scholarly journals Detection of rtxA Gene as a Biomarker of Seafood-Borne Pathogen Vibrio cholerae using In Silico PCR Assay

2020 ◽  
Vol 15 (2) ◽  
pp. 91
Author(s):  
Stalis Norma Ethica ◽  
Nur Hidayati ◽  
Hayatun Fuad ◽  
Chaerul Arham ◽  
Rivana Ariyadi ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
In Silico ◽  
Genomic Dna ◽  
Virulence Gene ◽  
Pcr Assay ◽  
Reading Frame ◽  
Marine Products ◽  
In Silico Pcr ◽  
Food Safety Risk ◽  
El Tor

Seafood-borne outbreaks caused by Vibrio cholerae have led to the increased need for food safety risk assessment of marine products. An in silico investigation about the potential of virulence gene of V. cholerae, rtxA, as a DNA biomarker of the toxigenic bacterium has been carried out. The aim of this study was to use the bacterial DNA biomarker sequence as a tool to facilitate early rapid detection of cholera infection. Five specific pairs of primers were designed from the rtxA open reading frame DNA of V. cholerae O1 biovar El Tor str. N16961 genomic DNA using Primer3Plus. Next, in silico Polymerase Chain Reaction (PCR) assay was carried out using the newly designed primers and 25 genomic DNA of vibrio spp. retrieved from the in silico database. One of the five designed pairs of primers, RtxAOF-RtxAOR: ‘5-CGCAAAACAGTTTCAGCCGA-3’ and 5’-AGGTTGGTCTTTTGTGGCCA-3’, could result in single DNA amplicon sized 518 bp only from V. cholerae species. No amplicon bands were produced from 17 other vibrio genomes studied using similar RtxAF-RtxAR primers. A further check showed that the amplicon was indeed part of the rtxA gene of V. cholerae. Based on this in silico study, rtxA gene appeared to be a DNA biomarker of V. cholerae, which is potential to facilitate rapid diagnosis of the virulence bacterium using in silico PCR assay.

scholarly journals The Cpx System Regulates Virulence Gene Expression in Vibrio cholerae

2015 ◽  
Vol 83 (6) ◽  
pp. 2396-2408 ◽  
Author(s):  
Nicole Acosta ◽  
Stefan Pukatzki ◽  
Tracy L. Raivio
Keyword(s):  
Vibrio Cholerae ◽  
Response Regulator ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Receptor Protein ◽  
Virulence Determinants ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Envelope Stress ◽  
El Tor

Bacteria possess signal transduction pathways capable of sensing and responding to a wide variety of signals. The Cpx envelope stress response, composed of the sensor histidine kinase CpxA and the response regulator CpxR, senses and mediates adaptation to insults to the bacterial envelope. The Cpx response has been implicated in the regulation of a number of envelope-localized virulence determinants across bacterial species. Here, we show that activation of the Cpx pathway inVibrio choleraeEl Tor strain C6706 leads to a decrease in expression of the major virulence factors in this organism, cholera toxin (CT) and the toxin-coregulated pilus (TCP). Our results indicate that this occurs through the repression of production of the ToxT regulator and an additional upstream transcription factor, TcpP. The effect of the Cpx response on CT and TCP expression is mostly abrogated in a cyclic AMP receptor protein (CRP) mutant, although expression of thecrpgene is unaltered. Since TcpP production is controlled by CRP, our data suggest a model whereby the Cpx response affects CRP function, which leads to diminished TcpP, ToxT, CT, and TCP production.

scholarly journals A Vibrio cholerae LysR Homolog, AphB, Cooperates with AphA at the tcpPH Promoter To Activate Expression of the ToxR Virulence Cascade

1999 ◽  
Vol 181 (14) ◽  
pp. 4250-4256 ◽  
Author(s):  
Gabriela Kovacikova ◽  
Karen Skorupski
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Growth Conditions ◽  
Culture Conditions ◽  
Virulence Gene Expression ◽  
Environmental Stimuli ◽  
High Level ◽  
El Tor

