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Infection and Immunity, August 2002, p. 4735-4742, Vol. 70, No. 8
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.8.4735-4742.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Vibrio Pathogenicity Island and Cholera Toxin Genetic Element-Associated Virulence Genes and Their Expression in Non-O1 Non-O139 Strains of Vibrio cholerae

Department of Microbiology, Bose Institute, Calcutta-700 054,¹ National Institute of Cholera and Enteric Diseases, Calcutta-700 010, India²

Received 9 October 2001/ Returned for modification 4 March 2002/ Accepted 9 May 2002

	ABSTRACT

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A non-O1 non-O139 Vibrio cholerae strain, 10259, belonging to the serogroup O53 was shown to harbor genes related to the vibrio pathogenicity island (VPI) and a cholera toxin (CT) genetic element called CTX. While the nucleotide sequence of the strain 10259 tcpA gene differed significantly (26 and 28%) from those of O1 classical and El Tor biotype strains, respectively, partial sequence analysis data of certain other VPI-associated genes (aldA, tagA, tcpP/H, toxT, acfB/C, and int) and intergenic regions (tcpF to toxT and tcpH to tcpA) of the strain showed only minor variations (0.4 to 4.8%) from corresponding sequences in O1 strains. Strain 10259 also contained CTX element-associated toxin genes with sequences almost identical to those of O1 strains. Growth of the organism in Luria broth (LB) under ToxR inducing conditions (30°C and pH 6.5) led to transcriptional activation of tcpP/H, toxR, toxT, and tcpA genes, but not of ctxA, as determined by reverse transcription-PCR (RT-PCR). Subsequent analysis revealed that strain 10259 possessed only two copies (instead of three or more copies found in epidemic-causing O1 or O139 strains) of the heptanucleotide (TTTTGAT) repeats in the intergenic region upstream of ctxAB. Therefore, a strain 10259 mutant was generated by replacement of this region with a homologous region (1.4 kb) derived from a V. cholerae O1 classical biotype strain (O395) that contained seven such repeats. The resultant recombinant strain (10259R) was found to be capable of coordinately regulated expression of toxT, ctxA, and tcpA when grown under the ToxR inducing conditions. Serological studies also demonstrated that the recombinant strain produced TcpA and a significantly (1,000-fold) higher level of CT in vitro compared to that of the parent strain. Virulence gene expression in two other non-O1 non-O139 strains (serogroup O37) containing VPI and the CTX element was studied by RT-PCR and serological assay. One strain (S7, which was involved in an epidemic in Sudan in 1968) showed coordinately regulated expression of virulence genes leading to the production of both CT and TcpA in LB medium. However, the other strain, V2, produced RT-PCR-detectable transcripts of toxT, ctxA, or tcpA genes in the early phase (6 h), but not in the late phase (16 h) of growth in LB medium. These results are consistent with the low levels of production of CT and TcpA by the strain that were serologically detectable. The significance of these results is discussed in relation to the role of virulence genes and their expression to the pathogenic potential of V. cholerae strains belonging to non-O1 serogroups.

