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ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host range mutants within infant rabbits infected with a mixture of wild-type and OmpU mutant strains. ICP2 host range mutants that can now infect OmpU mutant strains have missense mutations in the putative tail fiber gene gp25 and the putative adhesin gene gp23. Using site-specific mutagenesis, we show that single or double mutations in gp25 are sufficient to generate the host range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to produce a host range mutant phenotype. All ICP2 host range mutants retained the ability to form plaques on wild-type V. cholerae cells. The strength of binding of host range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host range mutants evolve by a two-step process. First, gp25 mutations are selected for their broad host range, albeit accompanied by low-level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near-wild-type efficiencies of adsorption and subsequent phage multiplication. IMPORTANCE Concern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to renewed interest in phage biology and the potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail that have been shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies an ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular coevolutionary arms race presents fitness costs to both ICP2 and V. cholerae.

The aim of the work was to study surface antigenic determinants of V. cholerae R-variant strains using enzyme immunoassay and a panel of monoclonal antibodies (MAbs).Materials and methods. 60 strains of V. cholerae R-variant isolated from ambient environment objects in the territories of the former USSR and the constituent entities of the Russian Federation over a 30-year period (1988–2019) were investigated in the slide agglutination reaction with cholera diagnostic sera, enzyme immunoassay (ELISA) using the panel of MAbs specific to membrane proteins and a set of reagents “Monoclonal diagnostic immunoglobulins labeled with horseradish peroxidase, dry, for serological identification of V. cholerae O1 and O139 (in vitro) through ELISA and dot-ELISA”.Results and discussion. The analysis of the surface structures of V. cholerae R-variant strains with atypical agglutinability has been carried out applying enzyme immunoassay. It showed that individual strains with different amounts of O-antigen are registered among the studied strains identified at isolation as V. cholerae R-variant (the optical density range is from 0.261±0.002 to 1.312±0.003). Epitopes of specific O-antigen were found in some “conservative” strains (30 %) that are agglutinated only with RO serum, and in several strains (20 %) that do not have the wbeT gene that determines its synthesis, and lost agglutinability with all diagnostic cholera sera, including RO. The protein epitopes recognized by complementary MAbs are represented with varying frequency in the composition of surface antigens of R-vibrios; a decrease in their representation or absence on the cell surface correlates with the modification or loss of R-LPS and is accompanied by a negative agglutination reaction.

Intestinal colonization and virulence factor production in response to environmental cues is mediated through several regulatory factors in Vibrio cholerae , including the highly conserved RNA-binding global regulatory protein CsrA. We have shown previously that CsrA increases synthesis of the virulence-associated transcription factor ToxR in response to specific amino acids (NRES) and is required for the virulence of V. cholerae in the infant mouse model of cholera. In this study, we mapped the 5' untranslated region (5' UTR) of toxR and showed that CsrA can bind directly to an RNA sequence encompassing the 5' UTR, indicating that the regulation of ToxR levels by CsrA is direct. Consistent with this observation, the 5' UTR of toxR contains multiple putative CsrA binding sequences (GGA motifs), and mutating these motifs disrupted the CsrA-mediated increase in ToxR. Optimal binding of CsrA to a defined RNA oligonucleotide required the bridging of two GGA motifs within a single RNA strand. To determine the mechanism of regulation by CsrA, we assayed toxR transcript levels, stability, and efficiency of translation. Both the amount of toxR mRNA in NRES and the stability of the toxR transcript were increased by CsrA. Using an in vitro translation assay, we further showed that synthesis of ToxR was greatly enhanced in the presence of purified CsrA, suggesting a direct role for CsrA in the translation of toxR mRNA. We propose a model in which CsrA binding to the 5' UTR of the toxR transcript promotes ribosomal access while precluding interactions with RNA-degrading enzymes.IMPORTANCE Vibrio cholerae is uniquely adapted to marine environments as well as the human intestinal tract. Global regulators, such as CsrA, which help translate environmental cues into an appropriate cellular response, are critical for switching between these distinct environments. Understanding the pathways involved in relaying environmental signals is essential for understanding both the environmental persistence and the intestinal pathogenesis of this devastating human pathogen. In this study, we demonstrate that CsrA directly regulates the synthesis of ToxR, a key virulence factor of V. cholerae . Under conditions favoring high levels of active CsrA in the cell, such as in the presence of particular amino acids, CsrA increases ToxR protein levels by binding to the toxR transcript and enhancing both its stability and translation. By responding to nutrient availability, CsrA is perfectly poised to activate the virulence gene regulatory cascade at the preferred site of colonization in the human host, the nutrient-rich small intestinal mucosa.

