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1.
Res Microbiol ; 175(4): 104179, 2024.
Article in English | MEDLINE | ID: mdl-38185395

ABSTRACT

Vibrio cholerae can form biofilms in the aquatic environment and in the human intestine, facilitating the release of hyper-infectious aggregates. Due to the increasing antibiotic resistance, alternatives need to be found. One of these alternatives is antimicrobial peptides, including polymyxin B (PmB). In this study, we first investigated the resistance of V. cholerae O1 El Tor strain A1552 to various antimicrobials under aerobic and anaerobic conditions. An increased resistance to PmB is observed in anaerobiosis, with a 3-fold increase in the dose required for 50 % growth inhibition. We then studied the impact of the PmB on the formation and the degradation of V. cholerae biofilms to PmB. Our results show that PmB affects more efficiently biofilm formation under anaerobic conditions. On the other hand, preformed biofilms are susceptible to degradation by PmB at concentrations close to the minimal inhibitory concentration. At higher concentrations, we observe an opacification of the biofilm structures within 20 min post-treatment, suggesting a densification of the structure. This densification does not seem to result from the overexpression of matrix genes but rather from DNA release through massive cell lysis, likely forming a protective shield that limits the penetration of the PmB into the biofilm.


Subject(s)
Anti-Bacterial Agents , Biofilms , Drug Resistance, Bacterial , Microbial Sensitivity Tests , Polymyxin B , Biofilms/drug effects , Biofilms/growth & development , Polymyxin B/pharmacology , Anti-Bacterial Agents/pharmacology , Vibrio cholerae/drug effects , Vibrio cholerae/genetics , Vibrio cholerae/physiology , Anaerobiosis , Humans , Vibrio cholerae O1/drug effects , Vibrio cholerae O1/genetics , Vibrio cholerae O1/physiology , Vibrio cholerae O1/growth & development
2.
ACS Appl Bio Mater ; 4(4): 3089-3100, 2021 04 19.
Article in English | MEDLINE | ID: mdl-35014397

ABSTRACT

Because of the emergence of multidrug-resistant pathogenic bacteria, there is a growing interest for the development of an efficient alternative to antibiotics. Gold nanoparticles (AuNPs) are promising candidates due to their inherent non-toxicity and can be used as effective carriers of drugs. Cholera caused by Gram-negative Vibrio cholerae is still a potential threat in many developing countries. Virstatin, a small molecule, has been reported to inhibit virulence regulation in V. cholerae. Herein, we report an efficient synthesis of virstatin-conjugated gold nanoparticles (VL-AuNPs) and their antibacterial efficacy against the El Tor biotype of V. cholerae (VcN16961). The spherical-shaped NPs have an average diameter of ∼17 nm. The uniqueness of VL-AuNPs relies in the enhanced antibacterial efficacy compared to virstatin, as evidenced from the inhibitory concentration obtained from growth kinetics, and attributed to the inhibition of ATPase activity and DNA damage. More importantly, the expression of cholera toxin, the most important virulence factor of V. cholera, is reduced to a far greater extent than by any of the component molecules. The effect of VL-AuNPs on VcN16961 was monitored using various assays such as confocal microscopy, FACS, fluorescence spectroscopy, and so on. Overall, VL-AuNPs could be a potential candidate for the use as an effective agent for combating diarrheal diseases caused by V. cholera.


Subject(s)
Anti-Bacterial Agents/pharmacology , Biocompatible Materials/pharmacology , Butyrates/pharmacology , Gold/pharmacology , Metal Nanoparticles/chemistry , Naphthalimides/pharmacology , Vibrio cholerae O1/drug effects , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Biocompatible Materials/chemical synthesis , Biocompatible Materials/chemistry , Butyrates/chemistry , Gold/chemistry , Kinetics , Materials Testing , Microbial Sensitivity Tests , Molecular Structure , Naphthalimides/chemistry , Particle Size , Vibrio cholerae O1/growth & development
3.
Afr J Prim Health Care Fam Med ; 12(1): e1-e6, 2020 Dec 08.
Article in English | MEDLINE | ID: mdl-33354982

