Strains of Vibrio cholerae serogroups O1 and O139 that produce the basic protective antigens

To find out stable and effective producers of major protective antigens intended for use as components of cholera chemical vaccine against V. cholerae strains of serogroups O and O139, the comparative analysis of the production of cholera toxin, toxin-coregulated pili (TCP), antigens O1 and O139, polysaccharide capsule and outer membrane protein OmpU in different V. cholerae strains groups O1 and O139 has been made. V. cholerae strain KM68, serogroup O1, has been found capable of the production of antigen O1, serovar Ogawa, protein OmpU at a sufficiently high level and the hyperproduction of cholera toxin and TCP, and thus suitable for use in the manufacture of cholera bivalent vaccine as the source of these antigens. Specially selected alysogenic noncapsular strain KM137 of serogroup O139, characterized by a high and stable level of the biosynthesis of this somatic antigen when grown in both laboratory and production conditions, may serve as the produces of antigen O139.     

Use of signature-tagged transposon mutagenesis to identify Vibrio cholerae genes critical for colonization

The pathogenesis of cholera begins with colonization of the host intestine by Vibrio cholerae . The toxin co-regulated pilus (TCP), a fimbrial structure produced by V . cholerae , is absolutely required for colonization (i.e. the persistence, survival and growth of V . cholerae in the upper intestinal milieu), but many other aspects of the colonization process are not well understood. In this study, we use signature-tagged transposon mutagenesis (STM) to conduct a screen for random insertion mutations that affect colonization in the suckling mouse model for cholera. Of approximately 1100 mutants screened, five mutants (approximately 0.5%) with transposon insertions in TCP biogenesis genes were isolated, validating the use of STM to identify attenuated mutants. Insertions in lipopolysaccharide, biotin and purine biosynthetic genes were also found to cause colonization defects. Similar results were observed for mutations in homologues of pta and ptfA , two genes involved in phosphate transfer. Finally, our screen identified several novel genes, disruption of which also caused colonization defects in the mouse model. These results demonstrate that STM is a powerful method for isolating colonization-defective mutants of V . cholerae .     

Genome sequencing reveals unique mutations in characteristic metabolic pathways and the transfer of virulence genes between V. mimicus and V. cholerae

Vibrio mimicus, the species most similar to V. cholerae, is a microbe present in the natural environmental and sometimes causes diarrhea and internal infections in humans. It shows similar phenotypes to V. cholerae but differs in some biochemical characteristics. The molecular mechanisms underlying the differences in biochemical metabolism between V. mimicus and V. cholerae are currently unclear. Several V. mimicus isolates have been found that carry cholera toxin genes (ctxAB) and cause cholera-like diarrhea in humans. Here, the genome of the V. mimicus isolate SX-4, which carries an intact CTX element, was sequenced and annotated. Analysis of its genome, together with those of other Vibrio species, revealed extensive differences within the Vibrionaceae. Common mutations in gene clusters involved in three biochemical metabolism pathways that are used for discrimination between V. mimicus and V. cholerae were found in V. mimicus strains. We also constructed detailed genomic structures and evolution maps for the general types of genomic drift associated with pathogenic characters in polysaccharides, CTX elements and toxin co-regulated pilus (TCP) gene clusters. Overall, the whole-genome sequencing of the V. mimicus strain carrying the cholera toxin gene provides detailed information for understanding genomic differences among Vibrio spp. V. mimicus has a large number of diverse gene and nucleotide differences from its nearest neighbor, V. cholerae. The observed mutations in the characteristic metabolism pathways may indicate different adaptations to different niches for these species and may be caused by ancient events in evolution before the divergence of V. cholerae and V. mimicus. Horizontal transfers of virulence-related genes from an uncommon clone of V. cholerae, rather than the seventh pandemic strains, have generated the pathogenic V. mimicus strain carrying cholera toxin genes.     

Method of DNA extraction and application of multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae O1 and O139 from aquatic ecosystems

Vibrio cholerae is a free-living bacterium found in water and in association with plankton. V. cholerae non-O1/non-O139 strains are frequently isolated from aquatic ecosystems worldwide. Less frequently isolated are V. cholerae O1 and V. cholerae O139, the aetiological agents of cholera. These strains have two main virulence-associated factors, cholera toxin (CT) and toxin co-regulated pilus (TCP). By extracting total DNA from aquatic samples, the presence of pathogenic strains can be determined quickly and used to improve a microbiological risk assessment for cholera in coastal areas. Some methods suggested for DNA extraction from water samples are not applicable to all water types. We describe here a method for DNA extraction from coastal water and a multiplex polymerase chain reaction (PCR) for O1 and O139 serogroups. DNA extraction was successfully accomplished from 117 sea water samples collected from coastal areas of Perú, Brazil and the USA. DNA concentration in all samples varied from 20 ng to 480 micro g micro l-1. The sensitivity of the DNA extraction method was 100 V. cholerae cells in 250 ml of water. The specificity of multiplex O1/O139 PCR was investigated by analysing 120 strains of V. cholerae, Vibrio and other Bacteria species. All V. cholerae O1 and O139 tested were positive. For cholera surveillance of aquatic environments and ballast water, total DNA extraction, followed by V. cholerae PCR, and O1/O139 serogroup and tcpA/ctxA genes by multiplex PCR offers an efficient system, permitting risk analysis for cholera in coastal areas.     