Bile resistance is essential for enteric pathogens, as exemplified by Vibrio cholerae, the causative agent of cholera. The outer membrane porin OmpU confers bacterial survival and colonization advantages in the presence of host‐derived antimicrobial peptides as well as bile. Expression of ompU is controlled by the virulence regulator ToxR. rpoE knockouts are accompanied by suppressor mutations causing ompU downregulation. Therefore, OmpU constitutes an intersection of the ToxR regulon and the σE‐pathway in V. cholerae. To understand the mechanism by which the sigma factor σE regulates OmpU synthesis, we performed transcription studies using ompU reporter fusions and immunoblot analysis. Our data revealed an increase in ompU promoter activity in ΔrpoE strains, as well as in a ΔompU background, indicating a negative feedback regulation circuit of ompU expression. This regulation seems necessary, since elevated lethality rates of ΔrpoE strains occur upon ompU overexpression. Manipulation of OmpU's C‐terminal portion revealed its relevance for protein stability and potency of σE release. Furthermore, ΔrpoE strains are still capable of elevating OmpU levels under membrane stress conditions triggered by the bile salt sodium deoxycholate. This study provides new details about the impact of σE on ompU regulation, which is critical to the pathogen's intestinal survival. [ABSTRACT FROM AUTHOR]

Cholera is a bacterial disease featured by dehydration and severe diarrhea. It is mainly caused by alimentary infection with Vibrio cholerae. Due to the wide applicability of quinazolin-2,4-dione compounds in medicinal and pharmaceutical chemistry, a new series of N -containing heterocyclic compounds was synthesized. We used the in silico docking method to test the efficacy of quinazolin-2,4-dione compounds in the prevention of cholera in humans. The newly synthesized compounds showed strong interactions and good binding affinity to outer membrane protein OmpU. Moreover, the pharmacokinetic properties of the newly synthesized compounds, such as absorption, distribution, metabolic, excretion, and toxicity (ADMET), were predicted through in silico methods. Compounds with acceptable pharmacokinetic properties were tested as novel ligand molecules. The synthesized compounds were evaluated in vitro for their antibacterial activity properties against Gram-negative Escherichia coli O78 strain using the minimum inhibition concentration (MIC) method. Compounds 2 and 6 showed reproducible, effective antibacterial activity. Hence, our study concludes that the quinazolin-2,4-dione derivatives 1 to 8 may be used as promising drug candidates with potential value for the treatment of cholera disease. [ABSTRACT FROM AUTHOR]

Vibrio cholerae causes the diarrheal disease cholera which affects millions of people globally. The outer membrane protein U (OmpU) is the outer membrane protein that is most prevalent in V. cholerae and has already been recognized as a critical component of pathogenicity involved in host cell contact and as being necessary for the survival of pathogenic V. cholerae in the host body. Computational approaches were used in this study to screen a total of 37,709 natural compounds from the traditional Chinese medicine (TCM) database against the active site of OmpU. Following a sequential screening of the TCM database, we report three lead compounds—ZINC06494587, ZINC85510056, and ZINC95910434—that bind strongly to OmpU, with binding affinity values of −8.92, −8.12, and −8.78 kcal/mol, which were higher than the control ligand (−7.0 kcal/mol). To optimize the interaction, several 100 ns molecular dynamics simulations were performed, and the resulting complexes were shown to be stable in their vicinity. Additionally, these compounds were predicted to have good drug-like properties based on physicochemical properties and ADMET assessments. This study suggests that further research be conducted on these compounds to determine their potential use as cholera disease treatment.

Vibrio parahaemolyticus is a seafood-borne Gram-negative bacteria causing diarrheal diseases in humans world wide. ToxR is a membrane-associated transcriptional factor which plays an important role in acid stress tolerance and regulates the expression of virulence genes including type III secretion system 1 (T3SS1) and type VI secretion system 1 (T6SS1) in V. parahaemolyticus. However, possible mechanisms of ToxR mediating virulence gene expression have not been fully understood. In this study, we demonstrated that ToxR is essential for V. parahaemolyticus to tolerate acid stress by constructing a ToxR deletion mutant (Δ toxR) and its complemented strain (toxR +). Quantitative PCR showed that the expression of toxR was up regulated under acid stress condition. RNA-seq analysis showed that ompU encoding one of outer membrane proteins was dramatically down regulated in Δ toxR. Furthermore, the mutation of ompU also led to a significant reduction in tolerating acid stress indicating that ToxR mediated acid stress through regulating ompU expression. RNA-seq results further confirmed that acid stress condition could alter multiple signaling pathways either depending on ToxR (e.g., quorum sensing, fatty acid metabolism) or independent of ToxR (e.g., biosynthesis of secondary metabolites, microbial metabolism in diverse environment, biosynthesis of antibiotics, biosynthesis of amino acids and carbon metabolism pathways). We also for the first time demonstrated that ToxR positively regulated the expression of T6SS2 gene and the interbacteria killing activity. Our study provides comprehensive understanding of signaling pathways which are regulated by both acid stress and ToxR. [ABSTRACT FROM AUTHOR]