ABSTRACT

BACKGROUND: Multiple drug resistance has become a major threat to the treatment of cholera. Recent studies in Kenya have described the epidemiology, especially the risk factors, of cholera; however, there is little information on the phenotypic and drug susceptibility patterns of Vibrio cholerae (V. cholerae) in outbreaks that in the recent past have occurred in western Kenya. AIM: To characterise and determine the antibiotics' susceptibility profiling of toxigenic V. cholerae isolates from Kisumu County. SETTING: The project was conducted in Kisumu County, Kenya. METHODS: A total of 119 V. cholerae O1, biotype El Tor, isolates collected during 2017 cholera outbreak in Kisumu County were used for this study. The samples were cultured on thiosulphate-citrate-bile salts sucrose (TCBS) agar and biochemical tests were carried out using standard procedures. Susceptibility tests were conducted by using various conventional antibiotics against standard procedures. RESULTS: Of the 119 isolates, 101 were confirmed to be V. cholerae belonging to serotypes Inaba and Ogawa, with Inaba being the predominant serotype (73.95%). The isolates were susceptible to ciprofloxacin (100%), ofloxacin (100%), gentamycin (100%), doxycycline (99%), ceftriaxone (99%) and streptomycin (96.04%) antimicrobials, and resistant to erythromycin (53.47%), amoxicillin (64.4%), nalidixic acid (83.2%) and ampicillin (89.11%), with high resistance to cotrimoxazole (99%) and tetracycline (97%). CONCLUSION: Vibrio cholerae was resistant to multiple antibiotics, including those commonly used in the management of cholera. Taken together, there is a need to carry out regular surveillance on antimicrobial drug resistance during outbreaks.


Subject(s)
Anti-Bacterial Agents/pharmacology , Cholera/microbiology , Drug Resistance, Bacterial , Drug Resistance, Multiple , Vibrio cholerae O1/drug effects , Anti-Bacterial Agents/therapeutic use , Cholera/drug therapy , Disease Outbreaks , Humans , Kenya , Microbial Sensitivity Tests , Phenotype , Serogroup , Spatial Analysis , Vibrio cholerae O1/classification , Vibrio cholerae O1/growth & development
4.
ACS Appl Mater Interfaces ; 12(42): 47278-47288, 2020 Oct 21.
Article in English | MEDLINE | ID: mdl-32990431

ABSTRACT

Due to increasing reports of multidrug-resistant (MDR) Vibrio cholerae O1, the goal of this study was to characterize the in vitro antimicrobial activity of chitosan microparticles (CMs) to evaluate their potential as a novel therapeutic agent for cholera. We examined the antimicrobial activity of CMs against toxigenic V. cholerae O1 using direct enumeration, microscopy, and fluorescence microplate assays. Bacterial viability kinetics were measured with different concentrations of CMs, solution pH, and salt content using a live/dead staining technique. Growth inhibition of CM-exposed V. cholerae strains was conducted using a redox-sensitive stain and compared between wild-type and isogenic outer membrane (OM) mutants. CM concentrations above 0.1 wt % were sufficient to kill V. cholerae O1 suspensions with approximately 108 CFU/mL within 3 h. The nonviable cells demonstrated increased OM permeability that corresponded to gross morphological changes observed through scanning electron microscopy. CMs exhibited dose-dependent bactericidal activity that increased predictably at lower pH and decreased with salt addition. V. cholerae O1 strains lacking O-antigen were twice as susceptible to growth inhibition by CMs, whereas those with glycine modification to lipid A were ten times more resistant. We propose that CMs exert vibriocidal activity via electrostatic surface interactions between their positively charged amine groups and the negatively charged Gram-negative bacterial OM, resulting in disruption, increased permeability, decreased redox metabolism, and subsequent loss of cellular viability. Further research should be conducted in vivo to evaluate the efficacy of CMs as luminal agents to treat infections caused by MDR, toxigenic V. cholerae and other diarrheal pathogens.


Subject(s)
Anti-Bacterial Agents/pharmacology , Chitosan/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Vibrio cholerae O1/drug effects , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Carbohydrate Conformation , Cell Survival/drug effects , Chitosan/chemical synthesis , Chitosan/chemistry , Microbial Sensitivity Tests , Particle Size , Surface Properties , Vibrio cholerae O1/cytology , Vibrio cholerae O1/growth & development
5.
Biol Pharm Bull ; 43(8): 1288-1291, 2020 Aug 01.
Article in English | MEDLINE | ID: mdl-32522950

ABSTRACT

It has been well known that Vibrio cholerae inhabit in environmental water. As many patients infected with cholera toxin-producing V. cholerae O1 (toxigenic V. cholerae O1) emerge in Kolkata, India, it has been thought that toxigenic V. cholerae O1 is easily detected in environmental water in Kolkata. However, we could not isolate toxigenic V. cholerae O1 from environmental water in Kolkata, though NAG Vibrio (generic name of V. cholerae non-O1/non-O139) is constantly detected. To clear the reason for the non-isolation of toxigenic V. cholerae O1, we examined the viability of V. cholera O1 and NAG Vibrios in the artificial low ionic strength aquatic solution. We found that the viability of toxigenic V. cholerae O1 in the solution is low, but that of NAG Vibrios is high. Subsequently, we examined the viability of NAG Vibrios possessing cholera toxin gene (ctx) in the same condition and found that the viability of these NAG Vibrios is low. These results indicate that the existence of ctx in V. cholerae affects the viability of V. cholerae in the aquatic solution used in this experiment. We thought that there was closely relation between the low viability of toxigenic V. cholerae O1 in the artificial low ionic strength aquatic solution and the low frequency of isolation of the strain from environmental water.


