ompU genes in non-toxigenic Vibrio cholerae associated with aquaculture

AIMS: The study was undertaken with the objective of understanding the virulence-associated genes of the CTX and TCP gene clusters in environmental isolates of Vibrio cholerae, an important human pathogen, isolated from the aquaculture environment. The involvement of the ompU gene in conferring bile resistance in these isolates was also evaluated. METHODS AND RESULTS: The V. cholerae isolates were tested by PCR and fluorescent antibody test for O1 (Ogawa and Inaba) and O139 serotypes. All isolates were found to be non-toxigenic V. cholerae confirmed by their positive PCR reaction for toxR but negative for ctx, zot and tcp gene. The hlyA gene was detected in 85% of the strains and ompU in 77%. The results on the bactericidal effect of bile salts suggest that ompU may play a role in conferring bile resistance in non-O1/non-O139 strains. CONCLUSION: The results of the study indicate that most environmental strains lacked the CTX and TCP gene clusters. However, most isolates had the hlyA gene indicating the potential of these environmental strains to cause mild gastroenteritis. It was also observed that strains lacking ompU showed less tolerance to bile salts. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on virulence factors of V. cholerae associated with aquaculture environment and products would be of value in risk assessment for human health.     

Effects of Polyamines on Vibrio cholerae Virulence Properties

Vibrio cholerae is the causative agent of the severe enteric disease cholera. To cause cholera the bacterium must be able to synthesize both cholera toxin (CT) and toxin-coregulated pilus (TCP) which mediates autoagglutination and is required for colonization of the small intestine. Only a few environmental signals have been shown to regulate V. cholerae virulence gene expression. Polyamines, which are ubiquitous in nature, and have been implicated in regulating virulence gene expression in other bacteria, have not been extensively studied for their effect on V. cholerae virulence properties. The objective of this study was to test the effect of several polyamines that are abundant in the human intestine on V. cholerae virulence properties. All of the polyamines tested inhibited autoagglutination of V. cholerae O1 classical strain in a concentration dependent manner. Putrescine and cadaverine decreased the synthesis of the major pilin subunit, TcpA, spermidine increased its production, and spermine had no effect. Putrescine and spermidine led to a decrease and increase, respectively, on the relative abundance of TCP found on the cell surface. Spermine led to a small reduction in cholera toxin synthesis whereas none of the other polyamines had an effect. The polyamines did not affect pili bundling morphology, but caused a small reduction in CTXφ transduction, indicating that the TCP present on the cell surface may not be fully functional. We hypothesize the inhibition of autoagglutination is likely to be caused by the positively charged amine groups on the polyamines electrostatically disrupting the pili-pili interactions which mediate autoagglutination. Our results implicate that polyamines may have a protective function against colonization of the small intestine by V. cholerae.     

Virulence genes in environmental strains of Vibrio cholerae

The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulated expression. The ctxAB and tcpA genes are known to play a cardinal role in maintaining virulence in Vibrio cholerae, and these genes are believed to be exclusively associated with clinical strains of O1 and O139 serogroups. In this study, we examined the presence of five virulence genes, including ctxAB and tcpA, as well as toxR and toxT, which are involved in the regulation of virulence, in environmental strains of V. cholerae cultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India. PCR analysis revealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes of environmental V. cholerae strains. Sequencing of a part of the tcpA gene carried by an environmental strain showed 97.7% homology to the tcpA gene of the classical biotype of V. cholerae O1. Strains carrying the tcpA gene expressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electron microscopy of the TCP pili. Strains carrying ctxAB genes also produced cholera toxin, determined by monosialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits. Thus, this study demonstrates the presence and expression of critical virulence genes or their homologues in diverse environmental strains of V. cholerae, which appear to constitute an environmental reservoir of virulence genes, thereby providing new insights into the ecology of V. cholerae.     

Molecular analysis of VCA1008: a putative phosphoporin of Vibrio cholerae

The PhoB/PhoR-dependent response to inorganic phosphate (Pi)-starvation in Vibrio cholerae O1 includes the expression of vc0719 for the response regulator PhoB, vca0033 for an alkaline phosphatase and vca1008 for an outer membrane protein (OMP). Sequences with high identity to these genes have been found in the genome of clinical and environmental strains, suggesting that the Pi-starvation response in V. cholerae is well conserved. VCA1008, an uncharacterized OMP involved in V. cholerae pathogenicity, presents sequence similarity to porins of Gram-negative bacteria such as phosphoporin PhoE from Escherichia coli . A three-dimensional model shows that VCA1008 is a 16-stranded pore-forming β-barrel protein that shares three of the four conserved lysine residues responsible for PhoE anionic specificity with PhoE. VCA1008 β-barrel apparently forms trimers that collapse into monomers by heating. Properties such as heat modifiability and resistance to denaturation by sodium dodecyl sulfate at lower temperatures permitted us to suggest that VCA1008 is a classical porin, more precisely, a phosphoporin due to its Pi starvation-induced PhoB-dependent expression, demonstrated by electrophoretic mobility shift assay and promoter fusion- lacZ assays.     