Aims: Vibrio vulnificus is a common bacterial pathogen causing haemorrhagic septicaemia in eel farming. This study investigates the immunogenicity of an outer membrane protein U (OmpU) of V. vulnificus and the feasibility of the protein as a new subunit vaccine against V. vulnificus. Methods and Results: Partial gene sequence of the OmpU of V. vulnificus was cloned, and then the OmpU was expressed and purified. Three groups of Japanese eels (Anguilla japonica) were intraperitoneally (i.p) injected with bovine serum albumin (BSA group), formalin‐killed whole cell of V. vulnificus (FKC group) or the expressed OmpU of V. vulnificus (OMP group). On 14, 21, 28 and 42 days postimmunization (dpi), the whole blood cells were collected to evaluate the stimulation index (SI) and bactericidal activity. The serum was obtained to assess the titres of specific antibody, lysozyme activity, complement activity and bactericidal activity. The lysozyme activities in the suspension of kidney, skin mucus and liver in eels were also ascertained. The results showed that the SI and the titres of anti‐V. vulnificus antibody in the OMP group was significantly increased on 28 dpi; lysozyme activity in the kidney and skin mucus of OMP group on 42 and 14 dpi were both significantly higher than BSA group; eels in OMP group showed strong bactericidal capacity on 21 and 28 days; and the relative percent survival of OMP vs BSA group after challenged by V. vulnificus on 28 dpi was 80%. Conclusions: These results showed that the expressed OmpU of V. vulnificus could significantly improve the immune function of Japanese eel and the resistance of eels to the infection of V. vulnificus. Significance and Impact of the Study: This study offered an alternative preliminary strategy of making aquaculture vaccines against V. vulnificus for eel farming. [ABSTRACT FROM AUTHOR]

Espinoza-Vergara et al. unveiled a novel transmission mode of Vibrio cholerae based on environmental protozoan predation, which the bacterial pathogen evades via its release in 'expelled food vacuoles.' Vacuole-enclosed bacteria are not only fairly protected against environmental stressors, but also show enhanced intestinal colonization fitness upon oral ingestion. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Vibrio cholerae causes a severe disease that kills thousands of people annually. The outer membrane protein OmpU is the most abundant outer membrane protein in V. cholerae, and has been identified as an important virulence factor that is involved in host‐cell interaction and recognition, as well as being critical for the survival of the pathogenic V. cholerae in the host body and in harsh environments. The mechanism of these processes is not well understood owing to a lack of the structure of V. cholerae OmpU. Here, the crystal structure of the V. cholerae OmpU trimer is reported to a resolution of 2.2 Å. The protomer forms a 16‐β‐stranded barrel with a noncanonical N‐terminal coil located in the lumen of the barrel that consists of residues Gly32–Ser42 and is observed to participate in forming the second gate in the pore. By mapping the published functional data onto the OmpU structure, the OmpU structure reinforces the notion that the long extracellular loop L4 with a β‐hairpin‐like motif may be critical for host‐cell binding and invasion, while L3, L4 and L8 are crucially implicated in phage recognition by V. cholerae. [ABSTRACT FROM AUTHOR]

Porins can act as pathogen-associated molecular patterns, can be recognized by the host immune system and modulate immune responses. Vibrio cholerae porin OmpU aids in bacterial survival in the human gut by increasing resistance against bile acids and anti-microbial peptides. V. cholerae OmpU is pro-inflammatory in nature. However, interestingly, it can also down-regulate LPS-mediated pro-inflammatory responses. In this study, we have explored how OmpU-pretreatment affects LPS-mediated responses. Our study indicates that OmpU-pretreatment followed by LPS-activation does not induce M2-polarization of macrophages/monocytes. Further, OmpU attenuates LPS-mediated TLR2/TLR6 signaling by decreasing the association of TLRs along with recruitment of MyD88 and IRAKs to the receptor complex. This results in decreased translocation of NFκB in the nucleus. Additionally, OmpU-pretreatment up-regulates expression of IRAK-M, a negative regulator of TLR signaling, in RAW 264.7 mouse macrophage cells upon LPS-stimulation. Suppressor cytokine IL-10 is partially involved in OmpU-induced down-regulation of LPS-mediated TNFα production in human PBMCs. Furthermore, OmpU-pretreatment also affects macrophage function, by enhancing phagocytosis in LPS-treated RAW 264.7 cells, and down-regulates LPS-induced cell surface expression of co-stimulatory molecules. Altogether, OmpU causes suppression of LPS-mediated responses by attenuating the LPS-mediated TLR signaling pathway. [ABSTRACT FROM AUTHOR]

The bacteria that infect humans and cause cholera are themselves infected by viruses, which have the potential to influence the course of a cholera infection.

