El Tor Strains Research Articles

RecA mutations reduce adherence and colonization by classical and El Tor strains of Vibrio cholerae

Two recA mutants of Vibrio cholerae (classical and El Tor biotypes) were constructed by disruption of the wild-type recA gene with mutated recA sequences of V. cholerae cloned in the suicide vector pGP704. Mutants defective in the recA gene were compared with their respective RecA+ parent strains with regard to their adherence to isolated rabbit intestine and colonization of intestine of infant mice. The recA mutation in V. cholerae was found to diminish adherence and markedly affected colonization.

Molecular analysis of rRNA and cholera toxin genes carried by the new epidemic strain of toxigenic Vibrio cholerae O139 synonym Bengal.

Vibrio cholerae O139 synonym Bengal recently caused large epidemics of cholera-like disease in Bangladesh and India. We compared the restriction fragment length polymorphisms of ctxA and rRNA genes (ribotypes) in 27 isolates of V. cholerae O139 from patients in Bangladesh and India with those of 48 isolates of V. cholerae O1 from patients and 21 V. cholerae isolates from surface waters in Bangladesh, which included 2 O139 and 19 other non-O1 isolates. Ribotyping of the isolates with BglI revealed that all 29 isolates of O139 vibrios belonged to a single ribotype, suggesting a clonal nature of the infection. However, the O139 vibrios comprised two ctxA genotypes and carried three or more copies of the ctxA gene, and the chromosomal locations of these copies were unlike those of the El Tor or classical vibrios. Analysis of the restriction fragment length polymorphisms of the rRNA genes suggested that V. cholerae O139 isolates are more closely related to El Tor strains of V. cholerae O1 than were 19 other non-O1 vibrios and 33 classical V. cholerae O1 isolates that were studied. However, further studies are needed to determine whether V. cholerae O139 originated from mutations and genetic changes in a V. cholerae O1 strain or was due to the acquisition of virulence genes by a previously unknown V. cholerae non-O1 strain.

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.

Comparative efficacy of biodegradable liposomes and microspheres as carriers for delivery of Vibrio cholerae antigens in the intestine

The effect of the encapsulated antigens of Vibrio cholerae and their route of administration in induction of immune response was studied in experimental cholera. The antigenic proteins of V. cholerae El Tor strain KB207 were obtained by fractionation of cell-free lysate by high-performance liquid chromatography. The antigenic proteins were pooled and encapsulated in biodegradable liposomes and poly( d,l) lactic co-glycolic acid microspheres. Rabbits were immunized with free as well as encapsulated antigens by different routes. Liposome-encapsulated antigens delivered intraintestinally offered maximum protection. Orally or intraintestinally delivered antigens in microspheres failed to elicit a significant immune response, although as a carrier microspheres were comparable to liposomes when judged by the subcutaneous route. The results suggested that liposomes and microspheres could be used as carriers of protective antigens of V. cholerae for effective immunization.

Modified CAMP test for biogrouping Vibrio cholerae O1 strains and distinguishing them from strains of V. cholerae non-O1

A modified CAMP test was used to identify 973 Vibrio cholerae isolates by phenotype. Eltor and non-O1 strains were CAMP positive; classical strains were CAMP negative. Sausage-shaped zones of hemolysis of eltor strains were easily distinguished from narrower bands of non-O1 isolates. For O1 isolates, there was 100% agreement between the CAMP test and inhibition by polymyxin B.

The role of toxin-coregulated pili in the pathogenesis of Vibrio cholerae O1 El Tor

Studies in the infant mouse cholera model have evaluated the significance of toxin-coregulated pili (TCP) in the pathogenesis of Vibrio cholerae strains of El Tor biotype. Four El Tor strains—two which produce TCP during in vitro growth and two which do not—were mutated by the insertion of an antibiotic-resistance cartridge into the tcpA gene (encoding the pilin monomer). The resulting mutants were otherwise indistinguishable from wild-type and in particular were unaltered in their sensitivity to antibody-dependent, complement-mediated bacteriolysis. All were dramatically attenuated and showed a marked impairment in terms of in vivo persistence in mixed-infection competition experiments. Virulence was restored by provision of a functional tcp operon in trans, confirming that the pathogenic potential of El Tor strains is critically dependent upon product(s) of this operon.