Subject(s)
Cholera Toxin/biosynthesis , Vibrio cholerae O1/growth & development , Cholera Toxin/genetics , Osmolar Concentration , Vibrio cholerae O1/metabolism
6.
Environ Microbiol Rep ; 12(4): 454-465, 2020 08.
Article in English | MEDLINE | ID: mdl-32542975

ABSTRACT

Vibrio cholerae O1, a natural inhabitant of estuarine environments, is found in a dormant, viable but non-culturable (VBNC) state during interepidemic periods. Although the individual roles of abiotic factors affecting VBNC formation have been extensively studied, their interplay in driving this phenomenon remains largely unaddressed. Here, we identified that major abiotic factors synergize with low nutrient conditions governing entry of cells into the VBNC state. Specifically, V. cholerae cells exposed to a combination of alkaline pH and high salinity under aeration at low temperatures (VBNC-inducing conditions) synergize to facilitate rapid entry into VBNC, whereas the opposite conditions prevented entry into the state. The major virulence regulator ToxR, and the stringent response protein RelA played opposing roles, repressing and facilitating VBNC entry respectively. Further, VBNC-inducing conditions negated the effects of ToxR and RelA, facilitating rapid formation of VBNC cells. In summary, this study highlights the synergy between critical abiotic factors and identified ToxR and RelA as two associated regulators, allowing for the persistence of V. cholerae in aquatic environments. Insights obtained in this study will help better understand environmental survival non-sporulating bacteria and transmission of facultative bacterial pathogens.


Subject(s)
Vibrio cholerae O1/growth & development , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cold Temperature , Gene Expression Regulation, Bacterial , Microbial Viability , Stress, Physiological , Vibrio cholerae O1/genetics , Vibrio cholerae O1/metabolism
7.
Environ Microbiol ; 22(10): 4314-4322, 2020 10.
Article in English | MEDLINE | ID: mdl-32319181

ABSTRACT

Vibrio cholerae can enter a viable but non-culturable (VBNC) state when it encounters unfavourable environments; VBNC cells serve as important reservoirs and still pose threats to public health. The genetic regulation of V. cholerae entering its VBNC state is not well understood. Here, we show a confrontation strategy adapted by V. cholerae O1 in which it utilizes a quorum sensing (QS) system to prevent transition into a VBNC state under low nutrition and temperature conditions. The upregulation of hapR resulted in a prolonged culturable state of V. cholerae in artificial sea water at 4°C, whereas the mutation of hapR led to fast entry into the VBNC state. We also observed that different V. cholerae O1 natural isolates with distinct QS functions present a variety of abilities to maintain culturability during the transition to a VBNC state. The strain groups with higher or constitutive expression of QS genes exhibit a greater tendency to maintain the culturable state during VBNC induction than those lacking QS functional groups. In summary, HapR-mediated QS regulation is associated with the transition to the VBNC state in V. cholerae. HapR expression causes V. cholerae to resist VBNC induction and become dominant over competitors in changing environments.


Subject(s)
Quorum Sensing/genetics , Quorum Sensing/physiology , Transcription Factors/metabolism , Vibrio cholerae O1/genetics , Vibrio cholerae O1/metabolism , Cell Line , Seawater , Temperature , Up-Regulation , Vibrio cholerae O1/growth & development , Vibrio cholerae O1/isolation & purification
8.
Sci Rep ; 10(1): 308, 2020 01 15.
Article in English | MEDLINE | ID: mdl-31941909

ABSTRACT

The 2 biotypes of Vibrio cholerae O1 serogroup strains-classical and El Tor-use glucose in distinct ways. Classical biotype strains perform organic acid-producing fermentation and eventually lose viability due to the self-induced creation of an acidic environment, whereas El Tor biotype strains use an alternative neutral fermentation pathway, which confers them with survival advantages. However, we report that the neutral fermentation pathway has only been recruited in prototype Wave 1 El Tor biotype strains, which have not been isolated since the mid-1990s. Current Wave 2 and Wave 3 atypical El Tor strains contain a single-base deletion in a gene that directs bacteria toward neutral fermentation, resulting in the loss of neutral fermentation and an appearance that is similar to classical biotype strains. Moreover, when sufficient glucose was supplied, Wave 1 El Tor strains maintained their use of acid-producing fermentation, in parallel with neutral fermentation, and thus lost viability in the late stationary phase. The global replacement of Wave 1 El Tor strains by Wave 2 and 3 atypical El Tor strains implies that the acidic fermentation pathway may not be disadvantageous to V. cholerae. The characteristics that we have reported might improve oral rehydration in the treatment of cholera.