ABSTRACT We describe here a new member of the LysR family of transcriptional regulators, AphB, which is required for activation of the Vibrio cholerae ToxR virulence cascade. AphB activates the transcription of the tcpPH operon in response to environmental stimuli, and this process requires cooperation with a second protein, AphA. The expression of neither aphA or aphB is strongly regulated by environmental stimuli, raising the possibility that the activities of the proteins themselves may be influenced under various conditions. Strains of the El Tor biotype of V. choleraetypically exhibit lower expression of ToxR-regulated virulence genes in vitro than classical strains and require specialized culture conditions (AKI medium) to induce high-level expression. We show here that expression of aphB from the tac promoter in El Tor biotype strains dramatically increases virulence gene expression to levels similar to those observed in classical strains under all growth conditions examined. These results suggest that AphB plays a role in the differential regulation of virulence genes between the two disease-causing biotypes.

scholarly journals Bicarbonate Induces Vibrio cholerae Virulence Gene Expression by Enhancing ToxT Activity

2009 ◽  
Vol 77 (9) ◽  
pp. 4111-4120 ◽  
Author(s):  
Basel H. Abuaita ◽  
Jeffrey H. Withey
Keyword(s):  
Gene Expression ◽  
Carbonic Anhydrase ◽  
Vibrio Cholerae ◽  
Regulatory Protein ◽  
Virulence Gene ◽  
Chemical Stimulus ◽  
High Concentration ◽  
Virulence Gene Expression ◽  
Regulatory Cascade ◽  
El Tor

ABSTRACT Vibrio cholerae is a gram-negative bacterium that is the causative agent of cholera, a severe diarrheal illness. The two biotypes of V. cholerae O1 capable of causing cholera, classical and El Tor, require different in vitro growth conditions for induction of virulence gene expression. Growth under the inducing conditions or infection of a host initiates a complex regulatory cascade that results in production of ToxT, a regulatory protein that directly activates transcription of the genes encoding cholera toxin (CT), toxin-coregulated pilus (TCP), and other virulence genes. Previous studies have shown that sodium bicarbonate induces CT expression in the V. cholerae El Tor biotype. However, the mechanism for bicarbonate-mediated CT induction has not been defined. In this study, we demonstrate that bicarbonate stimulates virulence gene expression by enhancing ToxT activity. Both the classical and El Tor biotypes produce inactive ToxT protein when they are cultured statically in the absence of bicarbonate. Addition of bicarbonate to the culture medium does not affect ToxT production but causes a significant increase in CT and TCP expression in both biotypes. Ethoxyzolamide, a potent carbonic anhydrase inhibitor, inhibits bicarbonate-mediated virulence induction, suggesting that conversion of CO2 into bicarbonate by carbonic anhydrase plays a role in virulence induction. Thus, bicarbonate is the first positive effector for ToxT activity to be identified. Given that bicarbonate is present at high concentration in the upper small intestine where V. cholerae colonizes, bicarbonate is likely an important chemical stimulus that V. cholerae senses and that induces virulence during the natural course of infection.

scholarly journals The Fatty Acid Regulator FadR Influences the Expression of the Virulence Cascade in the El Tor Biotype of Vibrio cholerae by Modulating the Levels of ToxT via Two Different Mechanisms

2017 ◽  
Vol 199 (7) ◽  
Author(s):  
Gabriela Kovacikova ◽  
Wei Lin ◽  
Ronald K. Taylor ◽  
Karen Skorupski
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Virulence Gene ◽  
Enteric Bacteria ◽  
Posttranslational Regulation ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Expression Of Genes ◽  
El Tor ◽  
Virulence Regulator