In vitro production of toxin-coregulated pili by Vibrio cholerae El Tor

Toxin-coregulated pili (TCP) have been shown to be a virulence determinant and protective antigen for Vibrio cholerae strains of classical biotype, but their role in infection by strains of the alternative El Tor biotype remains uncertain. In an attempt to demonstrate TCP production by El Tor vibrios we have over-expressed the El Tor tcpA gene in Escherichia coli, in order to prepare a biotype-specific anti-TcpA serum. This reagent proved to be a very sensitive indicator of TcpA production in immunoblotting studies, but failed to detect polymerized pili on the bacterial surface by immuno-electron microscopy (IEM). However, results with an analogous reagent which detects classical TcpA suggested that antisera to unprocessed TcpA do not efficiently recognize epitopes on native proteins. Accordingly we prepared a serum against a cell envelope fraction rich in processed El Tor TcpA. After extensive absorption this reagent reacted almost exclusively with TcpA by immunoblotting; when used in IEM, it allowed visualization of typical TCP bundles on the surfaces of each of five El Tor strains known to produce TcpA in vitro .We have previously reported that the El Tor strain O17 does not synthesize TcpA during growth in vitro, but that an O17 clone carrying a cosmid of classical origin expresses surface TCP. Using the biotype-specific anti-TcpA reagents in immunoblotting studies it has been possible to detect the product of the host chromosomal tcpA gene in such constructs; transcription of this gene was confirmed using biotype-specific tcpA probes. IEM revealed that El Tor TcpA was present in the TCP bundles associated with the O17 cosmid clones. Further studies suggest that regulation of the genes encoding TcpA and cholera toxin varies between different strains of El Tor biotype.

New phage typing scheme for Vibrio cholerae O1 biotype El Tor strains.

The conventional phage typing scheme proposed by S. Basu and S. Mukerjee (Experientia 24:299-300, 1968) has been used routinely for identification of the strains at the Vibrio Phage Reference Laboratory since 1968. However, because of limitations of this scheme, a new phage typing scheme using five newly isolated phages was incorporated into the conventional scheme. A different definition of routine test dilution (almost confluent lysis) was found to be more useful than the one previously used (confluent lysis). The 1,000 strains tested could be clustered into 27 types with the five new phages. With the new scheme of 10 phages (5 new phages and 5 phages of Basu and Mukerjee), the 1,000 strains could be grouped into 146 types. The new phages were different from each other and also from those of Basu and Mukerjee, as revealed by lytic pattern, electron microscopy, restriction endonuclease digestion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antiphage antiserum studies. With the new typing scheme, 99.6% of the strains were typeable. Phage type 115 was the most common and includes 119 (11.9%) of the 1,000 strains tested. Next most common were phage types 142 (9.4%), 143 (7.0%), 104 and 116 (both 5.4%), 3 (5.3%), 5 (4.1%), 4 (3.9%), 24 (2.1%), and 100 (1.7%). The larger number of types would be useful for further classification of the strains for epidemiological purposes. This newly developed scheme is highly applicable to, and could be widely adopted for, phage typing of Vibrio cholerae O1 biotype El Tor strains.

Induction of SOS like responses by nitrofurantoin in Vibrio cholerae el tor cells

Treatment of Vibrio cholerae el tor strain SLH22(J) with nitrofurantoin induced dose-dependent prophage 'kappa', the maximum induction being 6-fold the spontaneous induction level. UV-inactivated 'kappa' phages were Weigle reactivated, the maximum Weigle factor being 1.8 and 2.0 respectively in nitrofurantoin and UV pretreated el tor strain H218 Smr. Nitrofurantoin treatment also caused significant filamentation of the el tor strain H218 Smr and mutation of these cells from ampicillin sensitivity to ampicillin resistance. The levels of the four SOS-like responses induced by this drug were low but significant.