Subject(s)
Glucose/metabolism , Vibrio cholerae O1/metabolism , Batch Cell Culture Techniques , Glucose/pharmacology , Hydrogen-Ion Concentration , Microbial Viability/drug effects , Serogroup , Vibrio cholerae O1/drug effects , Vibrio cholerae O1/genetics , Vibrio cholerae O1/growth & development
9.
PLoS One ; 14(8): e0221431, 2019.
Article in English | MEDLINE | ID: mdl-31430343

ABSTRACT

Biofilm formation is a common strategy used by bacteria in order to survive and persist in the environment. In Vibrio cholerae (V. cholerae), a Gram-negative pathogen responsible for the cholera disease, biofilm-like aggregates are important for the pathogenesis and disease transmission. Biofilm formation is initiated by the attachment of the bacteria to a surface, followed by maturation stages involving the formation of a biofilm matrix. In V. cholerae, flagella are essential for the initial step of biofilm formation, allowing the bacteria to swim and to detect a surface. In this study, we explored the effect of polymyxin B (PmB), a cationic bacterial antimicrobial peptide, on biofilm formation in pathogenic V. cholerae strains belonging to the O1 and O139 serotypes. We found that sub-inhibitory concentration of PmB induces a reduction of the biofilm formation by V. cholerae O1 and O139. Experiment on preformed biofilm demonstrated that the biofilm formation inhibition occurs at the initial step of biofilm formation, where the flagella are essential. We further characterize the effect of PmB on V. cholerae flagellation. Our results demonstrate that the flagellin expression is not reduced in presence of sub-inhibitory concentration of PmB. However, a decrease of the abundance of flagellin associated with the bacterial cells together with an increase in the secretome was observed. Electron microscopy observations also suggest that the abundance of aflagellated bacteria increases upon PmB supplementation. Finally, in agreement with the effect on the flagellation, a reduction of the bacterial motility is observed. Altogether, our results suggest that the PmB affect V. cholerae flagella resulting in a decrease of the motility and a compromised ability to form biofilm.


Subject(s)
Biofilms/growth & development , Flagella/metabolism , Polymyxin B/pharmacology , Vibrio cholerae O1/physiology , Antimicrobial Cationic Peptides/pharmacology , Bacterial Adhesion/drug effects , Biofilms/drug effects , Flagellin/metabolism , Genes, Bacterial , Movement , Vibrio cholerae O1/drug effects , Vibrio cholerae O1/growth & development , Vibrio cholerae O1/ultrastructure
10.
Environ Microbiol Rep ; 10(5): 594-604, 2018 10.
Article in English | MEDLINE | ID: mdl-30058121

ABSTRACT

Vibrio cholerae O1 is a natural inhabitant of aquatic environments and causes the acute diarrheal disease cholera. Entry into a viable but non-culturable (VBNC) state is a survival strategy by which V. cholerae withstands natural stresses and is important for the transition between the aquatic and host environments during the V. cholerae life cycle. In this study, the formation of VBNC V. cholerae induced by cold seawater exposure was investigated using RNA sequencing (RNA-seq). The analysis revealed that the expression of 1420 genes was changed on VBNC state formation. In the VBNC cells, genes related to biofilm formation, chitin utilization and stress responses were upregulated, whereas those related to cell division, morphology and ribosomal activity were mainly downregulated. The concurrent acquisition of a carbon source and the arrest of cell division in cells with low metabolic activity help bacteria increase their resistance to unfavourable environments. Moreover, two transcriptional regulators, SlmA and MetJ, were found to play roles in both VBNC formation and intestinal colonization, suggesting that some genes may function in both processes. This acquired knowledge will improve our understanding of the molecular mechanisms of stress tolerance and may help control future cholera infections and outbreaks.