Proteolysis of Virulence Regulator ToxR Is Associated with Entry of Vibrio cholerae into a Dormant State

Vibrio cholerae O1 is a natural inhabitant of aquatic environments and causes the diarrheal disease, cholera. Two of its primary virulence regulators, TcpP and ToxR, are localized in the inner membrane. TcpP is encoded on the Vibrio Pathogenicity Island (VPI), a horizontally acquired mobile genetic element, and functions primarily in virulence gene regulation. TcpP has been shown to undergo regulated intramembrane proteolysis (RIP) in response to environmental conditions that are unfavorable for virulence gene expression. ToxR is encoded in the ancestral genome and is present in non-pathogenic strains of V. cholerae, indicating it has roles outside of the human host. In this study, we show that ToxR undergoes RIP in V. cholerae in response to nutrient limitation at alkaline pH, a condition that occurs during the stationary phase of growth. This process involves the site-2 protease RseP (YaeL), and is dependent upon the RpoE-mediated periplasmic stress response, as deletion mutants for the genes encoding these two proteins cannot proteolyze ToxR under nutrient limitation at alkaline pH. We determined that the loss of ToxR, genetically or by proteolysis, is associated with entry of V. cholerae into a dormant state in which the bacterium is normally found in the aquatic environment called viable but nonculturable (VBNC). Strains that can proteolyze ToxR, or do not encode it, lose culturability, experience a change in morphology associated with cells in VBNC, yet remain viable under nutrient limitation at alkaline pH. On the other hand, mutant strains that cannot proteolyze ToxR remain culturable and maintain the morphology of cells in an active state of growth. Overall, our findings provide a link between the proteolysis of a virulence regulator and the entry of a pathogen into an environmentally persistent state.     

Genotypes Associated with Virulence in Environmental Isolates of Vibrio cholerae

Vibrio cholerae is an autochthonous inhabitant of riverine and estuarine environments and also is a facultative pathogen for humans. Genotyping can be useful in assessing the risk of contracting cholera, intestinal, or extraintestinal infections via drinking water and/or seafood. In this study, environmental isolates of V. cholerae were examined for the presence of ctxA, hlyA, ompU, stn/sto, tcpA, tcpI, toxR, and zot genes, using multiplex PCR. Based on tcpA and hlyA gene comparisons, the strains could be grouped into Classical and El Tor biotypes. The toxR, hlyA, and ompU genes were present in 100, 98.6, and 87.0% of the V. cholerae isolates, respectively. The CTX genetic element and toxin-coregulated pilus El Tor (tcpA ET) gene were present in all toxigenic V. cholerae O1 and V. cholerae O139 strains examined in this study. Three of four nontoxigenic V. cholerae O1 strains contained tcpA ET. Interestingly, among the isolates of V. cholerae non-O1/non-O139, two had tcpA Classical, nine contained tcpA El Tor, three showed homology with both biotype genes, and four carried the ctxA gene. The stn/sto genes were present in 28.2% of the non-O1/non-O139 strains, in 10.5% of the toxigenic V. cholerae O1, and in 14.3% of the O139 serogroups. Except for stn/sto genes, all of the other genes studied occurred with high frequency in toxigenic V. cholerae O1 and O139 strains. Based on results of this study, surveillance of non-O1/non-O139 V. cholerae in the aquatic environment, combined with genotype monitoring using ctxA, stn/sto, and tcpA ET genes, could be valuable in human health risk assessment.     

Virulence of pre-CTXphi-carrying Vibrio cholerae: genotypic and phenotypic characteristics

