O1 El Tor Strains Research Articles

Identification of a vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage.

We identify and characterize a gene cluster in El Tor Vibrio cholerae that encodes a cytotoxic activity for HEp-2 cells in vitro. This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the V. cholerae genome. We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity. The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif. Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD. In V. cholerae strains of the classical biotype, a deletion within the gene cluster removes rtxC and eliminates cytotoxic activity. Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity. Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence. Furthermore, the RTX toxin of V. cholerae may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines.

Resurgent Vibrio cholerae O139: rearrangement of cholera toxin genetic elements and amplification of rrn operon.

The unprecedented genesis of a novel non-O1 Vibrio cholerae strain belonging to serogroup O139, which caused an epidemic in late 1992 in the Indian subcontinent, and its subsequent displacement by El Tor O1 vibrios after 18 months initiated a renewed investigation of the aspects of the organism that are related to pathogenesis. The reappearance of V. cholerae O139 with altered antibiotic sensitivity compared to O139 Bengal (O139B) in late 1996 has complicated the epidemiological scenario of V. cholerae and has necessitated an examination of possible rearrangements in the genome underlying such rapid changes in the phenotypic traits. With a view to investigating whether the phenotypic changes that have occurred are associated with alteration in the genome, the genome of the resurgent V. cholerae O139 (O139R) strains were examined. Pulsed-field gel electrophoresis analysis of NotI- and SfiI-digested genomic DNA of O139R isolates showed restriction fragment length polymorphism including in the cholera toxin (CTX) genetic element locus and with O139B isolates. Analyses of the organization of the CTX genetic elements in O139R strains showed that in contrast to two copies of the elements connected by two direct-repeat sequences (RS) in most of the genomes of O139B isolates, the genomes of all O139R strains examined, except strain AS192, have three such elements connected by a single RS. While the RS present in the upstream of the CTX genetic elements in the genome of O139R is of O139B origin, the RS connecting the cores of the elements has several new restriction sites and has lost the BglII site which is supposed to be conserved in all O1 strains and O139B. The endonuclease I-CeuI, which has sites only in the rrn operons in the genomes of all organisms examined so far, has 10 sites in the genomes of O139R strains, compared to 9 in the genomes of O139B strains. The recent isolates of V. cholerae O139 have thus gained one rrn operon. This variation in the number of rrn operons within a serogroup has not been reported for any other organism. The results presented in this report suggest that like the pathogenic El Tor O1 strains, the genomes of O139 strains are undergoing rapid alterations.

The tcp gene cluster of Vibrio cholerae.

The toxin co-regulated pilus (TCP) has been identified as a critical colonization factor in both animal models and humans for Vibrio cholerae O1. The major pilin subunit, TcpA (and also TcpB), is similar to type-4 pilins but TCP probably more appropriately belongs to a sub-class which includes the bundle-forming pilus of enteropathogenic Escherichia coli. The genes for TCP biosynthesis and assembly are clustered with the exception of housekeeping functions such as TcpG (=DsbA, a periplasmic disulfide bond epimerase). The nt sequences from El Tor and classical strains show only minor differences corresponding to the major regulatory regions and in TcpA itself. These differences are thought to account for the alternate conditions required for expression of TCP by the two biotypes and the antigenic variation and lack of cross-protection. Aside from the TcpA only a few of the proteins have had their roles in TCP biogenesis defined. Regulation of TCP is controlled by the ToxR regulon via ToxT with a possible involvement of TcpP and the cAMP-CRP system. Experiments using the infant mouse cholera model have now shown that TCP is a colonization factor and protective antigen for both classical and El Tor O1 strains and in the O139 Bengal serotype and that the mannose-sensitive haemagglutinin pilus does not appear to play a comparable role.

Molecular Characterization ofVibrio choleraeO1 Biotype El Tor Strains Isolated between 1992 and 1995 in Calcutta, India: Evidence for the Emergence of a New Clone of the El Tor Biotype

Sixty-one clinical strains of Vibrio cholerae O1 El Tor isolated in Calcutta before, during, and after the V. cholerae O139 Bengal outbreak were examined to see if the O1 strains of the post-O139 period were different from those in existence before. Comparison of the restriction fragment length polymorphism of the rRNA genes (ribotyping) and the CTX genetic element revealed that all "before" strains except 1 belonged to a single known ribotype, whereas all "after" strains except 2 belonged to a hitherto undescribed ribotype. Also, 23 of 25 "before" strains harbored two or more copies of CTX in tandem and also a "free" RS1 element away from CTX, whereas 19 of 21 "after" strains had a single copy of CTX and no free RS1 element. CTX occupied different chromosomal locations in "before" and "after" strains. These studies clearly showed that El Tor O1 strains, which displaced V. cholerae O139 in Calcutta, belonged to a new clone and suggested that there is a continuous genetic reassortment among El Tor strains of V. cholerae O1.

Characterization of Vibrio cholerae bacteriophage K139 and use of a novel mini-transposon to identify a phage-encoded virulence factor.

