechanisms of FDA-approved CaSR activator cinacalcet and tested its efficacy in clinically relevant human cell, mouse and intestinal organoid models of secretory diarrhea. By using selective inhibitors, we found that cAMP agonists-induced secretory short-circuit currents (Isc) in human intestinal T84 cells are mediated by collective actions of apical membrane cystic fibrosis transmembrane conductance regulator (CFTR) and Clc-2 Cl- channels, and basolateral membrane K+ channels. 30 µM cinacalcet pretreatment inhibited all 3 components of forskolin and cholera toxin-induced secretory Isc by ∼75%. In mouse jejunal mucosa, cinacalcet inhibited forskolin-induced secretory Isc by ∼60% in wild type mice, with no antisecretory effect in intestinal epithelia-specific Casr knockout mice (Casr-flox; Vil1-cre). In suckling mouse model of cholera induced by oral cholera toxin, single dose (30 mg/kg) oral cinacalcet treatment reduced intestinal fluid accumulation by ∼55% at 20 hours. Lastly, cinacalcet inhibited forskolin-induced secretory Isc by ∼75% in human colonic and ileal organoids. Our findings suggest that CaSR activator cinacalcet has antidiarrheal efficacy in distinct human cell, organoid and mouse models of secretory diarrhea. Considering its excellent clinical safety profile, cinacalcet can be repurposed as a treatment for cyclic nucleotide-mediated secretory diarrheas including cholera.

Natural statin derivatives as potential therapy to reduce intestinal fluid loss in cholera.

As a leading cause of death in children under 5 years old, secretory diarrheas including cholera are characterized by excessive intestinal fluid secretion driven by enterotoxin-induced cAMP-dependent intestinal chloride transport. This study aimed to identify fungal bioactive metabolites possessing anti-secretory effects against cAMP-dependent chloride secretion in intestinal epithelial cells. Using electrophysiological analyses in human intestinal epithelial (T84) cells, five fungus-derived statin derivatives including α,ß-dehydrolovastatin (DHLV), α,ß-dehydrodihydromonacolin K, lovastatin, mevastatin and simvastatin were found to inhibit the cAMP-dependent chloride secretion with IC50 values of 1.8, 8.9, 11.9, 11.4 and 5 µM, respectively. Being the most potent statin derivatives, DHLV was evaluated for its pharmacological properties including cellular toxicity, mechanism of action, target specificity and in vivo efficacy. DHLV at concentrations up to 20 µM did not affect cell viability and barrier integrity of T84 cells. Electrophysiological analyses indicated that DHLV inhibited cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent apical chloride channel, via mechanisms not involving alteration of intracellular cAMP levels or its negative regulators including AMP-activated protein kinases and protein phosphatases. DHLV had no effect on Na+-K+ ATPase activities but inhibited Ca2+-dependent chloride secretion without affecting intracellular Ca2+ levels. Importantly, intraperitoneal (2 mg/kg) and intraluminal (20 µM) injections of DHLV reduced cholera toxin-induced intestinal fluid secretion in mice by 59% and 65%, respectively without affecting baseline intestinal fluid transport. This study identifies natural statin derivatives as novel natural product-derived CFTR inhibitors, which may be beneficial in the treatment of enterotoxin-induced secretory diarrheas including cholera.

A Phase 2a randomized, single-center, double-blind, placebo-controlled study to evaluate the safety and preliminary efficacy of oral iOWH032 against cholera diarrhea in a controlled human infection model.

Cholera remains a major cause of infectious diarrhea globally. Despite the increased availability of cholera vaccines, there is still an urgent need for other effective interventions to reduce morbidity and mortality. Furthermore, increased prevalence of antibiotic-resistant Vibrio cholerae threatens the use of many drugs commonly used to treat cholera. We developed iOWH032, a synthetic small molecule inhibitor of the cystic fibrosis transmembrane conductance regulator chloride channel, as an antisecretory, host-directed therapeutic for cholera. In the study reported here, we tested iOWH032 in a Phase 2a cholera controlled human infection model. Forty-seven subjects were experimentally infected with V. cholerae El Tor Inaba strain N16961 in an inpatient setting and randomized to receive 500 mg iOWH032 or placebo by mouth every 8 hours for 3 days to determine the safety and efficacy of the compound as a potential treatment for cholera. We found that iOWH032 was generally safe and achieved a mean (± standard deviation) plasma level of 4,270 ng/mL (±2,170) after 3 days of oral dosing. However, the median (95% confidence interval) diarrheal stool output rate for the iOWH032 group was 25.4 mL/hour (8.9, 58.3), compared to 32.6 mL/hour (15.8, 48.2) for the placebo group, a reduction of 23%, which was not statistically significant. There was also no significant decrease in diarrhea severity and number or frequency of stools associated with iOWH032 treatment. We conclude that iOWH032 does not merit future development for treatment of cholera and offer lessons learned for others developing antisecretory therapeutic candidates that seek to demonstrate proof of principle in a cholera controlled human infection model study. Trial registration: This study is registered with ClinicalTrials.gov as NCT04150250.

