The 1.9 Å crystal structure of the extracellular matrix protein Bap1 from Vibrio cholerae provides insights into bacterial biofilm adhesion.

Growth of the cholera bacterium Vibrio cholerae in a biofilm community contributes to both its pathogenicity and survival in aquatic environmental niches. The major components of V. cholerae biofilms include Vibriopolysaccharide (VPS) and the extracellular matrix proteins RbmA, RbmC, and Bap1. To further elucidate the previously observed overlapping roles of Bap1 and RbmC in biofilm architecture and surface attachment, here we investigated the structural and functional properties of Bap1. Soluble expression of Bap1 was possible only after the removal of an internal 57-amino-acid-long hydrophobic insertion sequence. The crystal structure of Bap1 at 1.9 Å resolution revealed a two-domain assembly made up of an eight-bladed ß-propeller interrupted by a ß-prism domain. The structure also revealed metal-binding sites within canonical calcium blade motifs, which appear to have structural rather than functional roles. Contrary to results previously observed with RbmC, the Bap1 ß-prism domain did not exhibit affinity for complex N-glycans, suggesting an altered role of this domain in biofilm-surface adhesion. Native polyacrylamide gel shift analysis did suggest that Bap1 exhibits lectin activity with a preference for anionic or linear polysaccharides. Our results suggest a model for V. cholerae biofilms in which Bap1 and RbmC play dominant but differing adhesive roles in biofilms, allowing bacterial attachment to diverse environmental or host surfaces.

Seeking new approach for therapeutic treatment of cholera disease via inhibition of bacterial carbonic anhydrases: experimental and theoretical studies for sixteen benzenesulfonamide derivatives.

A series of sixteen benzenesulfonamide derivatives has been synthesised and tested as inhibitors of Vibrio cholerae carbonic anhydrase (CA) enzymes, belonging to α-CA, ß-CA, and γ-CA classes (VchCAα, VchCAß, and VchCAγ). The determined Ki values were compared to those of selected human CA isoforms (hCA I and hCA II). Structure-affinity relationship analysis highlighted that all tested compounds proved to be active inhibitors of VchCAα at nanomolar concentration. The VchCAß activity was lower to respect inhibitory efficacy toward VchCAα, whereas, these benzenesulfonamide derivatives failed to inhibit VchCAγ. Interestingly, compound 7e combined the best activity toward VchCAα and VchCAß. In order to obtain a model for binding mode of our inhibitors toward bacterial CAs, we carried out docking simulations by using the available crystal structures of VchCAß.

Flufenamic acid protects against intestinal fluid secretion and barrier leakage in a mouse model of Vibrio cholerae infection through NF-κB inhibition and AMPK activation.

Nuclear factor kappa B (NF-κB)-mediated inflammatory responses play crucial roles in the pathogenesis of diarrhea caused by the Vibrio cholerae El Tor variant (EL), which is a major bacterial strain causing recent cholera outbreaks. Flufenamic acid (FFA) has previously been demonstrated to be a potent activator of AMP-activated protein kinase (AMPK), which is a negative regulator of NF-κB signaling. This study aimed to investigate the anti-diarrheal efficacy of FFA in a mouse model of EL infection and to investigate the mechanisms by which FFA activates AMPK in intestinal epithelial cells (IEC). In a mouse closed loop model of EL infection, FFA treatment (20mg/kg) significantly abrogated EL-induced intestinal fluid secretion and barrier disruption. In addition, FFA suppressed NF-κB nuclear translocation and expression of proinflammatory mediators and promoted AMPK phosphorylation in the EL-infected mouse intestine. In T84 cells, FFA induced AMPK activation. Furthermore, FFA promoted tight junction assembly and prevented interferon gamma (IFN-γ)-induced barrier disruption in an AMPK-dependent manner. Biochemical and molecular docking analyses indicated that FFA activates AMPK via a direct stimulation of calcium/calmodulin-dependent protein kinase kinase beta (CaMKKß) activity. Collectively, our data indicate that FFA represents a class of existing drugs that may be of potential utility in the treatment of cholera caused by EL infection via AMPK-mediated suppression of NF-κB signaling in IEC.

[N-ACETYL-ß-D-GLUCOSAMINIDASE OF VIBRIO CHOLERAE].