Biophysical characterization of Vibrio El Tor typing phage e5.

Vibrio cholerae typing phage e5, which can lyse only the El Tor strains of V. cholerae, was characterized. The phage had a polyhedral head 51 nm in diameter and a short tail 13 nm in length. It contained 13 structural polypeptides, with the molecular mass of the major component being 50 kDa. Phage chromosome comprised a 38.5-kb linear double-stranded DNA molecule with unique termini, as determined by restriction fragment analysis and electron microscopy, and had a G+C content of 35.5%. A physical map was constructed with the restriction endonucleases HaeII and HpaII. Adsorption of the phage to its host followed a biphasic kinetics and its intracellular growth was characterized by a latent period of 15 min and a burst size of 100 particles per infected cell. The phage was found to be moderately thermotolerant.

Analysis of expression of toxin-coregulated pili in classical and El Tor Vibrio cholerae O1 in vitro and in vivo.

The expression of toxin-coregulated pili (TCP) and their structural subunit TcpA was compared in 20 strains of Vibrio cholerae of the classical and El Tor biotypes. Bacteria were isolated from the intestines of rabbits with experimental cholera and compared with the same strains grown under optimal TCP expression conditions in vitro. Immunoblotting revealed that TcpA production was induced in both biotypes after vibrios entered the intestinal milieu; TcpA-negative inocula gave rise to TcpA-positive vibrios after multiplication in the gut. The levels of TcpA expressed during growth in the intestine were, for most strains, comparable to those attained under optimal growth conditions in vitro. Of 11 classical strains tested, 10 expressed TCP antigen on the bacterial surface at levels comparable to or exceeding those seen after growth in vitro as determined by an inhibition enzyme-linked immunosorbent assay. In contrast, only one of the nine El Tor strains studied produced detectable amounts of TCP surface antigen in vivo and no fimbriae or surface antigen reacting with anti-TCP serum was found on El Tor vibrios from human cholera stools. Distinct TCP fimbriae were observed by immunoelectron microscopy on classical-biotype vibrios grown either in rabbit intestines or in vitro but were not detected on El Tor vibrios. The results show that TCP is expressed on V. cholerae O1 of the classical biotype but not on V. cholerae O1 of the El Tor biotype in the intestines of rabbits with experimental cholera infection.

Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins.

IrgA is an iron-regulated virulence factor for infection in an animal model with classical Vibrio cholerae strain 0395. We detected gene sequences hybridizing to irgA at high stringency in clinical isolates in addition to 0395, including another classical strain of V. cholerae, three V. cholerae strains of the El Tor biotype, three non-O1 isolates of V. cholerae, and individual isolates of Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus. No hybridization to irgA was seen with chromosomal DNA from Vibrio vulnificus or Aeromonas hydrophila. To verify that irgA is the structural gene for the major iron-regulated outer membrane protein of V. cholerae, we determined the amino-terminal sequence of this protein recovered after gel electrophoresis and demonstrated that it corresponds to the amino acid sequence of IrgA deduced from the nucleotide sequence. Gel electrophoresis showed that two El Tor strains of V. cholerae had a major iron-regulated outer membrane protein identical in size and appearance to IrgA in strain 0395, consistent with the findings of DNA hybridization. We have previously suggested that IrgA might be the outer membrane receptor for the V. cholerae siderophore, vibriobactin. Biological data presented here, however, show that a mutation in irgA had no effect on the transport of vibriobactin and produced no defect in the utilization of iron from ferrichrome, ferric citrate, haemin or haemoglobin. The complete deduced amino acid sequence of IrgA demonstrated homology to the entire class of Escherichia coli TonB-dependent proteins, particularly Cir. Unlike the situation with Cir, however, we were unable to demonstrate a role for IrgA as a receptor for catechol-substituted cephalosporins. The role of IrgA in the pathogenesis of V. cholerae infection, its function as an outer membrane receptor, and its potential interaction with a TonB-like protein in V. cholerae remain to be determined.