The impact of phage predation on bacterial pathogens in the context of human disease is not currently appreciated. Here, we show that predatory interactions of a phage with an important environmentally transmitted pathogen, Vibrio cholerae, can modulate the evolutionary trajectory of this pathogen during the natural course of infection within individual patients. We analyzed geographically and temporally disparate cholera patient stool samples from Haiti and Bangladesh and found that phage predation can drive the genomic diversity of intra-patient V. cholerae populations. Intra-patient phage-sensitive and phage-resistant isolates were isogenic except for mutations conferring phage resistance, and moreover, phage-resistant V. cholerae populations were composed of a heterogeneous mix of many unique mutants. We also observed that phage predation can significantly alter the virulence potential of V. cholerae shed from cholera patients. We provide the first molecular evidence for predatory phage shaping microbial community structure during the natural course of infection in humans.

OmpU is one of the major porins of Vibrio cholerae, a Gram-negative human pathogen. Previously, we showed that OmpU stimulates host monocytes and macrophages and induces the production of proinflammatory mediators via activation of the Toll-like receptor 1/2 (TLR1/2)-MyD88-dependent pathways. In the present study, we show that OmpU activates murine dendritic cells (DCs) via activation of the TLR2-mediated pathway and the NLRP3 inflammasome, leading to the production of proinflammatory cytokines and DC maturation. Our data reveal that although TLR2 plays an important role in providing both priming and the activation signal for the NLRP3 inflammasome in OmpU-activated DCs, OmpU is capable of activating the NLRP3 inflammasome, even in the absence of TLR2, if a priming signal is given. Furthermore, we show that the OmpU-mediated interleukin-1β (IL-1β) production in DCs depends on calcium flux and mitochondrial reactive oxygen species (mitoROS) generation. Interestingly, both OmpU translocation to the mitochondria of DCs as well as calcium signaling contribute to mitoROS production and prompt NLRP3 inflammasome activation. We also demonstrate that OmpU induces downstream signaling via activation of phosphoinositide-3-kinase (PI3K)-AKT, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and transcription factor NF-κB. Furthermore, our data reveal that OmpU-mediated activation of TLR2 induces signaling via PKC, MAPKs p38 and extracellular signal-regulated kinase (ERK), and transcription factor NF-κB; however, PI3K and MAPK Jun N-terminal protein kinase (JNK) are activated in TLR2 independent manner.

Background: Vibrio cholerae, the causative agent of cholera, has been responsible for global epidemics and many other problems over the centuries. It is one of the main public health issues in less-developed and developing countries and is considered one of the deadliest infectious agents. Therefore, precise and susceptible detection of V. cholerae from environmental and biological samples is critical. Aptamers provide a rapid, sensitive, highly specific, and inexpensive alternative to traditional methods., Objective: The present study develops a new protocol inspired by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to implement an in silico aptamer selection against V. cholerae, which can also be employed in the case of other pathogenic microorganisms., Methods: First, we built an oligonucleotide pool and screened it based on the secondary structure. Following that, we modeled the tertiary structures of filtered sequences and performed RNAprotein dockings to assess binding affinities between RNA sequences and Outer Membrane Protein U (OmpU), an effective marker in distinguishing epidemic strains of V. cholerae, which constitute up to 60% of the total outer membrane protein. Finally, we used molecular dynamics simulation to validate the results., Results: Three sequences (ChOmpU apta ) were proposed as final aptameric candidates. Analysis of the top-ranked docking results revealed that these candidate aptamers bound to all subunits of OmpU at the extracellular side with high affinity. Moreover, ChOmpU apta -3 and ChOmpU apta -2 were fully stable and formed strong bonds under dynamic conditions., Conclusion: We propose incorporating these candidate sequences into aptasensors for V. cholerae detection., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Polarization of the monocytes and macrophages toward the M1 and M2 states is important for hosts’ defense against the pathogens. Moreover, it plays a crucial role to resolve the overwhelming inflammatory responses that can be harmful to the host. Polarization of macrophages/monocytes can be induced by pathogen-associated molecular patterns (PAMPs). PAMP-mediated monocyte/macrophage polarization is important during the infection, as pathogen can suppress host immune system by altering the polarization status of the macrophages/monocytes. OmpU, an outer membrane porin protein of Vibrio cholerae , possesses the ability to induce pro-inflammatory responses in monocytes/macrophages. It is also able to down-regulate the LPS-mediated activation of the monocytes/macrophages. Such observation leads us to believe that OmpU may induce a state that can be called as M1/M2-intermediate state. In the present study, we evaluated a set of M1 and M2 markers in RAW 264.7 murine macrophage cell line, and THP-1 human monocytic cell line, in response to the purified OmpU protein. We observed that OmpU, as a PAMP, induced M1-polarization by activating the Toll-like receptor (TLR) signaling pathway. OmpU induced formation of TLR1/TLR2-heterodimers. OmpU-mediated TLR-activation led to the MyD88 recruitment to the TLR1/TLR2 complex. MyD88, in turn, recruited IRAK1. Ultimately, OmpU-mediated signaling led to the activation and subsequent nuclear translocation of the NFκB p65 subunit. We also observed that blocking of the TLR1, TLR2, IRAK1, and NFκB affected OmpU-mediated production of M1-associated pro-inflammatory cytokines such as TNFα and IL-6. [ABSTRACT FROM AUTHOR]