AIM: Study N-acetyl-ß-D-glucosaminidase (chitobiase) (EC 3.2.1.30) in strains of Vibrio cholerae of O1/non-O1 serogroups of various origin, that is a component of chitinolytic complex taking into account object of isolation and epidemiologic significance of strains. MATERIALS AND METHODS: Cultures of V. cholerae O1/non-O1 serogroup strains were obtained from the museum of live culture of Rostov RIPC. Enzymatic activity analysis was carried out in Hitachi F-2500 fluorescent spectrophotometer using FL Solutions licensed software. NCBI databases were used during enzyme characteristics. RESULTS: N-acetyl-ß-D-glucosaminidase in Vcholerae O1/non-O1 serogroup strains was detected, purified by column chromatography, studied and characterized by a number of physical-chemical and biological properties. Comparative computer analysis of amino acid sequence of N-acetyl-ß-D-glucosaminidases of V. cholerae (VC2217 gene), Serratia marcescens etc. has allowed. to attribute the enzyme from V. cholerae to glycosyl-hydrolases (chitobiases) of family 20 and classify it according to enzyme nomenclature as EC 3.2.1.30. CONCLUSION: N-acetyl-ß-D-glucosaminidase in V. cholerae of O1/non-O1 serogroups of various origin and epidemiologic significance, participating in chitin utilization was studied and characterized for the first time, and its possible role in biology of cholera causative agent was shown.

Chemoproteomic profiling of host and pathogen enzymes active in cholera.

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

Central role of the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) in sodium bioenergetics of Vibrio cholerae.

Vibrio cholerae is a Gram-negative bacterium that lives in brackish or sea water environments. Strains of V. cholerae carrying the pathogenicity islands infect the human gut and cause the fatal disease cholera. Vibrio cholerae maintains a Na(+) gradient at its cytoplasmic membrane that drives substrate uptake, motility, and efflux of antibiotics. Here, we summarize the major Na(+)-dependent transport processes and describe the central role of the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), a primary Na(+) pump, in maintaining a Na(+)-motive force. The Na(+)-NQR is a membrane protein complex with a mass of about 220 kDa that couples the exergonic oxidation of NADH to the transport of Na(+) across the cytoplasmic membrane. We describe the molecular architecture of this respiratory complex and summarize the findings how electron transport might be coupled to Na(+)-translocation. Moreover, recent advances in the determination of the three-dimensional structure of this complex are reported.

The highly conserved bacterial RNase YbeY is essential in Vibrio cholerae, playing a critical role in virulence, stress regulation, and RNA processing.

YbeY, a highly conserved protein, is an RNase in E. coli and plays key roles in both processing of the critical 3' end of 16 S rRNA and in 70 S ribosome quality control under stress. These central roles account for YbeY's inclusion in the postulated minimal bacterial genome. However, YbeY is not essential in E. coli although loss of ybeY severely sensitizes it to multiple physiological stresses. Here, we show that YbeY is an essential endoribonuclease in Vibrio cholerae and is crucial for virulence, stress regulation, RNA processing and ribosome quality control, and is part of a core set of RNases essential in most representative pathogens. To understand its function, we analyzed the rRNA and ribosome profiles of a V. cholerae strain partially depleted for YbeY and other RNase mutants associated with 16 S rRNA processing; our results demonstrate that YbeY is also crucial for 16 S rRNA 3' end maturation in V. cholerae and that its depletion impedes subunit assembly into 70 S ribosomes. YbeY's importance to V. cholerae pathogenesis was demonstrated by the complete loss of mice colonization and biofilm formation, reduced cholera toxin production, and altered expression levels of virulence-associated small RNAs of a V. cholerae strain partially depleted for YbeY. Notably, the ybeY genes of several distantly related pathogens can fully complement an E. coli ΔybeY strain under various stress conditions, demonstrating the high conservation of YbeY's activity in stress regulation. Taken together, this work provides the first comprehensive exploration of YbeY's physiological role in a human pathogen, showing its conserved function across species in essential cellular processes.

Vibrio cholerae evades neutrophil extracellular traps by the activity of two extracellular nucleases.

The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.

Activation of cholera toxin production by anaerobic respiration of trimethylamine N-oxide in Vibrio cholerae.