AIM: Complex assessment of virulence of cholera vibrios carrying the truncated CTX element (pre-CTXphi prophage). MATERIALS AND METHODS: Twenty-two strainsof Vibriocholerae O1 and non-O1/non-O139 were studied by PCR and laboratory models. RESULTS: Genomes of all strains, besides pre-CTXphi genes, contained genes hapA (hemagglutinin/proteases), cef (CHO cell elongating factor), rtxA (high-molecular cytotoxin), and rtxC (its activator). Nucleotide sequences of rtxA and vgrG genes from ACD domains, genes VPI and VPI-2 from islands of pathogenicity, mshA (mannose-sensitive pili) gene were presented in different combinations. None strains contained shiga-like toxin (slt1) aswell as thermostable direct (tdh) and thermostable direct-related (trh) hemolysin genes of V. parahaemoliticus. On the model of infant rabbits almost all strains caused a significant enteropathogenic effect sometimes resembling cholera effect and in a number of cases dissemination of bacteria into various organs and tissues took place. Cultural supernatants of the majority of strains stipulated cell rounding in CHO cultures (one of them caused cell destruction) and disconnection of cells in McCoy and L-929 dense monolayers as well as increase of skin permeability in Craig's test. Conclusion. Apparently, diarrhea of different severity observed in patients from whom these strains were isolated as well as signs of virulence revealed in the laboratory models were determined by the expression of genes of accessory pathogenicity factors including those detected in the present study.     

Role of rpoS in Stress Survival and Virulence of Vibrio cholerae

Vibrio cholerae is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded by rpoS, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a V. cholerae rpoS homolog was cloned by functional complementation of an Escherichia coli mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported rpoS gene products. To determine the functional role of rpoS in V. cholerae, we inactivated rpoS by homologous recombination. V. cholerae strains lacking rpoS are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that rpoS may be required for the persistence of V. cholerae in aquatic habitats. In addition, the rpoS mutation led to reduced production or secretion of hemagglutinin/protease. However, rpoS is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.     

Virulence properties of rough and smooth strains of Vibrio cholerae O1

A comparative study was carried out to see the differences in pathogenicity of rough and smooth strains. A total of 10 strains including 5 each of rough and smooth strains of Vibrio cholerae O1 were tested and found positive for toxin production by enzyme-linked immunosorbent assay (ELISA) in Richardson's and AKI media. All the smooth and rough strains, except one, showed a titre of 1: 10 and 1: 100 in Richardson's and AKI media, respectively. Both types of strains produced enterotoxin in rabbit ileal loop (RIL). The differences in multiplication abilities of smooth and rough strains in RIL were statistically significant (P <0.05). However, these differences in multiplying abilities did not influence the adherence potential or enterotoxin production as there was no significant difference (P >0.05) between these properties. This study demonstrated that the rough strains are equally pathogenic and as important as smooth strains.     

The Zymovars of Vibrio cholerae: multilocus enzyme electrophoresis of Vibrio cholerae

Zymovars analysis also known as multilocus enzyme electrophoresis is applied here to investigate the genetic variation of Vibrio cholerae strains and characterise strains or group of strains of medical and epidemiological interest. Fourteen loci were analyzed in 171 strains of non-O1 non-O139, 32 classical and 61 El Tor from America, Africa, Europe and Asia. The mean genetic diversity was 0.339. It is shown that the same O antigen (both O1 and non-O1) may be present in several genetically diverse (different zymovars) strains. Conversely the same zymovar may contain more than one serogroup. It is confirmed that the South American epidemic strain differs from the 7th pandemic El Tor strain in locus LAP (leucyl leucyl aminopeptidase). Here it is shown that this rare allele is present in 1 V. mimicus and 4 non-O1 V. cholerae. Non toxigenic O1 strains from South India epidemic share zymovar 14A with the epidemic El Tor from the 7th pandemic, while another group have diverse zymovars. The sucrose negative epidemic strains isolated in French Guiana and Brazil have the same zymovar of the current American epidemic V. cholerae.     

The Aspergillus fumigatus Dihydroxyacid Dehydratase Ilv3A/IlvC Is Required for Full Virulence

Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC.     

Vibrio cholerae Hemolysin Is Required for Lethality,Developmental Delay,and Intestinal Vacuolation in Caenorhabditis elegans

Background

Cholera toxin (CT) and toxin-co-regulated pili (TCP) are the major virulence factors of Vibrio cholerae O1 and O139 strains that contribute to the pathogenesis of disease during devastating cholera pandemics. However, CT and TCP negative V. cholerae strains are still able to cause severe diarrheal disease in humans through mechanisms that are not well understood.

Methodology/Principal Findings

To determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA) gene as a factor responsible for animal death and developmental delay. We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes. At the cellular level, V. cholerae infection induces formation of vacuoles in the intestinal cells in a hlyA dependent manner, consistent with the previous in vitro observations.

Conclusions/Significance

Our data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes.     

Distribution of Virulence Genes in Clinical and Environmental Vibrio cholerae Strains in Bangladesh

Vibrio cholerae, an environmental organism, is a facultative human pathogen. Here, we report the virulence profiles, comprising 18 genetic markers, of 102 clinical and 692 environmental V. cholerae strains isolated in Bangladesh between March 2004 and January 2006, showing the variability of virulence determinants within the context of public health.