The Vibrio splendidus clade has previously been associated with epidemic outbreaks of various aquatic animals, as in the case of the cupped oyster, Crassostrea gigas. To investigate whether involved strains could present a clonal origin and to identify possible alternative background carriage animals or zooplankton, a large epidemiological survey was conducted on isolates of the splendidus clade. For this purpose, Vibrio strains were isolated from various samples including oysters, mussels, sediments, zooplankton, and sea water on the basis of a North/South gradient of the European sea water zone (Ireland, The Netherlands, France, Italy, and Spain). A total of 435 isolates were successfully associated to the V. splendidus clade using real time polymerase chain reaction with 16S specific primers and probes. A multiple-locus variable-number tandem-repeat analysis (VNTR) was conducted on all isolates based on a multiplex PCR-VNTR with a set of primer pairs designed from the V. tasmaniensis LGP32 genome. Preliminary validation of the primers on a set of collection strains from the V. splendidus clade confirmed that the former V. splendidus-related LGP32 and relative strains were related to V. tasmaniensis rather than to the type strain V. splendidus LMG 4042. The VNTR analysis was then successfully conducted on 335 isolates which led to the characterization of 87 different profiles. Our results showed that (1) the high diversity of VNTR did not enlighten significant correlation between a specific pattern and the origin of collected samples. However, populations isolated from animal samples tend to differ from those of the background environment; (2) oyster mortality events could not be linked to the clonal proliferation of a particular VNTR type. However, few different patterns seemed successively associated with samples collected during peaks of oyster's mortality. (3) Finally, no correlation could be seen between specific VNTR patterns and sequence phylogeny of the virulence factors vsm and ompU that were detected among strains isolated during as well as outside mortality events. These results, combined with incongruence observed between the ompU and vsm phylogenetic trees, suggested both large diffusion of strains and massive lateral gene transfer within the V. splendidus clade. [ABSTRACT FROM AUTHOR]

The outer membrane proteins of the marine aquatic animal pathogen, Vibrio alginolyticus, play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein- Omp U was cloned and expressed in Escherichia coli. Polyclonal antibodies were raised in rabbits against the purified recombinant Omp U, and the reaction of the antibody was confirmed by Western blotting using the isolated Omp U and the recombinant Omp U of V. alginolyticus. To analyze the immunogenicity of the recombinant Omp U, crimson snapper, Lutjanus erythropterus Bloch, were immunized by intraperitoneal injection, and antibody response was assessed by the enzyme-linked immunosorbent assay ( ELISA). The results demonstrated that the recombinant Omp U produced an observable antibody response in all sera of the vaccinated fish. The vaccinated fish were challenged by virulent V. alginolyticus and observed to have high resistance to infection. These results indicate that the recombinant Omp U is an effective vaccine candidate against V. alginolyticus in L. erythropterus. [ABSTRACT FROM AUTHOR]

Abstract: OmpU is one of the major outer membrane porins of Vibrio cholerae. OmpU has been biochemically characterized previously for its ‘porin’-property. However, previous studies have used the OmpU protein extracted from the bacterial outer membrane envelope fractions. Such method of isolation imposes limitations on the availability of the protein reagent, and also enhances the possibility of the OmpU preparation being contaminated with lipid molecules of bacterial outer membrane origin, especially lipopolysaccharides (LPS). Here we report a strategy of purifying the V. cholerae OmpU protein recombinantly overexpressed in heterologous protein expression system in Escherichia coli, without its being incorporated into the bacterial membrane fraction. In our strategy, the majority of the protein was expressed as insoluble inclusion body in the E. coli cytoplasm, the protein was dissolved by denaturation in 8M urea, refolded, and purified to homogeneity in presence of detergent. Our strategy allowed isolation of the recombinant OmpU protein with significantly enhanced yield as compared to that of the wild type protein extracted from the V. cholerae membrane fraction. The recombinant V. cholerae OmpU protein generated in our study displayed functional channel-forming property in the synthetic liposome membrane, thus confirming its ‘porin’-property. To the best of our knowledge, this is the first report showing an efficient refolding and functional assembly of the V. cholerae OmpU porin recombinantly expressed as inclusion body in the cytoplasm of a heterologous host E. coli. [Copyright &y& Elsevier]

Vibrio cholerae is the etiological agent of epidemic cholera. Symptoms include stomach ache, bloody diarrhea, and vomiting, which lead to severe dehydration and even death. Environmental monitoring as well as rapid and accurate identification of this pathogen are important for public health protection. In this study, a real-time PCR method for the detection of toxigenic V. cholerae was developed. In total, 63 environmental and clinical strains were tested for the presence of seven targets, namely ompW, ompU, tcpA, ctxA, zot, rfbO1, and rfbO139. The proposed method is specific, simple, and fast, and can be used for detection of toxigenic and non-toxigenic V. cholerae strains. The minimum detection limit of this assay for V. cholerae in environmental water samples was 1.4 CFU/ml. [ABSTRACT FROM AUTHOR]