Vibrio cholerae is a gram-negative bacterium that causes cholera. Although the pathogenesis caused by this deadly pathogen takes place in the intestine, commonly thought to be anaerobic, anaerobiosis-induced virulence regulations are not fully elucidated. Anerobic growth of the V. cholerae strain, N16961, was promoted when trimethylamine N-oxide (TMAO) was used as an alternative electron acceptor. Strikingly, cholera toxin (CT) production was markedly induced during anaerobic TMAO respiration. N16961 mutants unable to metabolize TMAO were incapable of producing CT, suggesting a mechanistic link between anaerobic TMAO respiration and CT production. TMAO reductase is transported to the periplasm via the twin arginine transport (TAT) system. A similar defect in both anaerobic TMAO respiration and CT production was also observed in a N16961 TAT mutant. In contrast, the abilities to grow on TMAO and to produce CT were not affected in a mutant of the general secretion pathway. This suggests that V. cholerae may utilize the TAT system to secrete CT during TMAO respiration. During anaerobic growth with TMAO, N16961 cells exhibit green fluorescence when stained with 2',7'-dichlorofluorescein diacetate, a specific dye for reactive oxygen species (ROS). Furthermore, CT production was decreased in the presence of an ROS scavenger suggesting a positive role of ROS in regulating CT production. When TMAO was co-administered to infant mice infected with N16961, the mice exhibited more severe pathogenic symptoms. Together, our results reveal a novel anaerobic growth condition that stimulates V. cholerae to produce its major virulence factor.

[Point mutation of the Virbrio cholerae O139 cef (CHO cell elongating factor) gene alters the substrate specificity of its product].

Sequencing of the cef (CHO cell elongating factor) of Vibrio cholerae serogroup O139 revealed one nucleotide substitution (C for T in position 2015) in comparison with classical V. cholerae O1 and two substitutions (AC for GT in positions 2014-2015) in comparison with V. cholerae O1 E1 Tor. A comparative bioinformatic analysis showed that the substitution determines a threonine residue in position 672 of the Cefprotein, while the position is occupied by an isoleucine residue in the classical strains and a valine residue in the El Tor group. The last two amino acids are hydrophobic, while threonine is hydrophilic, having a polar R group. The non- synonymous substitution affects the predicted secondary and, probably, tertiary structures of the Cef-O139 protein and explained our previous finding that the protein fails to degrade tributyrin, while retaining the tweenase activity spectrum and all other characteristics. It cannot be excluded that the inability of Cef-O139 to cleave triglycerides, along with other genetic specifics, contribute to the fact that the O139 serogroup has been displaced from a dominating position in etiology of cholera by the El Tor genotype. The nucleotide sequence of the V. cholerae O139 cefgene and the deduced amino acid sequence of its product are reported for the first time and were deposited in GenBank under accession nos. JF499787 and AEC04822.1, respectively.

Contribution of hemagglutinin/protease and motility to the pathogenesis of El Tor biotype cholera.

Vibrio cholerae is a highly motile organism that secretes a Zn-dependent metalloprotease, hemagglutinin/protease (HapA). HapA has been shown to have mucinase activity and contribute to the reactogenicity of live vaccine candidates, but its role in cholera pathogenesis is not yet clear. The contribution of motility to pathogenesis is not fully understood, since conflicting results have been obtained with different strains, mutants, and animal models. The objective of this work was to determine the contribution of HapA and motility to the pathogenesis of El Tor biotype cholera. To this end we constructed isogenic motility (motY) and mucinase (hapA) single and double mutants of an El Tor biotype V. cholerae strain. Mutants were characterized for the expression of major virulence factors in vitro and in vivo. The motility mutant showed a remarkable increase in cholera toxin (CT), toxin coregulated pilus major subunit (TcpA), and HapA production in vitro. Increased TcpA and CT production could be explained by increased transcription of tcpA, ctxA, and toxT. No effect was detected on the transcription of hapA in the motility mutant. The sodium ionophore monensin diminished production of HapA in the parent but not in the motility mutant. Phenamil, a specific inhibitor of the flagellar motor, diminished CT production in the wild-type and motY strains. The hapA mutant showed increased binding to mucin. In contrast, the motY mutation diminished adherence to biotic and abiotic surfaces including mucin. Lack of HapA did not affect colonization in the suckling mouse model. The motility and mucinase defects did not prevent induction of ctxA and tcpA in the mouse intestine as measured by recombinase-based in vivo expression technology. Analysis of mutants in the rabbit ileal loop model showed that both V. cholerae motility and HapA were necessary for full expression of enterotoxicity.

Histopathological changes in experimental cholera with a non toxigenic non- O1 non-O139 Vibrio cholerae strain isolated from Kolkata, India.