Temperate bacteriophage K139 was isolated from a Vibrio cholerae O139 isolate and characterized in this study. The phage genome consists of a 35 kbp, double-stranded, linear DNA molecule that circularizes and integrates into the chromosome in a site-specific manner. DNA sequences that cross-hybridize with K139 phage DNA are present in all strains of V. cholerae serogroup O1 of the classical biotype examined and in some strains of the El Tor biotype. Phage K139 produces plaques on El Tor O1 strains that do not carry the K139-related sequences but does not plaque on O139 strains that lack detectable phage DNA. This results suggests that O139 strains arose in part by horizontal gene transfer of the O139 antigen genes into an El Tor O1 strain that harboured a K139 prophage. Consistent with this interpretation, the morphology of K139 phage particles is identical to that displayed by the widely distributed family of O1 phages referred to as 'kappa'. In order to test whether K139 phage is involved in lysogenic conversion of V. cholerae, we constructed a novel mini-transposon, Tn10d-bla, which was designed to produce beta-lactamase fusions to phage-encoded, exported proteins. All Tn10d-bla insertions obtained were closely linked to one location on the K139 phage genome. DNA sequence determination of the fusion joints revealed an open reading frame (ORF1), encoding a gene product of 137 amino acids with a typical N-terminal hydrophobic signal sequence. ORF1 was designated the glo gene (G protein-like ORF) because its amino acid sequence shows similarity to eukaryotic Gs(alpha) protein (34.5% identity over an 81-amino-acid overlap) and its C-terminus displays the consensus motif (CAAX) which is found in many small eukaryotic GTP-binding proteins. LD50 assays with isogenic Glo+ and Glo- K139 lysogens suggest that glo encodes a secreted virulence determinant of V. cholerae.

The Vibrio cholerae O139 serogroup antigen includes an O-antigen capsule and lipopolysaccharide virulence determinants.

Vibrio cholerae serogroup O139 emerged on the Indian subcontinent in October 1992 to become the first non-O1 V. cholerae serogroup documented to cause epidemic cholera. Although related to V. cholerae El Tor O1 strains, O139 strains have unique surface structures that include a capsular surface layer and lipopolysaccharide (LPS). Immunoblot analysis of either whole-cell lysates or LPS preparations revealed three electrophoretic forms of the O139 antigen: two slowly migrating forms and one rapidly migrating form that appeared identical to O139 LPS. All three forms of the antigen shared an epitope defined by an O139-specific monoclonal antibody. A serum-sensitive nonencapsulated mutant was isolated that lacks only the slow migrating forms. The slow migrating forms did not stain with silver whereas the rapidly migrating form did, suggesting that the former might constitute highly polymerized O-antigen side-chain molecules that were not covalently bound to core polysaccharide and lipid A (an "O-antigen capsule"). A single transposon insertion resulted in the loss of immunoreactivity of both the LPS and the O-antigen capsule, implying that there are genes common to the biosynthesis of both these macromolecules. The O139 LPS and O-antigen capsule were both important for colonization of the small intestine of the newborn mouse and for serum resistance, demonstrating that both of these forms of the O139 serogroup antigen are virulence factors.

Characterization of phenotypic, serological, and toxigenic traits of Vibrio cholerae O139 bengal

Biochemical and physiological traits of a collection of strains of Vibrio cholerae O139 Bengal isolated from India, Bangladesh, and Thailand showed that these strains formed a phenotypically homogeneous group with identical characteristics that were essentially similar to those of the O1 serogroup. Resistance to 150 micrograms of the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine) and Mukherjee's El Tor phage 5 and classical phage IV and the nonagglutinability of the strains with O1 antiserum were the only discernible differences between the O139 and O1 serogroups. Extensive serological characterization further revealed the O139 serogroup to be distinct from the existing 138 serogroups of V. cholerae. Antiserum raised against the O139 serogroup required absorption with the R reference strain CA385 and with the reference strain representing serogroup O22 to remove cross-reacting agglutinins. All of the 223 representative strains of V. cholerae O139 examined hybridized with DNA probes specific for the cholera toxin (CT) gene, zonula occludens toxin gene, and El Tor hemolysin gene but not with the probe specific for the heat-stable enterotoxin gene. The amount of CT present in stool samples of patients infected with the O139 serogroup was higher than that found in stools of patients infected with O1 El Tor, and this echoed findings that the amount of CT produced by O139 strains in vitro was higher than that produced by the O1 El Tor strains. The nucleotide sequences of the genes encoding the A and B subunits of CT of the O139 serogroup were identical to the sequences reported for the CT gene of O1 El Tor. The CT gene of O139 strains could be amplified by using primers developed for detection of the CT gene of the O1 serogroup by a PCR assay, which could also be used to detect the CT gene in stool samples of patients infected with strains of the O139 serogroup.