Subject(s)
Cold Temperature , Gene Expression Regulation, Bacterial , Seawater , Vibrio cholerae O1/growth & development , Vibrio cholerae O1/genetics , Animals , Bacterial Proteins/genetics , Gene Expression Profiling , Intestines/microbiology , Mice , Mutation , RNA, Bacterial/genetics , Stress, Physiological/genetics
11.
Mol Cell Probes ; 39: 14-24, 2018 06.
Article in English | MEDLINE | ID: mdl-29574083

ABSTRACT

This study aimed to determine the antibiofilm activity of seawater microbes against Vibrio cholerae (VCO1) through functional metagenomics approach. A metagenomic library was constructed from Palk Bay seawater and the library was screened to identify the biofilm inhibitory metaclone. Metaclone SWMC166 (harbouring ∼30 kb metagenomic insert) was found to exhibit antibiofilm activity against VCO1. The biofilm inhibitory potential of partially purified ethyl acetate extract of SWMC166 (EA166) was further evaluated through microscopic studies and biochemical assays. Further, EA166 treated VCO1 divulged up-regulation of genes involved in high cell density-mediated quorum sensing (QS) pathway which was analysed by real-time PCR. In order to identify the genes of interest (within ∼30 kb insert), subcloning was performed through shotgun approach. Small molecules from positive subclones SC5 and SC8 were identified through HRLC-MS analysis. Resulted small molecules were docked against QS receptors of V. cholerae to identify the bioactive metabolites. Docking studies revealed that totally seven metabolites were able to interact with QS receptors that can possibly trigger the QS cascade and sequentially inhibit the biofilm formation and virulence factors of VCO1.


Subject(s)
Biofilms/drug effects , Computer Simulation , Metagenome , Seawater/chemistry , Small Molecule Libraries/pharmacology , Vibrio cholerae O1/drug effects , Extracellular Polymeric Substance Matrix/metabolism , Gene Expression Regulation, Bacterial/drug effects , Hydrophobic and Hydrophilic Interactions , Molecular Docking Simulation , Peptide Hydrolases/metabolism , Quorum Sensing/drug effects , Quorum Sensing/genetics , Thermodynamics , Vibrio cholerae O1/genetics , Vibrio cholerae O1/growth & development
12.
Microbiologyopen ; 4(4): 589-96, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25974870

ABSTRACT

In our previous work, we demonstrated that viable but nonculturable (VBNC) Vibrio cholerae O1 and O139 were converted to culturable by coculture with eukaryotic cells. Furthermore, we isolated a factor converting VBNC V. cholerae to culturable (FCVC) from a eukaryotic cell line, HT-29. In this study, we purified FCVC by successive column chromatographies comprising UNO Q-6 anion exchange, Bio-Scale CHT2-1 hydroxyapatite, and Superdex 200 10/300 GL. Homogeneity of the purified FCVC was demonstrated by SDS-PAGE. Nano-LC MS/MS analysis showed that the purified FCVC was a human catalase. An experiment of RNAi knockdown of catalase mRNA from HT-29 cells and treatment of the purified FCVC with a catalase inhibitor, 3-amino-1,2,4-triazole confirmed that the FCVC was a catalase. A possible role of the catalase in converting a VBNC V. cholerae to a culturable state in the human intestine is discussed.


Subject(s)
Catalase/metabolism , Eukaryotic Cells/enzymology , Vibrio cholerae O1/physiology , Catalase/isolation & purification , Cell Line , Chromatography, Liquid , Coculture Techniques , Electrophoresis, Polyacrylamide Gel , Humans , Tandem Mass Spectrometry , Vibrio cholerae O1/growth & development
13.
Article in Russian | MEDLINE | ID: mdl-25842946

ABSTRACT

AIM: Experimental production, characterization and evaluation of the role of cholera vibrio biofilm. MATERIALS AND METHODS: 33 strains of Vibrio cholerae eltor O1 and V. cholerae O139 of various epidemic significance and origin were studied in a series of experiments by bacteriologic, microscopic (light-optic, luminescent, scanning electron microscopy), molecular genetics, spectrophotometric and statistical methods. RESULTS: Formation of a biofilm involving inter-cellular bonds, pili and extracellular material and variability of the microorganism (RO-phenotype and transition into uncultivable forms) was shown at various temperature and substrate conditions. A more pronounced ability to form biofilms was detected for strains isolated from environmental samples compared with isolated from clinical material regardless of their epidemic significance. Toxigenic strains of eltor biovar (from surface reservoirs during cholera outbreaks) have demonstrated the highest parameters of optical density compared with toxigenic clinical isolates and non-toxigenic O1 and O139 serogroup cultures. The presence of mbaA1 and mbaA2, vpsR, toxR, hapA genes is common for strains that form a biofilm. CONCLUSION: The data obtained confirm the role of biofilm in reservation of cholera vibrio strains of various epidemic significance in saprophytic phase of microorganism existence.