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.

The heptameric structure of the flagellar regulatory protein FlrC is indispensable for ATPase activity and disassembled by cyclic-di-GMP.

The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. Although, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in "cis-mediated" style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration-dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram-negative bacteria.

Cyclic nucleotides, gut physiology and inflammation.

Misregulation of gut function and homeostasis impinges on the overall well-being of the entire organism. Diarrheal disease is the second leading cause of death in children under 5 years of age, and globally, 1.7 billion cases of childhood diarrhea are reported every year. Accompanying diarrheal episodes are a number of secondary effects in gut physiology and structure, such as erosion of the mucosal barrier that lines the gut, facilitating further inflammation of the gut in response to the normal microbiome. Here, we focus on pathogenic bacteria-mediated diarrhea, emphasizing the role of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate in driving signaling outputs that result in the secretion of water and ions from the epithelial cells of the gut. We also speculate on how this aberrant efflux and influx of ions could modulate inflammasome signaling, and therefore cell survival and maintenance of gut architecture and function.

El Tor Biotype Vibrio cholerae Activates the Caspase-11-Independent Canonical Nlrp3 and Pyrin Inflammasomes.

Vibrio cholerae is a Gram-negative enteropathogen causing potentially life-threatening cholera disease outbreaks, for which the World Health Organization currently registers 2-4 million cases and ~100.000 cholera-associated deaths annually worldwide. Genomic Vibrio cholerae research revealed that the strains causing this ongoing cholera pandemic are members of the El Tor biotype, which fully replaced the Classical biotype that caused former cholera pandemics. While both of these biotypes express the characteristic Cholera Toxin (CT), the El Tor biotype additionally expresses the accessory toxins hemolysin (hlyA) and multifunctional auto-processing repeat-in-toxin (MARTX). Previous studies demonstrated that the Classical biotype of Vibrio cholerae triggers caspase-11-dependent non-canonical inflammasome activation in macrophages following CT-mediated cytosolic delivery of LPS. In contrast to the Classical biotype, we here show that El Tor Vibrio cholerae induces IL-1ß maturation and secretion in a caspase-11- and CT-independent manner. Instead, we show that El Tor Vibrio cholerae engages the canonical Nlrp3 inflammasome for IL-1ß secretion through its accessory hlyA toxin. We further reveal the capacity of this enteropathogen to engage the canonical Pyrin inflammasome as an accessory mechanism for IL-1ß secretion in conditions when the pro-inflammatory hlyA-Nlrp3 axis is blocked. Thus, we show that the V. cholerae El Tor biotype does not trigger caspase-11 activation, but instead triggers parallel Nlrp3- and Pyrin-dependent pathways toward canonical inflammasome activation to induce IL-1ß-mediated inflammatory responses. These findings further unravel the complex inflammasome activating mechanisms that can be triggered when macrophages face the full arsenal of El Tor Vibrio cholerae toxins, and as such increase our understanding of host-pathogen interactions in the context of the Vibrio cholerae biotype associated with the ongoing cholera pandemic.

An elusive adenylate cyclase complicit in cholera is exposed.

The intestinal consequences of cholera enterotoxin are caused by activation of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel through the actions of an as-yet-unknown adenylate cyclase. A new study hunts down this elusive enzyme, showing that mouse and human intestinal epithelium functionally and structurally pair adenylate cyclase isoform 6 (AC6) with CFTR. These findings provide important insights into the molecular mechanisms underlying the robust pathological activation of CFTR activity and promise new opportunities to treat cholera.

Modifications of cholera toxin subunit B binding to human large intestinal epithelium. An immunohistochemical study.

Binding of cholera toxin subunit B (CTB) to its receptor and toxin transport into the intestinal epithelial cells are the causative events for the potentially lethal disease cholera. The five sugar mono-sialo ganglioside GM1 is the cell surface receptor for cholera toxin B-subunit. CTB binding was determined by use of immobilized GM1 to microtiter plates and by immunohistochemistry. Sections from the human colon and the human soft palate were incubated with FITC-conjugated CTB and with anti-MUC2. Both the luminal surface of the intestine and the secretory goblet cells exhibited strong binding. Addition of simple carbohydrates and milk to the incubation medium showed that a combination of lactose and non-fat dry milk was potent inhibitors of toxin- and mucin binding. Both CTB and ant-MUC2 stained to the cytoplasm (mucin granules) in the goblet cells from the human soft palate. In the colon CTB stained the entire cytoplasm of the goblet cells while anti-MUC2 detected only the supranuclear region of some cells, suggesting carbohydrate heterogeneity between goblet cell mucin granules in different regions of the human body. Both CTB- and MUC2 binding were inhibited when GM1 was added to the incubation medium. It is proposed that the human colonic goblet cells play a role in the secretory diarrhea in patients with cholera and that milk might have a prophylactic or therapeutic application in the management of cholera.