ABSTRACT FadR is a master regulator of fatty acid (FA) metabolism that coordinates the pathways of FA degradation and biosynthesis in enteric bacteria. We show here that a ΔfadR mutation in the El Tor biotype of Vibrio cholerae prevents the expression of the virulence cascade by influencing both the transcription and the posttranslational regulation of the master virulence regulator ToxT. FadR is a transcriptional regulator that represses the expression of genes involved in FA degradation, activates the expression of genes involved in unsaturated FA (UFA) biosynthesis, and also activates the expression of two operons involved in saturated FA (SFA) biosynthesis. Since FadR does not bind directly to the toxT promoter, we determined whether the regulation of any of its target genes indirectly influenced ToxT. This was accomplished by individually inserting a double point mutation into the FadR-binding site in the promoter of each target gene, thereby preventing their activation or repression. Although preventing FadR-mediated activation of fabA, which encodes the enzyme that carries out the first step in UFA biosynthesis, did not significantly influence either the transcription or the translation of ToxT, it reduced its levels and prevented virulence gene expression. In the mutant strain unable to carry out FadR-mediated activation of fabA, expressing fabA ectopically restored the levels of ToxT and virulence gene expression. Taken together, the results presented here indicate that V. cholerae FadR influences the virulence cascade in the El Tor biotype by modulating the levels of ToxT via two different mechanisms. IMPORTANCE Fatty acids (FAs) play important roles in membrane lipid homeostasis and energy metabolism in all organisms. In Vibrio cholerae, the causative agent of the acute intestinal disease cholera, they also influence virulence by binding into an N-terminal pocket of the master virulence regulator, ToxT, and modulating its activity. FadR is a transcription factor that coordinately controls the pathways of FA degradation and biosynthesis in enteric bacteria. This study identifies a new link between FA metabolism and virulence in the El Tor biotype by showing that FadR influences both the transcription and posttranslational regulation of the master virulence regulator ToxT by two distinct mechanisms.

scholarly journals ANTIBIOFILM FORMATION ACTIVITY, RESISTANT GENES PROFILING AND DETECTION OF VIRULENCE FACTORS OF TOXIGENIC Vibrio cholerae ISOLATES FROM KISUMU COUNTY, KENYA

2021 ◽  
Author(s):  
Silas O. Awuor ◽  
Omwenga O. Eric ◽  
Ibrahim I. Daud
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Factors ◽  
Tetracycline Resistance ◽  
Positive Control ◽  
Toxin Gene ◽  
Pcr Assay ◽  
Tetracycline Resistance Gene ◽  
Resistant Genes ◽  
Standard Procedures ◽  
El Tor

ABSTRACTIntroductionVibrio cholerae can switch between motile and biofilm lifestyles with some of its strains forming biofilms in addition to production of various virulence traits and possessing antimicrobial resistance traits. This study is aim to show antibiofilm formation activity, resistant genes profiling and detection of virulence factors of toxigenic vibrio cholerae isolates from Kisumu County.MethodologyA total of 119 Vibrio cholerae O1, biotype El Tor isolates collected during 2017 cholera outbreak in Kisumu County were used for this study. The samples were cultured on TCBS and PCR assay carried out using standard procedures. Biofilm assay tests and detection of virulence factors were also done by use of standard procedures.ResultsOf the 101 confirmed vibrio cholerae isolates, 80.2% possessed the cholera toxin gene (ctxA) whereas 19.8% did not. Analysis of the toxR gene revealed that 98.0% harbored the toxR gene and only 2.0% did not. It was also revealed that 80.2% harbored the class I integron (inDS gene) while 19.8% did not, 93.1% were confirmed to possess the SXT integrating conjugative element (ICE) while 7.0% did not. The tetracycline resistance gene was present in 96.0% of the isolates. In 7 isolants strains which were resistance to common used antibiotics were screened for biofilm formation. Three of the strains (04/17-07, 06/17-14, and 05/17-03) failed to form biofilm while four strains namely 03/17-16, 02/17-09, 04/17-13 and P. aeruginosa ATCC 10145 as a positive control formed biofilms. In addition, out of those 7 isolants 71.42% produced protease, 85.71% produced phospholipases, 71.42% of isolates has the ability to produce lipase and 100% were able to produce the haemolysin.ConclusionAn understanding of this intricate signaling pathway is essential for the development of methods to treat and prevent this devastating disease.