Subject(s)
Biofilms/growth & development , Cholera/genetics , Vibrio cholerae O1/growth & development , Cholera/microbiology , Cholera Toxin/biosynthesis , Cholera Toxin/genetics , Humans , Vibrio cholerae O1/genetics , Vibrio cholerae O1/pathogenicity , Water Microbiology
14.
Microbiol Immunol ; 59(5): 305-10, 2015 May.
Article in English | MEDLINE | ID: mdl-25664673

ABSTRACT

Many bacterial species are known to become viable but nonculturable (VBNC) under conditions that are unsuitable for growth. In this study, the requirements for resuscitation of VBNC-state Vibrio cholerae cells were found to change over time. Although VBNC cells could initially be converted to culturable by treatment with catalase or HT-29 cell extract, they subsequently entered a state that was not convertible to culturable by these factors. However, fluorescence microscopy revealed the presence of live cells in this state, from which VBNC cells were resuscitated by co-cultivation with HT-29 human colon adenocarcinoma cells. Ultimately, all cells entered a state from which they could not be resuscitated, even by co-cultivation with HT-29. These characteristic changes in VBNC-state cells were a common feature of strains in both V. cholerae O1 and O139 serogroups. Thus, the VBNC state of V. cholerae is not a single property but continues to change over time.


Subject(s)
Microbial Viability , Vibrio cholerae O139/physiology , Vibrio cholerae O1/physiology , Bacteriological Techniques/methods , Cell Line, Tumor , Coculture Techniques/methods , Epithelial Cells/microbiology , Epithelial Cells/physiology , Humans , Vibrio cholerae O1/growth & development , Vibrio cholerae O139/growth & development
15.
Infect Immun ; 83(3): 1089-103, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25561705

ABSTRACT

Vibrio cholerae O1 is a major cause of acute watery diarrhea in over 50 countries. Evidence suggests that V. cholerae O1 may activate inflammatory pathways, and a recent study of a Bangladeshi population showed that variants in innate immune genes play a role in mediating susceptibility to cholera. We analyzed human proteins present in the small intestine of patients infected with V. cholerae O1 to characterize the host response to this pathogen. We collected duodenal biopsy specimens from patients with acute cholera after stabilization and again 30 days after initial presentation. Peptides extracted from biopsy specimens were sequenced and quantified using label-free mass spectrometry and SEQUEST. Twenty-seven host proteins were differentially abundant between the acute and convalescent stages of infection; the majority of these have known roles in innate defense, cytokine production, and apoptosis. Immunostaining confirmed that two proteins, WARS and S100A8, were more abundant in lamina propria cells during the acute stage of cholera. Analysis of the differentially abundant proteins revealed the activation of key regulators of inflammation by the innate immune system, including Toll-like receptor 4, nuclear factor kappa-light-chain-enhancer of activated B cells, mitogen-activated protein kinases, and caspase-dependent inflammasomes. Interleukin-12ß (IL-12ß) was a regulator of several proteins that were activated during cholera, and we confirmed that IL-12ß was produced by lymphocytes recovered from duodenal biopsy specimens of cholera patients. Our study shows that a broad inflammatory response is generated in the gut early after onset of cholera, which may be critical in the development of long-term mucosal immunity against V. cholerae O1.


Subject(s)
Cholera/genetics , Convalescence , Duodenum/immunology , Immunity, Mucosal , Signal Transduction/immunology , Vibrio cholerae O1/pathogenicity , Acute Disease , Apoptosis/immunology , Biopsy , Calgranulin A/genetics , Calgranulin A/immunology , Cholera/immunology , Cholera/microbiology , Cholera/pathology , Duodenum/microbiology , Duodenum/pathology , Gene Expression Profiling , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Inflammasomes/genetics , Inflammasomes/immunology , Interleukin-12 Subunit p40/genetics , Interleukin-12 Subunit p40/immunology , Proteomics , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/immunology , Tryptophan-tRNA Ligase/genetics , Tryptophan-tRNA Ligase/immunology , Vibrio cholerae O1/growth & development , Vibrio cholerae O1/immunology
16.
Article in Russian | MEDLINE | ID: mdl-25536777

ABSTRACT

AIM: Analysis of antibiotic resistance profiles of Vibrio cholerae O1 strains isolated from the environmental objects in the territory of Russia in 2005 - 2012. MATERIALS AND METHODS: Antibiotocograms of 52 strains of V. cholerae were determined by serial dilution method in dense nutrient medium. Interpretation of the results was carried out in accordance with guidelines MI 4.2.2495-09 (2009). RESULTS: All the cultures turned out to be sensitive to tetracyclines, ciprofloxacin, cephalosporins: Isolates from Stavropol region were resistant to furazolidone (33.3%), trimethoprim/sulfamethoxazole (100%). Strains resistant to ampicillin, streptomycin, rifampicin (7%), furazolidone (43%), trimethoprim/sulfamethoxazole (100%) were isolated in Primorsky region. In Irkutsk region and Kalmykia--to furazolidone and trimethoprim/sulfamethoxazole (11 - 89%), ampicillin (8.3 - 11%). Analysis of antibioticograms gives evidence on the occurrence in the studied strains of 1 to 5 r-determinants of antibiotic resistance in various combinations. CONCLUSION: The data obtained give evidence on preservation of the tendency to expand the specter of antibiotic resistance in V. cholerae O1 isolated from environmental objects that necessitates a more rational and effective use of antibacterial preparations, determination of antiobioticogram for every isolated culture, strict bacteriologic control during the course of etiotropic therapy for the prevention of increase of number of resistant strains.