Epitope differences in toxin-coregulated pili produced by classical and El Tor Vibrio cholerae O1

A toxin-coregulated pilus (TCP), that is important for intestinal colonization of Vibrio cholerae O1, may be produced by vibrios of both classical and El Tor biotypes. By comparing TCP produced by various strains of the two biotypes in immunoblotting and enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies (mAbs) and polyclonal antisera against TCP from classical vibrios, we have found biotype-related epitope differences in TCP. Our results indicate that TCP of classical strains has an epitope in the TcpA-subunit (20.5 kDa) that is missing in El Tor TcpA, and an additional epitope that is more strongly expressed in classical TcpA. A polyclonal antiserum reacted strongly with TcpA from strains of both biotypes in immunoblotting suggesting both the presence of major shared TcpA epitopes and that the low or absent reactivity of El Tor TcpA with the mAbs was not due to lower production of TcpA by El Tor strains. Whereas all the TcpA-positive classical strains inhibited the binding of polyclonal antiserum and mAbs to solid phase-bound TCP-positive bacteria in an inhibition ELISA, practically no inhibition was observed with TcpA-positive El Tor strains. This together with findings in immunoelectron microscopy studies that TCP ‘bundles’ were only detected on classical strains, suggest that TCP is poorly expressed on El Tor vibrios.

Roles of motility and flagellar structure in pathogenicity of Vibrio cholerae: analysis of motility mutants in three animal models

Wild-type Vibrio cholerae of both El Tor and classical biotypes (strains N16961 and 395, respectively) and nonmotile mutant derivatives with and without flagellar structures were characterized in three different animal models: (i) the rabbit ileal loop, (ii) the removable intestinal tie adult rabbit diarrhea (RITARD) model, and (iii) the suckling mouse model. Both the wild-type strains and nonmotile mutants were toxinogenic in the rabbit ileal loop and the suckling mouse models. However, all of the nonmotile mutants produced significantly less fluid accumulation than did the wild-type parental strains. The two nonmotile mutants of strain N16961 did not adhere to rabbit ileal mucosa, but both nonmotile mutants derived from strain 395 exhibited adherence. In the RITARD model, the motile El Tor strains were more virulent than both the flagellate and aflagellate nonmotile mutants (all infected rabbits died within 18 h), while the nonmotile mutants, when fatalities occurred, required 78 to 105 h to produce a fatal outcome. Likewise, the motile classical parent 395 produced a fatal outcome within ca. 25 h, while nonmotile mutants required 69 to 96 h. The nonmotile flagellate strain KR31 was not significantly more virulent than the nonmotile aflagellate strain KR26. Of the two classical nonmotile mutants, KR1, which produces a coreless sheathlike structure, was clearly more virulent (5 of 10 rabbits died within 96 h), while KR3 (nonmotile, aflagellate) did not produce fatalities in any of the 10 rabbits tested. Similarly, no significant difference in diarrheagenicity or colonizing ability was detected between the two nonmotile mutants derived from the El Tor strain, but the classical nonmotile mutant with the coreless sheath caused significantly greater diarrhea and colonized for a longer time than did the isogenic nonmotile aflagellate strain, KR3. No significant differences between the nonmotile mutants were detected in competition studies done with suckling mice. Analysis of the wild-type and mutant strains in these three animal models clearly demonstrated a role for motility in V. cholerae pathogenicity, while analysis of only the nonmotile mutants derived from the classical parent suggested a role for flagellar structures.

Identification of Vibrio cholerae by enzyme electrophoresis

Zymovar analysis of 260 strains of Vibrio cholerae plus 3 reference strains of V. mimicus, using 13 structural loci, led to the grouping of strains in 73 zymovars (strain or group of strains sharing the same alleles). Effective separation of strains, distinction of V. cholerae strains from closely related V. mimicus and the detection of 2 vibrio strains, including one with two O1 serovars, in supposedly pure collection cultures, illustrate the potential of zymovar analysis in the identification of V. cholerae isolates. Two El Tor strains from USA, one CT+ and the other CT−, shared the same zymovar 71, while 127 typical El Tor strains belonged to zymovar 14.