The article discusses a study which revealed the role of vaccination of a pathogen called Vibrio (V) alginolyticus with outer membrane protein (OmpU) in inducing antibody response in Lutjanus erythropterus. It is said that the entire sera of the vaccinated fish was determined through an antibody titer against OmpU. The research showed a lower mortality rate in vaccinated fishes compared with those in control group following a V. alginolyticus challenge. It also mentions the ability of the pathogen's OmpU to maintain natural antigenicity. Also noted is the efficacy of isolated OmpU as a vaccine candidate to resist infection caused by V. alginolyticus.

Autoagglutinable strains of Vibrio cholerae O1 (seven nonfimbriate strains and one fimbriate strain) were transformed to obtain resistance to ampicillin. Two distinct mechanisms were found in these strains. One was operating in nonfimbriate strains by reducing OmpU protein production and the other was operating in a fimbriate strain (Bgd17) by newly overproducing cpxP protein. The twitching motility in the fimbriate Bgd17 strain disappeared depending on the production of cpxP protein, suggesting that fimbriation of V. cholerae O1 is controlled by a two-component signal transduction system. [ABSTRACT FROM AUTHOR]

OmpU is an integral outer membrane protein belonging to the porine family, which is present in the outer membrane of bacteria of the genus Vibrio. This manuscript describes the construction of a recombinant plasmid coding for OmpU protein fused to a myc-hexa-histidine domain (myc-hexa-his) of 2 kDa. The plasmid encoded recombinant protein OmpU-myc-hexa-his, is expressed at high levels in the E. coli Top 10 strain, which is accumulated insoluble in inclusion bodies inside the cell. The recombinant protein is recovered in 7 mol/L urea from insoluble inclusion bodies. The denatured form was purified by immobilized metal-chelate affinity chromatography. SDS-PAGE analysis of the purified fractions detected a band of approximately 40 kDa, which is in correspondence with the molecular mass reported for the wild type monomer of OmpU (38 kDa) in Vibrio cholerae strains coupled to the recombinant c-myc-hexa-his domain. The recombinant protein reacted with anti-OmpU y anti-hexa-his monoclonal antibodies in western blotting. The detection of comigrating bands with these two antibodies indicates the presence of OmpU protein fused to hexa-hystidine tag in the same peptide. The protocol described allowed obtaining this protein in a single chromatographic step and increased the purity up to 90 %, yielding on average a 23 % of the initial amount. Consequently, a host -vector system is available to express this protein. [ABSTRACT FROM AUTHOR]

Vibrio cholerae causes the diarrheal disease cholera which affects millions of people globally. The outer membrane protein U (OmpU) is the outer membrane protein that is most prevalent in V. cholerae and has already been recognized as a critical component of pathogenicity involved in host cell contact and as being necessary for the survival of pathogenic V. cholerae in the host body. Computational approaches were used in this study to screen a total of 37,709 natural compounds from the traditional Chinese medicine (TCM) database against the active site of OmpU. Following a sequential screening of the TCM database, we report three lead compounds—ZINC06494587, ZINC85510056, and ZINC95910434—that bind strongly to OmpU, with binding affinity values of −8.92, −8.12, and −8.78 kcal/mol, which were higher than the control ligand (−7.0 kcal/mol). To optimize the interaction, several 100 ns molecular dynamics simulations were performed, and the resulting complexes were shown to be stable in their vicinity. Additionally, these compounds were predicted to have good drug-like properties based on physicochemical properties and ADMET assessments. This study suggests that further research be conducted on these compounds to determine their potential use as cholera disease treatment. [ABSTRACT FROM AUTHOR]

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.

Bile resistance is essential for enteric pathogens, as exemplified by Vibrio cholerae, the causative agent of cholera. The outer membrane porin OmpU confers bacterial survival and colonization advantages in the presence of host-derived antimicrobial peptides as well as bile. Expression of ompU is controlled by the virulence regulator ToxR. rpoE knockouts are accompanied by suppressor mutations causing ompU downregulation. Therefore, OmpU constitutes an intersection of the ToxR regulon and the σ E -pathway in V. cholerae. To understand the mechanism by which the sigma factor σ E regulates OmpU synthesis, we performed transcription studies using ompU reporter fusions and immunoblot analysis. Our data revealed an increase in ompU promoter activity in ΔrpoE strains, as well as in a ΔompU background, indicating a negative feedback regulation circuit of ompU expression. This regulation seems necessary, since elevated lethality rates of ΔrpoE strains occur upon ompU overexpression. Manipulation of OmpU's C-terminal portion revealed its relevance for protein stability and potency of σ E release. Furthermore, ΔrpoE strains are still capable of elevating OmpU levels under membrane stress conditions triggered by the bile salt sodium deoxycholate. This study provides new details about the impact of σ E on ompU regulation, which is critical to the pathogen's intestinal survival., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