Subject(s)
Anti-Bacterial Agents/administration & dosage , Drug Resistance, Microbial/genetics , Environmental Microbiology , Vibrio cholerae O1/drug effects , Humans , Microbial Sensitivity Tests , Russia , Vibrio cholerae O1/genetics , Vibrio cholerae O1/growth & development
17.
mBio ; 5(6)2014 Dec 23.
Article in English | MEDLINE | ID: mdl-25538196

ABSTRACT

UNLABELLED: The bacterial cell surface is the first structure the host immune system targets to prevent infection. Cationic antimicrobial peptides of the innate immune system bind to the membrane of Gram-negative pathogens via conserved, surface-exposed lipopolysaccharide (LPS) molecules. We recently reported that modern strains of the global intestinal pathogen Vibrio cholerae modify the anionic lipid A domain of LPS with a novel moiety, amino acids. Remarkably, glycine or diglycine addition to lipid A alters the surface charge of the bacteria to help evade the cationic antimicrobial peptide polymyxin. However, the regulatory mechanisms of lipid A modification in V. cholerae are unknown. Here, we identify a novel two-component system that regulates lipid A glycine modification by responding to important biological cues associated with pathogenesis, including bile, mildly acidic pH, and cationic antimicrobial peptides. The histidine kinase Vc1319 (VprB) and the response regulator Vc1320 (VprA) respond to these signals and are required for the expression of the almEFG operon that encodes the genes essential for glycine modification of lipid A. Importantly, both the newly identified two-component system and the lipid A modification machinery are required for colonization of the mammalian host. This study demonstrates how V. cholerae uses a previously unknown regulatory network, independent of well-studied V. cholerae virulence factors and regulators, to respond to the host environment and cause infection. IMPORTANCE: Vibrio cholerae, the etiological agent of cholera disease, infects millions of people every year. V. cholerae El Tor and classical biotypes have been responsible for all cholera pandemics. The El Tor biotype responsible for the current seventh pandemic has displaced the classical biotype worldwide and is highly resistant to cationic antimicrobial peptides, like polymyxin B. This resistance arises from the attachment of one or two glycine residues to the lipid A domain of lipopolysaccharide, a major surface component of Gram-negative bacteria. Here, we identify the VprAB two-component system that regulates the charge of the bacterial surface by directly controlling the expression of genes required for glycine addition to lipid A. The VprAB-dependent lipid A modification confers polymyxin B resistance and contributes significantly to pathogenesis. This finding is relevant for understanding how Vibrio cholerae has evolved mechanisms to facilitate the evasion of the host immune system and increase bacterial fitness.


Subject(s)
Gene Expression Regulation, Bacterial , Lipid A/metabolism , Protein Kinases/metabolism , Signal Transduction/genetics , Transcription Factors/metabolism , Vibrio cholerae O1/genetics , Virulence Factors/metabolism , Antimicrobial Cationic Peptides/metabolism , Bile/metabolism , Histidine Kinase , Humans , Hydrogen-Ion Concentration , Lipid A/toxicity , Protein Kinases/genetics , Stress, Physiological , Transcription Factors/genetics , Vibrio cholerae O1/drug effects , Vibrio cholerae O1/growth & development , Vibrio cholerae O1/physiology , Virulence , Virulence Factors/toxicity
18.
ACS Chem Biol ; 9(10): 2382-92, 2014 Oct 17.
Article in English | MEDLINE | ID: mdl-25068415

ABSTRACT

The current pandemic El Tor biotype of O1 Vibrio cholerae is resistant to polymyxins, whereas the previous pandemic strain of the classical biotype is polymyxin sensitive. The almEFG operon found in El Tor V. cholerae confers >100-fold resistance to polymyxins through the glycylation of lipopolysaccharide. Here, we present the mechanistic determination of initial steps in the AlmEFG pathway. We verify that AlmF is an aminoacyl carrier protein and identify AlmE as the enzyme required to activate AlmF as a functional carrier protein. A combination of structural information and activity assays was used to identify a pair of active site residues that are important for mediating AlmE glycine specificity. Overall, the structure of AlmE in complex with its glycyl-adenylate intermediate reveals that AlmE is related to Gram-positive d-alanine/d-alanyl carrier protein ligase, while the trio of proteins in the AlmEFG system forms a chemical pathway that resembles the division of labor in nonribosomal peptide synthetases.


