Effect of anaerobiosis on expression of virulence factors in Vibrio cholerae

In Vibrio cholerae, the transmembrane DNA binding proteins, ToxR and TcpP, activate expression of the regulatory gene toxT in response to specific environmental signals. The resulting enhanced level of ToxT leads to a coordinated increase in the production of a subset of virulence factors, including cholera toxin (CT) and toxin-coregulated pilus (TCP). The effect of anaerobiosis on expression of the V. cholerae virulence regulatory cascade was examined. The expression of the major regulatory genes, tcpP, toxR, and toxT, in anaerobically grown V. cholerae was comparable to that in cells grown under aerobic conditions, and no significant difference in the ToxT-dependent expression of tcpA was detected when aerobic and anaerobic cultures were compared. However, in spite of the presence of functional ToxT, ctxAB expression was drastically reduced, and practically no CT was detected in cells grown under anaerobic conditions. In a V. cholerae hns mutant, however, high levels of ctxAB expression occurred even under anaerobic conditions. Also, deletion of the H-NS binding site from the ctxAB promoter eliminated anaerobic repression of ctxAB expression. These results suggest that H-NS directly represses ctxAB expression under anaerobic growth conditions. It has been reported that in the first stage of infection of infant mice by V. cholerae, tcpA is expressed but ctxAB expression is shut off (S. H. Lee, D. L. Hava, M. K. Waldor, and A. Camilli, Cell 99: 625-634, 1999). This pattern is similar to the pattern in anaerobic cultures of V. cholerae. Under all other in vitro conditions, ctxAB and tcpA are known to be coordinately expressed.     

Plasmid-mediated changes in virulence of Vibrio cholerae.

The effects of several plasmids, including cloning vectors and R factors, on the virulence of Vibrio cholerae CA401R were determined by measuring the dose-related diarrheal response in orally challenged infant mice. The plasmids were also examined for their effects on the colonization ability of strain CA401R by joint infection experiments with a spectinomycin-resistant CA401 strain as an internal standard. One V. cholerae R factor, pVH2, enhanced the diarrheal response, while R factors Rts1 and pVH1 reduced it; plasmids RP4, pRK290, Sa, pSJ8, pSJ5, and pBR328 had no effect. The ability of the plasmids to affect in vitro toxin production by CA401R was variable. Cells containing large plasmids all showed a modest decrease in colonization ability. These results showed that some plasmids affected V. cholerae virulence, but that the cloning vectors pBR328, RP4, and pRK290 did not.     

In vivo and in vitro characterization of virulence-deficient mutants of Vibrio cholerae.

In vitro and in vivo interactions between Vibrio cholerae and the infant mouse intestinal environment were examined by using a number of virulence-deficient mutants of strain CA401 which are unable to induce a typical diarrheal response. In vitro interactions with upper bowel sections were evaluated by determining percent association of radiolabeled organisms with sections. In vivo behavior was evaluated in the upper bowel early in infection with radiolabeled inocula. Ths relative degree of mechanical clearance was indicated by the percent recovery of input label. The relative degree of multiplication and killing was determined by changes in the specific activities (counts per minute per colony-forming unit) of inocula compared with recovered viable organisms. The results indicated that, whereas some virulence-deficient mutant classes exhibit net multiplication in the upper bowel, other classes show net killing in and accelerated clearance from the upper bowel. The in vitro association patterns failed to correlate with in vivo upper bowel recovery.     

Plasmids and factors associated with virulence in environmental isolates of Vibrio cholerae non-O1 in Bangladesh

Plasmid profiles and factors associated with toxigenicity in 51 strains of Vibrio cholerae non-O1 isolated from water samples collected in Bangladesh were analysed. Eleven (21.5%) strains were found to harbour at least one plasmid of 1.7-115 Mda; seven of these strains shared a 115-Mda plasmid. Six of 13 strains tested gave positive cytotoxic and enterotoxic responses. However, two non-cytotoxic strains were enterotoxigenic. Only three of the six cytotoxic and enterotoxic strains caused haemagglutination of human erythrocytes which indicated that toxin production and haemagglutinating activity were unrelated in these V. cholerae non-O1 strains. Conjugal transfer assays demonstrated that the 115-Mda plasmid harboured by some of the toxigenic V. cholerae non-O1 strains carried genes coding for antibiotic resistance and cytotoxin production but not for enterotoxin production. However, this plasmid was also carried by non-toxigenic strains. Some other strains carrying no plasmids or only small-mol.-wt plasmids, were found to be toxigenic. Therefore, toxin production is not plasmid-mediated in all V. cholerae non-O1 strains. Regardless of their pathogenic potential, V. cholerae non-O1 strains possessed the capacity to grow in conditions of iron limitation and, under these conditions, synthesis of at least two new outer-membrane proteins was induced.     

Role of the histone-like nucleoid structuring protein in colonization, motility, and bile-dependent repression of virulence gene expression in Vibrio cholerae

Bile-mediated repression of virulence gene expression is relieved in a Vibrio cholerae hns mutant. The mutant also exhibited reduced motility due to lower flrA expression, higher in vivo production of the virulence factors, and lower colonization efficiency. The colonization defect of the mutant was due to low FlrA production.     

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.     