Toxin Production by Atypical Strains of Vibrio cholerae El Tor Under Different Cultural Conditions

It was observed that at 37 degrees C under in vitro conditions, aerobic culture filtrates of a few strains of Vibrio cholerae biotype El Tor isolated from diarrhoeal cases produced a minute amount of toxin which failed to elicit a positive ileal loop reaction like toxigenic strains. Thus, these strains showed an atypical behaviour in their toxin producing ability. At 25 degrees C and 30 degrees C under aerobic cultural conditions enhanced toxin production was noticed in toxigenic strains, but these temperatures did not affect the toxigenicity of the atypical strains. The atypical Vibrio cholerae El Tor strains exhibited enhanced toxin production only at 37 degrees C under anaerobic conditions and the amount of toxin produced was akin to those of the toxigenic strains. In comparison to aerobic conditions, growth was observed to be comparatively lower under anaerobiosis both in the toxigenic and atpyical V. cholerae strains. Moreover, in contrast to the toxigenic strains, the toxin did not remain membrane-bound in these atypical strains at 37 degrees C and aerobic cultural conditions.

Molecular cloning and nucleotide sequence analysis of cholera toxin genes of the CtxA- Vibrio cholerae strain Texas Star-SR

The ctx operons from the Vibrio cholerae El Tor strain 3083 and its CtxA- derivative Texas Star-SR were cloned, and their nucleotide sequences were compared. A single missense mutation in the Texas Star-SR ctxA cistron which results in the substitution of threonine for alanine at position 191 of the 258-amino-acid CtxA precursor was identified. Immunoblot analysis detected the mutant CtxA intracellularly early in the culture cycle but not extracellularly at any growth stage.

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.

High-frequency spontaneous mutation of classical Vibrio cholerae to a nonmotile phenotype

The species Vibrio cholerae contains within it two biotypes, classical and El Tor, both of which are motile. Phenotypic expression of motility was unaffected by type of growth medium, salt concentration, pH, or temperature of incubation. However, seven strains of classical V. cholerae produced spontaneous nonmotile mutants at an unusually high frequency (ca. 10(-4)), while no mutants were detected for all three El Tor strains examined. No revertants of these nonmotile mutants were detected. Four independent mutants of classical strain 395 were isolated to characterize this phenomenon. By transmission electron microscopy, one of the nonmotile mutants was found to be flagellated, while the other three were found to be aflagellate. Chromosomal DNA from the mutants and parental wild-type strain 395 was examined by Southern blot analysis with, as probes, V. cholerae mutagenic prophages VcA-1 and VcA-2 and six cloned motility gene regions isolated from transposon insertion motility mutants of strains 395 and N16961 (El Tor, Inaba). The parental wild-type strain and all of the mutants exhibited the same pattern of bands when probed with VcA-1 and VcA-2 DNAs. Four of the cloned motility gene regions hybridized to the same fragments of DNA in both the wild-type and mutant isolates. However, two other probes detected a new fragment for a single aflagellate mutant. The observations that spontaneous nonmotile mutants occurred at a high frequency and that these mutants did not revert at a detectable frequency suggested that a genetic event is involved. The phenomenon appears to be limited to classical V. cholerae and may explain why classical V. cholerae is only sporadically associated with disease in the current pandemic.

Characterization of the hlyB gene and its role in the production of the EI Tor haemolysin of Vibrio choierae O1

El Tor strains of Vibrio cholerae are capable of producing a haemolysin which they actively secrete into the growth medium. This requires translation to produce the protein at the surface of the cytoplasmic membrane and translocation across this membrane, the periplasmic space and the outer membrane. The mechanism by which this occurs is poorly understood. In addition to the structural gene for the haemolysin (hlyA), we have cloned a second adjacent gene, hlyB. By site-directed mutagenesis, specific hlyB mutants have been constructed. These mutants are defective in the secretion of HlyA in the early to mid-exponential phase of growth and the haemolysin becomes trapped within the cell and is only released in stationary phase. Nucleotide sequence analysis and cell fractionations reveal HlyB to be a 60.3 kD putative outer membrane-associated protein.