Subject(s)
Anti-Infective Agents/pharmacology , Bacterial Proteins/metabolism , Lipopolysaccharides/pharmacology , Peptide Fragments/pharmacology , Peptide Synthases/chemistry , Peptide Synthases/metabolism , Vibrio cholerae O1/drug effects , Cholera/drug therapy , Cholera/microbiology , Crystallography, X-Ray , Drug Resistance, Bacterial/drug effects , Gene Expression Regulation, Bacterial/drug effects , Polymyxin B/pharmacology , Protein Conformation , Signal Transduction , Substrate Specificity , Tandem Mass Spectrometry , Vibrio cholerae O1/enzymology , Vibrio cholerae O1/growth & development
19.
BMC Microbiol ; 13: 96, 2013 May 03.
Article in English | MEDLINE | ID: mdl-23642157

ABSTRACT

BACKGROUND: Many Gram-negative bacteria rely on a type VI secretion system (T6SS) to infect eukaryotic cells or to compete against other microbes. Common to these systems is the presence of two conserved proteins, in Vibrio cholerae denoted VipA and VipB, which have been shown to interact in many clinically relevant pathogens. In this study, mutagenesis of a defined region within the VipA protein was used to identify residues important for VipB binding in V. cholerae O1 strain A1552. RESULTS: A dramatically diminished interaction was shown to correlate with a decrease in VipB stability and a loss of hemolysin co-regulated protein (Hcp) secretion and rendered the bacterium unable to compete with Escherichia coli in a competition assay. CONCLUSIONS: This confirms the biological relevance of the VipA-VipB interaction, which is essential for the T6SS activity of many important human pathogens.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Secretion Systems , Protein Interaction Mapping , Vibrio cholerae O1/metabolism , Bacterial Proteins/genetics , DNA Mutational Analysis , Escherichia coli/growth & development , Humans , Mutant Proteins/genetics , Mutant Proteins/metabolism , Vibrio cholerae O1/genetics , Vibrio cholerae O1/growth & development
20.
J Proteomics ; 86: 1-15, 2013 Jun 28.
Article in English | MEDLINE | ID: mdl-23665147

ABSTRACT

PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. In order to disclose other roles of this system, a proteomic analysis of Vibrio cholerae 569BSR and its phoB/phoR mutant under high Pi levels was performed. Most of the proteins downregulated by the mutant have roles in energy production and conversion and in amino acid transport and metabolism. In contrast, the phoB/phoR mutant upregulated genes mainly involved in adaptation to atypical conditions, indicating that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways. This might be a strategy to overcome the lack of RpoS, whose expression in the stationary phase cells of V. cholerae seems to be controlled by PhoB/PhoR. Moreover, compared to the wild-type strain the phoB/phoR mutant presented a reduced cell density at stationary phase of culture in Pi abundance, lower resistance to acid shock, but higher tolerance to thermal and osmotic stresses. Together our findings show, for the first time, the requirement of PhoB/PhoR for full growth under high Pi level and for the accumulation of RpoS, indicating that PhoB/PhoR is a fundamental system for the biology of V. cholerae. BIOLOGICAL SIGNIFICANCE: Certain V. cholerae strains are pathogenic to humans, causing cholera, an acute dehydrating diarrhoeal disease endemic in Southern Asia, parts of Africa and Latin America, where it has been responsible for significant mortality and economical damage. Its ability to grow within distinct niches is dependent on gene expression regulation. PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. However, Pho regulon genes also play roles in virulence, motility and biofilm formation, among others. In this paper we report that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways, in Pi abundance. Moreover, we showed, for the first time, that the interrelationship between PhoB-RpoS-(p)ppGpp-poly(P) in V. cholerae, is somewhat diverse from the model of inter-regulation between those systems, described in Escherichia coli. The V. cholerae dependence on PhoB/PhoR for the RpoS mediated stress response and cellular growth under Pi abundance, suggests that this system's roles are broader than previously thought.


Subject(s)
Bacterial Proteins/genetics , Phosphates/metabolism , Proteomics , Vibrio cholerae O1/genetics , Bacterial Proteins/biosynthesis , Bacterial Proteins/physiology , Down-Regulation , Gene Expression Regulation, Bacterial , Guanine Nucleotides/metabolism , Mutation , Polyphosphates/metabolism , Sigma Factor/biosynthesis , Transcriptome , Up-Regulation , Vibrio cholerae O1/growth & development
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