Novel type of specialized transduction for CTX phi or its satellite phage RS1 mediated by filamentous phage VGJ phi in Vibrio cholerae

The main virulence factor of Vibrio cholerae, the cholera toxin, is encoded by the ctxAB operon, which is contained in the genome of the lysogenic filamentous phage CTX phi. This phage transmits ctxAB genes between V. cholerae bacterial populations that express toxin-coregulated pilus (TCP), the CTX phi receptor. In investigating new forms of ctxAB transmission, we found that V. cholerae filamentous phage VGJ phi, which uses the mannose-sensitive hemagglutinin (MSHA) pilus as a receptor, transmits CTX phi or its satellite phage RS1 by an efficient and highly specific TCP-independent mechanism. This is a novel type of specialized transduction consisting in the site-specific cointegration of VGJ phi and CTX phi (or RS1) replicative forms to produce a single hybrid molecule, which generates a single-stranded DNA hybrid genome that is packaged into hybrid viral particles designated HybP phi (for the VGJ phi/CTX phi hybrid) and HybRS phi (for the VGJ phi/RS1 hybrid). The hybrid phages replicate by using the VGJ phi replicating functions and use the VGJ phi capsid, retaining the ability to infect via MSHA. The hybrid phages infect most tested strains more efficiently than CTX phi, even under in vitro optimal conditions for TCP expression. Infection and lysogenization with HybP phi revert the V. cholerae live attenuated vaccine strain 1333 to virulence. Our results reinforce that TCP is not indispensable for the acquisition of CTX phi. Thus, we discuss an alternative to the current accepted evolutionary model for the emergence of new toxigenic strains of V. cholerae and the importance of our findings for the development of an environmentally safer live attenuated cholera vaccine.     

Virulence and the environment: a novel role for Vibrio cholerae toxin-coregulated pili in biofilm formation on chitin

The toxin-coregulated pilus (TCP) of Vibrio cholerae is required for intestinal colonization and cholera toxin acquisition. Here we report that TCP mediates bacterial interactions required for biofilm differentiation on chitinaceous surfaces. We also show that undifferentiated TCP- biofilms have reduced ecological fitness and, thus, that chitin colonization may represent an ecological setting outside the host in which selection for a host colonization factor may take place.     

Progress towards development of a cholera subunit vaccine

Cholera, an enteric disease that can reach pandemic proportions, remains a world-wide problem that is positioned to increase in incidence as changes in global climate or armed conflict spawn the conditions that enhance transmission to humans and, thus, precipitate epidemic cholera. An effective subunit cholera vaccine that can provide protective immunity with one parenteral immunization would be a major advantage over the existing oral vaccines that can require two doses for optimal protection. The existing vaccines are clearly effective in some settings, but are less so in others, especially with respect to specific groups such as young (2-5 years) children. In our efforts to develop a cholera subunit vaccine, we focused on two Vibrio cholerae antigens, LPS (lipopolysaccharide) and TCP (toxin co-regulated pilus), that are known to induce protective antibodies in animal models and, in the case of anti-LPS antibodies, to be associated with clinical protection of V. cholerae exposed or vaccinated individuals. This review discusses the current cholera vaccines and compares the advantages of a cholera subunit vaccine to that of the whole cell vaccines. We discuss the possible subunit antigens and prospective targeted use of a subunit cholera vaccine.     

Filamentous phages linked to virulence of Vibrio cholerae

The pathogenicity of Vibrio cholerae depends upon its production of two key virulence factors: the toxin co-regulated pilus (TCP), a colonization factor, and cholera toxin, an exotoxin. Genes encoding both virulence factors were introduced into V. cholerae by horizontal gene transfer. The toxin genes are contained within the genome of CTXphi, an integrated filamentous phage identified in 1996. In the past few years, it has been shown that CTXphi relies on novel processes for phage DNA integration, replication and secretion. In addition, expression of CTXphi genes--including the toxin genes--and transmission of CTXphi were recently found to be promoted by the antirepressor RstC, which is encoded within RS1, a newly described satellite phage of CTXphi. The genetic island that encodes TCP has also been described as a filamentous phage; however, these sequences are unlike the genome of any previously characterized filamentous phage.     