• Altered antibiotic susceptibility associated with OmpU downregulation in outbreak strains of Vibrio cholerae. • Emergence of hypervirulent cholera outbreak strains in Solapur, Maharashtra, India. It is evident from previous cholera epidemics/outbreaks in India, Africa and America that isolates of the seventh pandemic Vibrio cholerae El Tor (7PET) with Haitian cholera toxin (HCT) genotype were associated with increased mortality. The present study highlights the emergence of 7PET-HCT isolates causing two cholera outbreaks in Walsang and Wagdari (Solapur, India) in 2016. Molecular analyses revealed that 7PET strains from earlier outbreaks (2010 and 2012) were progenitors of the current 7PET-HCT isolates. Isolates from the 2016 outbreaks carried qnrVC and floR genes and showed reduced susceptibility to tetracycline, ciprofloxacin and azithromycin, drugs recommended by the World Health Organization (WHO) for the treatment of cholera. Remarkably, protein profiling and mass spectrometry revealed disappearance of the outer membrane protein U (OmpU) porin in 7PET-HCT isolates from the second outbreak in 2016. Downregulation of ompU gene expression was also confirmed at the transcriptional level. Strains with downregulated OmpU showed reduced minimum inhibitory concentrations (MICs) for polymyxin B, which is a pore-forming antimicrobial agent. A multipronged approach is of utmost importance to prevent further spread of circulating 7PET-HCT strains. There is a pressing need for the formulation and implementation of international policies to closely monitor the effective use of antibiotics in order to prevent the further rise and spread of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria release outer membrane vesicles into the external milieu to deliver effector molecules that alter the host and facilitate virulence. Vesicle formation is driven by phospholipid accumulation in the outer membrane and regulated by the phospholipid transporter VacJ/Yrb. We use the facultative human pathogen Vibrio cholerae to show that VacJ/Yrb is silenced early during mammalian infection, which stimulates vesiculation that expedites bacterial surface exchange and adaptation to the host environment. Hypervesiculating strains rapidly alter their bacterial membrane composition and exhibit enhanced intestinal colonization fitness. This adaptation is exemplified by faster accumulation of glycine-modified lipopolysaccharide (LPS) and depletion of outer membrane porin OmpT, which confers resistance to host-derived antimicrobial peptides and bile, respectively. The competitive advantage of hypervesiculation is lost upon pre-adaptation to bile and antimicrobial peptides, indicating the importance of these adaptive processes. Thus, bacteria use outer membrane vesiculation to exchange cell surface components, thereby increasing survival during mammalian infection. • Phospholipid transport of Vibrio cholerae is silenced upon host entry • Reduced transporter activity triggers OMV release and increases colonization fitness • Increased vesiculation accelerates modulation of cell surface composition • Hypervesiculation leads to faster adaptation to host defense mediators Upon infection, Vibrio cholerae must alter its surface profile to evade host defenses and adapt to the gastrointestinal environment. Zingl et al. show that increased release of outer membrane vesicles upon host entry allows the bacteria to rapidly modify their cell surface, thus increasing in vivo adaptation and colonization fitness. [ABSTRACT FROM AUTHOR]

Outer membrane protein U (OmpU) is a major porin from Vibrio alginolyticus and has been considered a vaccine candidate against infection by V. alginolyticus. After pre-incubated with polyclonal antibody against rOmpU, V. alginolyticus showed a 78% decrease in extracellular iron level, suggesting that interruption of OmpU could increase intracellular iron level. The mRNA expression of ompU under iron-limited conditions was determined using real-time reverse transcriptase PCR. The mRNA level of ompU was downregulated to 0.27-, 0.036- and 0.019-fold after the addition of the iron chelator 2,2′-bipyridyl for 10, 30 and 60 min, respectively. In addition, the promoter of ompU contained a ferric uptake regulator (Fur) binding site, which revealed the potential regulation of ompU by Fur and iron. Fur from V. alginolyticus was purified and used for electrophoretic mobility shift assay. The result showed that in the absence of Fe2+, purified recombinant Fur could specifically bind to the promoter DNA of ompU , while in the presence of Fe2+, the binding of Fur and the promoter DNA was suppressed. Our study preliminarily explored the function of OmpU in iron balance in V. alginolyticus , and these findings were helpful in understanding iron metabolism in V. alginolyticus. [ABSTRACT FROM AUTHOR]

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. After having been alerted by an anonymous reader the authors found out that in order to substantiate one of their conclusions (DeltaToxR-reduced killing activity is mediated via T6SS2) more experiments are needed. To avoid any potentially wrong conclusions being published, the authors decided to retract the article and to resubmit their manuscript once the additional experiments have been completed. The Editor-in-Chief agreed to the retraction. The authors wish to apologize for any inconvenience caused., (Copyright © 2019. Published by Elsevier GmbH.)