This study was conducted to understand the pathophysiological changes in experimental rabbit ileal loop model using the Vibrio cholerae strain non-O1non-O139, isolated as sole pathogen from clinically diagnosed cholera patients in Kolkata. Significant amount of haemorrhagic fluid accumulation was observed in all the test loops of rabbit model where the strain of V.cholerae was inoculated as compared to control loops. Microscopic examination of the accumulated fluid showed the presence of erythrocytes and pus cells. Histology revealed structural alteration of the villous epithelium with inflammatory cells infiltration in all the layers of the gut mucosa including the nerve plexus region. Preliminary observation with a haemagglutinin protease extracted from the non-O1 non-O139 strain, was also studied in different concentrations in the same animal model which showed similar type of macroscopic and microscopic response in the ileal loops as seen with the original strain. The results highlight that along with other pathways, inflammatory cells and the enteric neurons have an important role in the pathophysiology of diarrhoea and the isolated protease may be the probable virulence factor in initiating the disease process in this non-O1non-O139 strain induced cholera.

New drug targets for cholera therapy.

Intestinal infection with Vibrio cholerae results in secretory diarrhea with potentially massive fluid losses and volume depletion. Morbidity and mortality associated with cholera remain a major problem in the developing world despite the success of oral rehydration therapy. New research aiming to inhibit cholera toxin binding to receptors in the intestine provides an attractive strategy for cholera therapy. Together with anti-secretory agents, including inhibitors of enkephalinase and of the cystic fibrosis transmembrane conductance regulator, new treatment options for managing severe diarrhea in cholera could soon be available.

Preserved exocrine function in patients with acute cholera and acute non-cholera diarrhoea.

Exocrine pancreatic function was assessed by means of the Lundh test in 14 patients with acute cholera and 18 patients with acute infectious non-cholera diarrhoea within the first 24 h of their admission. Mean tryptic activity amounted to 39.8 +/- 4.8 microEq/min/ml in the cholera group and to 64.4 +/- 11.0 microEq/min/ml in the non-cholera group. None of these patients shared a value below the lower limit of normal. In fact, the mean tryptic activity per 2 h was significantly higher than that reported previously in a control group from the Bengal area. It is therefore concluded that the exocrine pancreatic function is preserved and responds to food stimulation in various types of acute infectious diarrhoea, including cholera. These findings provide the pathophysiological background for the recent observation that oral rehydration solutions containing high-molecular-weight nutrients such as rice powder are at least as efficient or even more potent than the WHO-recommended glucose-electrolyte formula in acute diarrhoea.

Elevated cAMP in intestinal epithelial cells during experimental cholera and salmonellosis.

Cholera and salmonellosis are two diarrheal diseases in which intestinal tissue cyclic adenosine monophosphate (cAMP) concentrations are elevated. Investigations of each experimental disease were initiated to identify the specific intestinal cells containing the elevated cAMP. Epithelial cells were eluted from the mucosa of infected and control intestinal loops of adult rabbits, after which the cAMP content of the epithelial cell fractions and the lamina propria cells was extracted and assayed. The identity of the epithelial cells (in the villus tip-to-crypt cell gradient) was monitored by measuring their intracellular alkaline phosphatase activity, while scanning electron microscopy was used to visualize the effects of infection and cell elution techniques. Clearly, in both experimental cholera and salmonellosis, elevated cAMP levels were associated with crypt epithelial cells. Villus tip epithelial cells from either infection tended to contain less cAMP than those of noninfected control tissue. In Salmonella-infected loops, it was apparent that cAMP was also elevated in lamina propria cell fractions. Lamina propria cells from V. cholerae-infected intestinal loops contained only basal levels of cAMP. In vitro exposure of isolated intestinal cells from normal rabbit intestine to a cell-free lysate of Salmonella resulted in elevation of cAMP in the epithelial cells and lamina propria cells. We conclude that in experimental cholera and salmonellosis, significant elevation of the cAMP levels occurred in intestinal crypt cells, consistent with an enterotoxin-mediated mechanism. In Salmonella-infected loops, it was unclear if the increased concentration of cAMP in lamina propria cells was generated by enterotoxin released from the invasive salmonellae or by prostaglandins formed during the inflammatory response to the bacteria, or by both mechanisms.

[Electron microscopic demonstration of adenylate cyclase in rabbit small intestine enterocytes doubly stimulated by cholera toxin and sodium fluoride]. / Elektronno-mikroskopicheskoe vyiavlenie adenilattsiklazy énterotsitov tonkogo kishechnika krolikov pri dvoinom stimulirovanii kholernym toksinom i ftoristym natriem.

Cytochemical investigations showed adenylate cyclase in the rabbit small intestine enterocytes to be activated both with cholera toxin and sodium fluoride. Following double stimulation of adenylate cyclase in the intestinal enterocytes by the mentioned two substances maximal critical levels of cAMP were attained resulting in self-inhibition of adenylate cyclase; in this case only a low adenylate cyclase activity, if any, could be demonstrated by electron microscopy.