In vitro and in vivo cholera toxin production by classical and El Tor isolates of Vibrio cholerae

A comparative study was carried out on the in vitro production of cholera toxin by 19 Vibrio cholerae El Tor isolates from patients with cholera in South Africa, one El Tor isolate from a patient in Malawi (a country approximately 1000 km north-northeast of South Africa), 6 El Tor and 12 classical type isolates from patients in Bangladesh, and 5 culture collection classical strains. Identical phage types and indistinguishable toxigenicities among the South African and Malawi V. cholerae, representing isolations obtained over a 10-year period, indicated that essentially a single strain was involved in the cholera of these regions. Similarly, phage typing and toxin profiles indicated that the 12 classical and 6 El Tor V. cholerae cultures in Bangladesh, all isolated in November 1983, represented just two strains. As assessed by titrations in Y-1 mouse adrenal and Chinese hamster ovary cell lines, the general order of toxigenicities was Bangladesh and culture collection classical greater than Bangladesh El Tor greater than southern African El Tor. The African isolates consistently gave rise to very low titers. Their relative reluctance to produce the toxin in vitro compared with the culture collection classical strains, particularly strain 569B, was confirmed by rocket electrophoresis. In somewhat of a contrast, maximum in vivo titers in rice water stools from cholera patients in South Africa and from both classical and El Tor type cholera patients in Bangladesh were essentially equal. It is postulated that under the continuous culture conditions that occur in vivo, cholera toxin concentrations can accumulate to a maximum level, depending on the rate of purging by the diarrheal fluid rather than the toxigenicity of the infecting stain. The relevance of these findings to the relative severities of classical and El Tor types of cholera is discussed.     

Vibrio cholerae expresses iron-regulated outer membrane proteins in vivo

A comparison was made, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of the outer membrane proteins of four strains of Vibrio cholerae grown in vivo in infant rabbits and in vitro in low-iron and iron-supplemented defined media. In vivo-grown V. cholerae expressed novel outer membrane-associated proteins which, in part, were similar to those observed on V. cholerae grown in vitro under conditions of iron deprivation.     

Iron and fur regulation in Vibrio cholerae and the role of fur in virulence

Regulation of iron uptake and utilization is critical for bacterial growth and for prevention of iron toxicity. In many bacterial species, this regulation depends on the iron-responsive master regulator Fur. In this study we report the effects of iron and Fur on gene expression in Vibrio cholerae. We show that Fur has both positive and negative regulatory functions, and we demonstrate Fur-independent regulation of gene expression by iron. Nearly all of the known iron acquisition genes were repressed by Fur under iron-replete conditions. In addition, genes for two newly identified iron transport systems, Feo and Fbp, were found to be negatively regulated by iron and Fur. Other genes identified in this study as being induced in low iron and in the fur mutant include those encoding superoxide dismutase (sodA), fumarate dehydratase (fumC), bacterioferritin (bfr), bacterioferritin-associated ferredoxin (bfd), and multiple genes of unknown function. Several genes encoding iron-containing proteins were repressed in low iron and in the fur mutant, possibly reflecting the need to reserve available iron for the most critical functions. Also repressed in the fur mutant, but independently of iron, were genes located in the V. cholerae pathogenicity island, encoding the toxin-coregulated pilus (TCP), and genes within the V. cholerae mega-integron. The fur mutant exhibited very weak autoagglutination, indicating a possible defect in expression or assembly of the TCP, a major virulence factor of V. cholerae. Consistent with this observation, the fur mutant competed poorly with its wild-type parental strain for colonization of the infant mouse gut.     

Development of an in vitro model for study of non-O1 Vibrio cholerae virulence using Caco-2 cells.

Non-O1 Vibrio cholerae strains have been reported as a causative agent of diarrhea throughout the world. We recently reported that non-O1 V. cholerae strains cause diarrhea in human volunteers. In this study we evaluated the virulence of three strains of non-O1 V. cholerae in a Caco-2 cell adherence assay by light and electron microscopy. A-5 is an environmental isolate which failed to colonized volunteers and did not cause diarrhea. It exhibited low numbers of organisms adherent to Caco-2 cells, leaving the microvilli intact. Strain 2076-79, isolated from a patient with diarrhea, colonized human volunteers without producing disease. It adhered to Caco-2 cells in moderate numbers without producing any damage to the microvilli. Strain NRT36S, a clinical isolate, colonized human volunteers and produced significant diarrhea disease. This strain adhered in very large numbers to Caco-2 cells and caused damage to the brush borders. Membrane-bound bacteria were also seen within the cytoplasm of these cells. Scanning electron microscopy confirmed the generalized adherence of NRT36S to the microvilli of Caco-2 cells. The three strains did not appear to compete with each other for binding sites on Caco-2 cells and were not adherent when assays were conducted at 4 degrees C. Our results with strains A-5, 2076-79, and NRT36S correlate well with observations in human volunteer studies, suggesting that Caco-2 cells provide an appropriate in vitro system for further investigation of the pathogenesis of non-O1 V. cholerae gastroenteritis.     

Iron-vibriobactin transport system is not required for virulence of Vibrio cholerae.

The possible requirement of a functional siderophore (vibriobactin)-mediated iron transport system in the pathogenicity of Vibrio cholerae was determined. Two mutants of V. cholerae defective in the iron-vibriobactin transport system were examined for their ability to multiply and elicit diarrhea in infant mice. One mutant, 40130, was unable to synthesize vibriobactin. The second mutant, 1510, was able to synthesize, but not transport, the siderophore. Both mutants retained the ability to multiply and produce disease in the infant mouse, and virulence was indistinguishable from the parent strains. This indicates that a functional iron-vibriobactin transport system is not essential for cholera pathogenesis. These mutants, like the wild-type strains, were found to have a ferric citrate iron uptake system and could utilize citrate or asparagine for growth in low-iron medium. Compounds of this type may increase the availability of iron to V. cholerae in the host.