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.

Killed Whole-Cell Oral Cholera Vaccine Induces CCL20 Secretion by Human Intestinal Epithelial Cells in the Presence of the Short-Chain Fatty Acid, Butyrate.

Short-chain fatty acids (SCFAs), such as acetate, butyrate, and propionate, modulate immune responses in the gut. However, the effect of SCFAs on mucosal vaccine-induced immune cell migration is poorly understood. Here, we investigated whether SCFAs modulate chemokine expression induced by the killed whole-cell oral cholera vaccine, Shanchol™, in human intestinal epithelial cells. Shanchol™ induced expression of CCL2, CCL5, CCL20, and CXCL10 at the mRNA level, but not at the protein level. Interestingly, CCL20 secretion was substantially increased by co-stimulation with Shanchol™ and butyrate, while neither acetate nor propionate showed such effect. Enhanced CCL20 secretion was associated with GPR109A activation, and histone deacetylase (HDAC) inhibition. In addition, co-treatment with Shanchol™ and butyrate synergistically increased the secretion of adenosine triphosphate (ATP). Moreover, CCL20 secretion was decreased by inhibiting the extracellular ATP receptor P2X7. However, neither inflammasomes nor caspases were involved in CCL20 production. The culture supernatant of cells treated with Shanchol™ and butyrate augmented human immature dendritic cell migration. Collectively, these results suggest that butyrate enhances Shanchol™-induced CCL20 production in human intestinal epithelial cells via HDAC inhibition and ATP-P2X7 signaling by activating GPR109A. These effects potentially enhance the mucosal immune responses in the gut induced by this oral cholera vaccine.

Anoctamin 6 Contributes to Cl- Secretion in Accessory Cholera Enterotoxin (Ace)-stimulated Diarrhea: AN ESSENTIAL ROLE FOR PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIP2) SIGNALING IN CHOLERA.

Accessory cholera enterotoxin (Ace) of Vibrio cholerae has been shown to contribute to diarrhea. However, the signaling mechanism and specific type of Cl- channel activated by Ace are still unknown. We have shown here that the recombinant Ace protein induced ICl of apical plasma membrane, which was inhibited by classical CaCC blockers. Surprisingly, an Ace-elicited rise of current was neither affected by ANO1 (TMEM16A)-specific inhibitor T16A(inh)-AO1(TAO1) nor by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker, CFTR inh-172. Ace stimulated whole-cell current in Caco-2 cells. However, the apical ICl was attenuated by knockdown of ANO6 (TMEM16F). This impaired phenotype was restored by re-expression of ANO6 in Caco-2 cells. Whole-cell patch clamp recordings of ANO currents in HEK293 cells transiently expressing mouse ANO1-mCherry or ANO6-GFP confirmed that Ace induced Cl- secretion. Application of Ace produced ANO6 but not the ANO1 currents. Ace was not able to induce a [Ca2+]i rise in Caco-2 cells, but cellular abundance of phosphatidylinositol 4,5-bisphosphate (PIP2) increased. Identification of the PIP2-binding motif at the N-terminal sequence among human and mouse ANO6 variants along with binding of PIP2 directly to ANO6 in HEK293 cells indicate likely PIP2 regulation of ANO6. The biophysical and pharmacological properties of Ace stimulated Cl- current along with intestinal fluid accumulation, and binding of PIP2 to the proximal KR motif of channel proteins, whose mutagenesis correlates with altered binding of PIP2, is comparable with ANO6 stimulation. We conclude that ANO6 is predominantly expressed in intestinal epithelia, where it contributes secretory diarrhea by Ace stimulation in a calcium-independent mechanism of RhoA-ROCK-PIP2 signaling.

Nonredundant Roles of Iron Acquisition Systems in Vibrio cholerae.

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, thrives in both marine environments and the human host. To do so, it must encode the tools necessary to acquire essential nutrients, including iron, under these vastly different conditions. A number of V. cholerae iron acquisition systems have been identified; however, the precise role of each system is not fully understood. To test the roles of individual systems, we generated a series of mutants in which only one of the four systems that support iron acquisition on unsupplemented LB agar, Feo, Fbp, Vct, and Vib, remains functional. Analysis of these mutants under different growth conditions showed that these systems are not redundant. The strain carrying only the ferrous iron transporter Feo grew well at acidic, but not alkaline, pH, whereas the ferric iron transporter Fbp promoted better growth at alkaline than at acidic pH. A strain defective in all four systems (null mutant) had a severe growth defect under aerobic conditions but accumulated iron and grew as well as the wild type in the absence of oxygen, suggesting the presence of an additional, unidentified iron transporter in V. cholerae. In support of this, the null mutant was only moderately attenuated in an infant mouse model of infection. While the null mutant used heme as an iron source in vitro, we demonstrate that heme is not available to V. cholerae in the infant mouse intestine.

Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria.

Porins, a major class of outer membrane proteins in Gram-negative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis, such as flipping of phosphatidylserine in the membrane as well as cell size shrinkage and increased cell granularity, it does not show the caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential, resulting in the release of cytochrome c and apoptosis-inducing factor (AIF). AIF translocates to the host cell nucleus, implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria, where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of the mitochondrial permeability transition pore. All of these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role.

Autophagy and endosomal trafficking inhibition by Vibrio cholerae MARTX toxin phosphatidylinositol-3-phosphate-specific phospholipase A1 activity.

Vibrio cholerae, responsible for acute gastroenteritis secretes a large multifunctional-autoprocessing repeat-in-toxin (MARTX) toxin linked to evasion of host immune system, facilitating colonization of small intestine. Unlike other effector domains of the multifunctional toxin that target cytoskeleton, the function of alpha-beta hydrolase (ABH) remained elusive. This study demonstrates that ABH is an esterase/lipase with catalytic Ser-His-Asp triad. ABH binds with high affinity to phosphatidylinositol-3-phosphate (PtdIns3P) and cleaves the fatty acid in PtdIns3P at the sn1 position in vitro making it the first PtdIns3P-specific phospholipase A1 (PLA1). Expression of ABH in vivo reduces intracellular PtdIns3P levels and its PtdIns3P-specific PLA1 activity blocks endosomal and autophagic pathways. In accordance with recent studies acknowledging the potential of extracellular pathogens to evade or exploit autophagy to prevent their clearance and facilitate survival, this is the first report highlighting the role of ABH in inhibiting autophagy and endosomal trafficking induced by extracellular V. cholerae.

The Effect of Solar Irradiated Vibrio cholerae on the Secretion of Pro-Inflammatory Cytokines and Chemokines by the JAWS II Dendritic Cell Line In Vitro.

The use of solar irradiation to sterilize water prior to its consumption has resulted in the reduction of water related illnesses in waterborne disease endemic communities worldwide. Currently, research on solar water disinfection (SODIS) has been directed towards understanding the underlying mechanisms through which solar irradiation inactivates the culturability of microorganisms in water, enhancement of the disinfection process, and the health impact of SODIS water consumption. However, the immunological consequences of SODIS water consumption have not been explored. In this study, we investigated the effect that solar irradiated V. cholerae may have had on the secretion of cytokines and chemokines by the JAWS II dendritic cell line in vitro. The JAWS II dendritic cell line was stimulated with the different strains of V. cholerae that had been: (i) prepared in PBS, (ii) inactivated through a combination of heat and chemical, (iii) solar irradiated, and (iv) non-solar irradiated, in bottled water. As controls, LPS (1 µg/ml) and CTB (1 µg/ml) were used as stimulants. After 48 hours of stimulation the tissue culture media from each treatment was qualitatively and quantitatively analysed for the presence of IL-1α, IL-1ß, IL-6, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, MIP-1α, MIP-1ß, MIP-2, RANTES, TNF-α, IL-23 and IL-27. Results showed that solar irradiated cultures of V. cholerae induced dendritic cells to secrete significant (p<0.05) levels of pro-inflammatory cytokines in comparison to the unstimulated dendritic cells. Furthermore, the amount of pro-inflammatory cytokines secreted by the dendritic cells in response to solar irradiated cultures of V. cholerae was not as high as observed in treatments involving non-solar irradiated cultures of V. cholerae or LPS. Our results suggest that solar irradiated microorganisms are capable of inducing the secretion of pro-inflammatory cytokines and chemokines. This novel finding is key towards understanding the possible immunological consequences of consuming SODIS treated water.

A Genome-Wide Screen Reveals that the Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System Modulates Virulence Gene Expression.

Diverse 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 tcpA expression, the screen yielded ptsI and ptsH, which encode the enzyme I (EI) and Hpr components of the V. cholerae phosphoenolpyruvate phosphotransferase system (PTS). In addition to reduced expression of TcpA, strains lacking EI, Hpr, or the associated EIIA(Glc) protein 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 tcpPH and 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. cholerae PTS is an additional modulator of the ToxT regulon and demonstrate the potency of loss-of-function TIS sequencing screens for defining regulatory networks.