Characterization and immunogenicity of Vibrio cholerae ghosts expressing toxin-coregulated pili

Bacterial ghosts are attractive for use as non-living vaccines and as carriers of heterologous antigens of vaccine relevance. Ghosts were prepared from Vibrio cholerae strains of O1 or O139 serogroup after growth under culture conditions, which favor or repress the production of toxin-coregulated pili (TCP). Immunoblotting confirmed the TCP status of these V. cholerae ghosts (VCG), which retained the cellular morphology and envelope sub-component profile of viable bacteria. Rabbits were immunized with VCGs prepared from O139 bacteria with TCP-positive or TCP-negative phenotypes and the resulting sera assayed for antibodies to lipopolysaccharide (LPS) and to TCP. Regardless of the TCP status of the VCG preparations used for immunization, all animals produced antibodies to LPS as demonstrated in bactericidal assays. These antibodies were probably responsible for the capacity of the antisera to confer passive immunity to challenge with the homologous O139 strain in the infant mouse cholera model (IMCM). Only following immunization with TCP-positive VCG, however, were antibodies to TCP generated, as judged by the potential of antisera to mediate protection against a challenge strain of heterologous serogroup.     

pIIICTX,a predicted CTXphi minor coat protein,can expand the host range of coliphage fd to include Vibrio cholerae

CTXphi is a filamentous bacteriophage that encodes cholera toxin. CTXphi infection of its host bacterium, Vibrio cholerae, requires the toxin-coregulated pilus (TCP) and the products of the V. cholerae tolQRA genes. Here, we have explored the role of OrfU, a predicted CTXphi minor coat protein, in CTXphi infection. Prior to the discovery that it was part of a prophage, orfU was initially described as an open reading frame of unknown function that lacked similarity to known protein sequences. Based on its size and position in the CTXphi genome, we hypothesized that OrfU may function in a manner similar to that of the coliphage fd protein pIII and mediate CTXphi infection as well as playing a role in CTXphi assembly and release. Deletion of orfU from CTXphi dramatically reduced the number of CTXphi virions detected in supernatants from CTXphi-bearing cells. This defect was complemented by expression of orfU in trans, thereby confirming a role for this gene in CTXphi assembly and/or release. To evaluate the requirement for OrfU in CTXphi infection, we introduced fragments of orfU into gIII in an fd derivative to create OrfU-pIII fusions. While fd is ordinarily unable to infect V. cholerae, an fd phage displaying the N-terminal 274 amino acids of OrfU could infect V. cholerae in a TCP- and TolA-dependent fashion. Since our findings indicate that OrfU functions as the CTXphi pIII, we propose to rename OrfU as pIII(CTX). Our data also provide new evidence for a conserved pathway for filamentous phage infection.     

CTXphi infection of Vibrio cholerae requires the tolQRA gene products

CTXphi is a lysogenic filamentous bacteriophage that encodes cholera toxin. Filamentous phages that infect Escherichia coli require both a pilus and the products of tolQRA in order to enter host cells. We have previously shown that toxin-coregulated pilus (TCP), a type IV pilus that is an essential Vibrio cholerae intestinal colonization factor, serves as a receptor for CTXphi. To test whether CTXphi also depends upon tol gene products to infect V. cholerae, we identified and inactivated the V. cholerae tolQRAB orthologues. The predicted amino acid sequences of V. cholerae TolQ, TolR, TolA, and TolB showed significant similarity to the corresponding E. coli sequences. V. cholerae strains with insertion mutations in tolQ, tolR, or tolA were reduced in their efficiency of CTXphi uptake by 4 orders of magnitude, whereas a strain with an insertion mutation in tolB showed no reduction in CTXphi entry. We could detect CTXphi infection of TCP(-) V. cholerae, albeit at very low frequencies. However, strains with mutations in both tcpA and either tolQ, tolR, or tolA were completely resistant to CTXphi infection. Thus, CTXphi, like the E. coli filamentous phages, uses both a pilus and TolQRA to enter its host. This suggests that the pathway for filamentous phage entry into cells is conserved between host bacterial species.     

Characterization of Novel Alleles of Toxin Co-Regulated Pilus A Gene (tcpA) from Environmental Isolates of Vibrio cholerae

Vibrio cholerae is causative agent of life threatening diarrheal disease, cholera. The toxin co-regulated pilus (TCP) is a critical colonization factor of V. cholerae and it also serves as receptor for CTXФ. In this study, we describe nucleotide sequence of four novel alleles of tcpA gene from toxigenic and non-toxigenic V. cholerae isolated from environmental sources. The phylogenetic analysis of tcpA revealed that it is related to tcpA of newly emerged O1 strain and unrelated to tcpA of wild type (classical and El Tor strains). All strains showed variant tcpA and also harbored intact Vibrio Pathogenicity Island (VPI). The expression of all variant alleles was demonstrated by RT-PCR.     

Temperate phage induction and filament formation in Vibrio cholerae by furazolidone

Furazolidone a synthetic nitrofuran used in the treatment of cholera, was found to cause prophage induction and filament formation in Vibrio cholerae. Maximum induction of phage beta was obtained at a drug concentration of 0.5 microgram/ml. Neither induction of prophage nor filamentation took place if the drug treatment was carried out in the presence of 2.5 micrograms/ml chloramphenicol, indicating a requirement for de novo protein synthesis. Our results strongly suggest the existence of "SOS" type functions in Vibrio cholerae.