scholarly journals Phylogenetic Affinity and Genome Structure Features of ctxAB– tcpA+ Vibrio cholerae from the Surface Waterbodies in the Territory that is Non-Endemic as Regards Cholera

2020 ◽  
pp. 115-123
Author(s):  
L. V. Mironova ◽  
N. O. Bochalgin ◽  
A. S. Gladkikh ◽  
S. I. Feranchuk ◽  
A. S. Ponomareva ◽  
...  
Keyword(s):  
Surface Water ◽  
Vibrio Cholerae ◽  
In Silico ◽  
Genome Structure ◽  
Far East ◽  
Water Sources ◽  
Water Bodies ◽  
Surface Water Bodies ◽  
Toxigenic Strains ◽  
El Tor

Objective is analyzing the origin of the ctxAB– tcpA+ Vibrio cholerae О1 El Tor strains isolated from the surface water bodies in the territory that is non-endemic of cholera; as well as investigating their phylogenetic relations to varying by epidemic significance groups of strains, based on the structure of housekeeping genes and whole genome structure. Materials and methods: we examined 25 V. cholerae strains, isolated in Siberia and Far East, including two ctxAB– tcpA+ strains from surface water sources (Altai Territory, 2011; Khabarovsk Territory, 2013). Phylogenetic analysis included genomes of 36 V. cholerae strains from GenBank. Multilocus sequence typing (MLST) was carried out based on dnaE, cat, lap, pgm, recA, gyrB, and chi genes; in silico MLST – adk, gyrB, metE, mdh, pntA, purM and pyrC genes. Reconstruction of phylogeny was performed based on the comparative analysis of core genome SNPs in PhyML 3.0. Results and discussion. MLST of ctxAB– tcpA+ V. cholerae О1 El Tor strains from the surface water bodies revealed that such strains form an individual genotype in the cluster of toxigenic strains and spontaneous mutants of toxigenic strains. According to results of in silico MLST, ctxAB– tcpA+ isolates belong to SТ75, common to US Gulf phylogenetic line. Based on SNP-typing, ctxAB– tcpA+ strains from the surface water sources were assigned to the group, originating from US Gulf V. cholerae, moreover isolate from Khabarovsk (2013) demonstrated high degree of genome homology with US Gulf-like strain from China (2009). For strains from Khabarovsk and China, we also showed the compositional identity of the pathogenicity island VPI-I and the presence of pandemicity island, VSP-1. The results testify to the fact that ctxAB– tcpA+ V. cholerae strains, isolated in Siberia and Far East, originate from the US Gulf phylogenetic line. Furthermore, taking into account the results of epidemiological analysis, we can deduce that these strains are imported ones.

scholarly journals Genetic Diversity of Clinical and Environmental Isolates of Vibrio cholerae Determined by Amplified Fragment Length Polymorphism Fingerprinting

2000 ◽  
Vol 66 (1) ◽  
pp. 148-153 ◽  
Author(s):  
Sunny C. Jiang ◽  
Maria Matte ◽  
Glavur Matte ◽  
Anwar Huq ◽  
Rita R. Colwell
Keyword(s):  
Vibrio Cholerae ◽  
Restriction Enzyme ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Genomic Dna ◽  
Fragment Length Polymorphism ◽  
Amplified Fragment Length Polymorphism ◽  
Clinical Isolates ◽  
Environmental Isolates ◽  
El Tor