Vibrio parahaemolyticus is a human pathogen, and it is a major cause of severe gastroenteritis in coastal areas. OmpU is one of the major outer membrane porins of V. parahaemolyticus Host-immunomodulatory effects of V. parahaemolyticus OmpU (VpOmpU) have not been elucidated yet. In this study, in an effort towards characterizing the effect of VpOmpU on innate immune responses of the host, we observed that VpOmpU is recognized by the Toll-like receptor 1/2 (TLR1/2) heterodimer in THP-1 monocytes but by both TLR1/2 and TLR2/6 heterodimers in RAW 264.7 macrophages. To the best of our knowledge, this is the first report of a natural pathogen-associated molecular pattern (PAMP) recognized by both TLR1/2 and TLR2/6 heterodimers; so far, mainly the synthetic ligand Pam 2 CSK4 has been known to be recognized by both the TLR1/2 and TLR2/6 heterodimers. We also have shown that VpOmpU can activate monocytes and macrophages, leading to the generation of proinflammatory responses as indicated by tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and NO production in macrophages and TNF-α and IL-6 production in monocytes. VpOmpU-mediated proinflammatory responses involve MyD88-IRAK-1 leading to the activation of mitogen-activated protein (MAP) kinases (p38 and Jun N-terminal protein kinase [JNK]) and transcription factors NF-κB and AP-1. Further, we have shown that for the activation of macrophages leading to the proinflammatory responses, the TLR2/6 heterodimer is preferred over the TLR1/2 heterodimer. We have also shown that MAP kinase activation is TLR2 mediated., (Copyright © 2019 American Society for Microbiology.)

Summary The outer membranes (OM) of many Gram-negative bacteria contain general porins, which form nonspecific, large-diameter channels for the diffusional uptake of small molecules required for cell growth and function. While the porins of Enterobacteriaceae (e.g., E. coli OmpF and OmpC) have been extensively characterized structurally and biochemically, much less is known about their counterparts in Vibrionaceae. Vibrio cholerae, the causative agent of cholera, has two major porins, OmpU and OmpT, for which no structural information is available despite their importance for the bacterium. Here we report high-resolution X-ray crystal structures of V. cholerae OmpU and OmpT complemented with molecular dynamics simulations. While similar overall to other general porins, the channels of OmpU and OmpT have unusual constrictions that create narrower barriers for small-molecule permeation and change the internal electric fields of the channels. Together with electrophysiological and in vitro transport data, our results illuminate small-molecule uptake within the Vibrionaceae. [ABSTRACT FROM AUTHOR]

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., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Vibrio cholerae can cause severe diarrhea and dehydration leading to high mortality and morbidity. Current cholera vaccines are formulated with KVC. Although the innate immune responses following vaccination deeply influence the induction of adaptive immunity, the initial recognition of cholera vaccines by the host innate immune system is not well characterized. In this study, the ability of KVC to induce innate immune responses was investigated. Unlike typical Gram-negative bacteria stimulating TLR2 and TLR4, KVC activated TLR2 but hardly TLR4. However, purified V. cholerae LPS preferentially stimulated TLR4, although not as potently as LPS of other Gram-negative bacteria, implying that LPS is not a major immunostimulatory component of KVC. Instead, MPFs were similar to KVC in the capacity to activate TLR2, transcription factors, and cytokine expression. Furthermore, OmpU is an abundant membrane protein of V. cholerae and could interact with TLR2 for inducing cytokine expression. Notably, cholera vaccine-induced immune responses are impaired in TLR2(-/-) mice. Conclusively, TLR2 is essential for the immune responses to cholera vaccination, and OmpU is the major immunostimulatory component of cholera vaccines., (© Society for Leukocyte Biology.)

The bacteria that infect humans and cause cholera are themselves infected by viruses, which have the potential to influence the course of a cholera infection., (Copyright © 2014, De Paepe and Petit.)

The impact of phage predation on bacterial pathogens in the context of human disease is not currently appreciated. Here, we show that predatory interactions of a phage with an important environmentally transmitted pathogen, Vibrio cholerae, can modulate the evolutionary trajectory of this pathogen during the natural course of infection within individual patients. We analyzed geographically and temporally disparate cholera patient stool samples from Haiti and Bangladesh and found that phage predation can drive the genomic diversity of intra-patient V. cholerae populations. Intra-patient phage-sensitive and phage-resistant isolates were isogenic except for mutations conferring phage resistance, and moreover, phage-resistant V. cholerae populations were composed of a heterogeneous mix of many unique mutants. We also observed that phage predation can significantly alter the virulence potential of V. cholerae shed from cholera patients. We provide the first molecular evidence for predatory phage shaping microbial community structure during the natural course of infection in humans., (Copyright © 2014, Seed et al.)