Emergence of a New Cholera Pandemic: Molecular Analysis of Virulence Determinants in Vibrio cholerae 0139 and Development of a Live Vaccine Prototype

In October 1992, a non-O1 strain of Vibrio cholerae emerged as a cause of epidemic cholera in India and Bangladesh. This antigenically novel clone has been designated serogroup O139 synonym Bengal. Since its emergence, V. cholerae O139 has caused a massive cholera epidemic throughout and beyond the Indian subcontinent. Molecular analysis of virulence determinants in clinical isolates suggests that O139 strains are highly related to El Tor O1 strains. Unlike other non-O1 strains, O139 strains carry multiple copies of the cholera toxin genetic element and also genes for the toxin-coregulated pilus. These results guided construction of a live V. cholerae O139 vaccine prototype through deletion of genes for at least four specific virulence determinants (ctxA, ace, zot, and cep) as well as other factors involved in site-specific and homologous recombination (RS1, attRS1, and recA). It is hoped that this attenuated live vaccine will help control the pandemic spread of V. cholerae O139.

ToxR regulates virulence gene expression in non-O1 strains of Vibrio cholerae that cause epidemic cholera

Vibrio cholerae serogroup O1 has historically been thought to be the exclusive cause of epidemic cholera. O139 is a novel serogroup of V. cholerae which emerged on the Indian subcontinent in the last few months of 1992 and is the first non-O1 serogroup of V. cholerae to cause epidemic cholera. We have investigated the expression of some of the known virulence factors of classical and El Tor O1 strains of V. cholerae in clinical isolates of O139 strains. We show that, in contrast to other non-O1 strains, O139 strains express TcpA, the major subunit of the toxin-coregulated pilus found in O1 strains. As in O1 strains, the expression of cholera toxin and TcpA is coordinately regulated by environmental parameters in O139 strains. Derivatives of O139 strains that contain a toxR null mutation were constructed and used to demonstrate that the expression of cholera toxin, TcpA, and the outer membrane protein OmpU in O139 strains, as in O1 strains, is dependent on ToxR. Two kinds of evidence suggest that O139 strains are closely related to El Tor strains of V. cholerae. First, both O139 and El Tor strains share a restriction fragment length polymorphism for tcpA, which distinguishes El Tor from classical strains of V. cholerae. Second, cholera toxin production in O139 strains is greatly enhanced by culture conditions that have been previously shown to promote production of cholera toxin in El Tor strains and not in classical strains of V. cholerae. Although O139 is a novel serotype of V. cholerae, O139 strains conform to a fundamental theme that has evolved from the study of O1 strains: ToxR mediates coordinate regulation of virulence gene expression.

Two-step processing for activation of the cytolysin/hemolysin of Vibrio cholerae O1 biotype El Tor: nucleotide sequence of the structural gene (hlyA) and characterization of the processed products

Vibrio cholerae O1 biotype El Tor produces and secretes a 65-kDa cytolysin/hemolysin into the culture medium. We cloned the structural gene (hlyA) for the cytolysin from the total DNA of a V. cholerae O1 El Tor strain, N86. Nucleotide sequence analysis of hlyA revealed an open reading frame consisting of 2,223 bp which can code for a protein of 741 amino acids with a molecular weight of 81,961. Consistent with this, a 79-kDa protein was identified as the product of hlyA by maxicell analysis in Escherichia coli. N-terminal amino acids of this 79-kDa HlyA protein and those of a 65-kDa El Tor cytolysin purified from V. cholerae were Asn-26 and Asn-158, respectively. The 82- and 79-kDa precursors of the 65-kDa mature cytolysin were found in V. cholerae by pulse-chase labeling and Western blot (immunoblot) analysis of hlyA products. Hemolytic activity of the 79-kDa HlyA protein from E. coli was less than 5% that for the 65-kDa cytolysin from V. cholerae. Our results suggest that in V. cholerae, the 82-kDa preprotoxin synthesized in the cytoplasm is secreted through the membranes into the culture medium as the 79-kDa inactive protoxin after cleavage of the signal peptide and is then further processed into the 65-kDa active cytolysin by release of the N-terminal 15-kDa fragment.

Haemolysin genes ofVibrio cholerae: presence of homologous DNA in non-haemolytic O1 and haemolytic non-O1 strains

The structural gene for the haemolysin and two accessory genes from a Vibrio cholerae O1 El Tor strain have previously been cloned in Escherichia coli K-12 to give the plasmid pPM431. This plasmid has been used as a probe with a variety of O1 and non-O1 Vibrio cholerae strains to examine by Southern DNA hybridisations for the presence of homologous DNA. Such experiments show that the DNA homologous to that present in pPM431 is present in all of the 20 strains examined, whether they were haemolytic or non-haemolytic, implying that the genes were present but not expressed in non-haemolytic strains. Using a variety of restriction enzymes to cut the chromosomal DNA of different V. cholerae strains and probing with pPM431, it was possible to distinguish O1 and non-O1 strains, as well as haemolytic or non-haemolytic strains. This variability between hly+ and hly− may be indicative of a change in the regulatory region of the haemolysin genes. The results also imply a high degree of homology of the haemolysin of O1 and non-O1 strains.