ABSTRACT Vibrio cholerae, the causative agent of major epidemics of diarrheal disease in Bangladesh, South America, Southeastern Asia, and Africa, was isolated from clinical samples and from aquatic environments during and between epidemics over the past 20 years. To determine the evolutionary relationships and molecular diversity of these strains, in order to understand sources, origin, and epidemiology, a novel DNA fingerprinting technique, amplified fragment length polymorphism (AFLP), was employed. Two sets of restriction enzyme-primer combinations were tested for fingerprinting of V. cholerae serogroup O1, O139, and non-O1, O139 isolates. Amplification of HindIII- and TaqI-digested genomic DNA produced 30 to 50 bands for each strain. However, this combination, although capable of separating environmental isolates of O1 and non-O1 strains, was unable to distinguish between O1 and O139 clinical strains. This result confirmed that clinical O1 and O139 strains are genetically closely related. On the other hand, AFLP analyses of restriction enzyme ApaI- andTaqI-digested genomic DNA yielded 20 to 30 bands for each strain, but were able to separate O1 from O139 strains. Of the 74 strains examined with the latter combination, 26 serogroup O1 strains showed identical banding patterns and were represented by the O1 El Tor strain of the seventh pandemic. A second group, represented by O139 Bengal, included 12 strains of O139 clinical isolates, with 7 from Thailand, 3 from Bangladesh, and 2 from India. Interestingly, an O1 clinical isolate from Africa also grouped with the O139 clinical isolates. Eight clinical O1 isolates from Mexico grouped separately from the O1 El Tor of the seventh pandemic, suggesting an independent origin of these isolates. Identical fingerprints were observed between an O1 environmental isolate from a river in Chile and an O1 clinical strain from Kenya, both isolated more than 10 years apart. Both strains were distinct from the O1 seventh pandemic strain. Two O139 clinical isolates from Africa clustered with environmental non-O1 isolates, independent of other O139 strains included in the study. These results suggest that although a single clone of pathogenic V. cholerae appears responsible for many cases of cholera in Asia, Africa, and Latin America during the seventh pandemic, other cases of clinical cholera were caused by toxigenic V. choleraestrains that appear to have been derived locally from environmental O1 or non-O1 strains.

scholarly journals The Cyclic Dipeptide Cyclo(Phe-Pro) Inhibits Cholera Toxin and Toxin-Coregulated Pilus Production in O1 El Tor Vibrio cholerae

2010 ◽  
Vol 192 (14) ◽  
pp. 3829-3832 ◽  
Author(s):  
Xiaowen R. Bina ◽  
James E. Bina
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Virulence Factors ◽  
Virulence Gene ◽  
Cyclic Dipeptide ◽  
Present Evidence ◽  
Toxin Coregulated Pilus ◽  
El Tor ◽  
Virulence Regulator

ABSTRACT Cyclo(Phe-Pro) is a cyclic dipeptide produced by multiple Vibrio species. In this work, we present evidence that cyclo(Phe-Pro) inhibits the production of the virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) in O1 El Tor Vibrio cholerae strain N16961 during growth under virulence gene-inducing conditions. The cyclo(Phe-Pro) inhibition of CT and TCP production correlated with reduced transcription of the virulence regulator tcpPH and was alleviated by overexpression of tcpPH.

scholarly journals DNA Binding Proteins of the Filamentous Phages CTXφ and VGJφ of Vibrio cholerae

2009 ◽  
Vol 191 (18) ◽  
pp. 5873-5876 ◽  
Author(s):  
Alina Falero ◽  
Andy Caballero ◽  
Beatriz Ferrán ◽  
Yovanny Izquierdo ◽  
Rafael Fando ◽  
...  
Keyword(s):  
Dna Binding ◽  
Vibrio Cholerae ◽  
Genomic Dna ◽  
Open Reading Frame ◽  
Rolling Circle Replication ◽  
Binding Properties ◽  
Rolling Circle ◽  
Reading Frame ◽  
Double Stranded Dna ◽  
Dna Binding Properties

ABSTRACT The native product of open reading frame 112 (orf112) and a recombinant variant of the RstB protein, encoded by Vibrio cholerae pathogen-specific bacteriophages VGJφ and CTXφ, respectively, were purified to more than 90% homogeneity. Orf112 protein was shown to specifically bind single-stranded genomic DNA of VGJφ; however, RstB protein unexpectedly bound double-stranded DNA in addition to the single-stranded genomic DNA. The DNA binding properties of these proteins may explain their requirement for the rolling circle replication of the respective phages and RstB's requirement for single-stranded-DNA chromosomal integration of CTXφ phage dependent on XerCD recombinases.

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