	TEXT

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In humans, the disease cholera is caused by strains of the gram-negative bacterium Vibrio cholerae that belong to the O1 or O139 serogroup. The organism enters into the host during ingestion of contaminated water or food material, colonizes the small intestine, and produces an enterotoxin (cholera toxin [CT]) that is primarily responsible for the induction of massive loss of salt and water in the form of diarrhea (18). Colonization of the gut is facilitated through the expression of bundle-forming pilus structures (toxin-coregulated pilus [TCP]) on the surface of the bacterium (48). The expression of both CT and TCP is coordinately regulated at the transcriptional level by a cascade of signaling pathways that involve several transmembrane and cytosolic regulatory proteins (23, 45). Briefly, in response to appropriate environmental stimuli, the transmembrane protein ToxR, in association with ToxS of the toxRS operon, activates toxT, the gene encoding the cytosolic protein ToxT, which in turn activates transcription of genes for CT (ctxAB), the major structural protein subunit gene tcpA, and several others involved in TCP biosynthesis and secretion (23, 45, 54). More recently, membrane-associated proteins (e.g., TcpP and TcpH of the tcpPH operon) have been shown to play an additional regulatory role in the transcriptional activation of toxT (6, 15). Furthermore, activation of tcpPH is controlled, either positively (46) or negatively (2), by several regulatory elements. The ctxAB operon is located within a larger genetic element called CTX, which also encodes genes for several other toxins and accessory virulence factors (38). CTX was likely to be integrated into the host chromosome following lysogenic conversion of a filamentous bacteriophage, CTX (51). Similarly, the tcp gene cluster has been shown to be a part of a 39-kb DNA region, referred to as a vibrio pathogenicity island (VPI), which contains a gene for the ToxT regulatory protein as well as several other clusters of genes of known and unknown function (19). Like CTX, the VPI has been proposed to be of another lysogenic bacteriophage origin (20). The acquisition of VPI by V. cholerae endows the organism with the ability to express TCP, which acts as a receptor for CTX (20). The VPI and CTX genetic elements are primarily found in V. cholerae strains of O1 and O139 serogroups, which are associated with epidemic cholera (18, 19). The majority of strains belonging to about 200 other non-O1 non-O139 serogroups (53) do not contain genes for CT and/or TCP (18, 31, 47), although the gene for ToxR is ubiquitously present in these strains (34). The non-O1 non-O139 strains, which are predominantly isolated from an aquatic environment, are largely nonpathogenic in nature, although some of these are known to cause sporadic cases or occasional outbreaks of diarrhea in humans (30). Recently, however, ctxAB- and tcp-related genes have been shown to be present in certain strains of non-O1 non-O139 V. cholerae of both clinical and environmental origins (7, 14, 35, 36, 40, 42). This has raised important issues related to their evolution as well as relevance from the public health point of view. The point assumes considerable significance in view of the fact that strains belonging to non-O1 non-O139 serogroups have recently been implicated as the causative agents of a large number of cases of diarrhea in various parts of the world (1, 9, 41, 43). Evidently, documentation of the mere presence of the virulence-associated genes is not likely to provide sufficient information on the pathogenic potential of these strains, which is likely to depend on the presence of complex signaling pathways to couple appropriate environmental signals to virulence gene expression. Although some of the non-O1 non-O139 strains described earlier were shown to express detectable amounts of CT and/or TcpA protein in vitro (10, 14, 31, 32, 35), only limited information about their VPI and CTX is available so far (7, 14, 20), and no data on the regulation of virulence gene expression are available. In an earlier study, we characterized a new type of TcpA protein in a toxigenic V. cholerae strain, 10259, belonging to serogroup O53 (35). In the present communication, we describe partial characterization of VPI and CTX genetic elements of strain 10259 and provide information on its pathogenic potential by determining its ability to express virulence genes when grown in vitro under conditions that favor the expression of the same genes in V. cholerae O1 strains. We have extended this study by including data obtained with two other non-O1 non-O139 strains harboring VPI and CTX-related genes.

The bacterial strains and plasmids used in this study are listed in Table 1. The presence of virulence-associated genes in V. cholerae strains was determined by PCR amplification experiments with the primers listed in Table 2. All of these target genes, except toxR, are located in the VPI or CTX genetic element of the V. cholerae chromosome. Bacterial cells were grown overnight in Luria broth (LB) at 37°C, and chromosomal DNA was isolated from the harvested bacteria by a standard protocol. PCR amplification of target DNA was carried out in a thermal cycler (Perkin-Elmer) by essentially following the methodology described earlier (34). The reaction mixture was subjected to 30 cycles of amplification. Each cycle consisted of three successive steps in the following order: denaturation at 94°C for 30 s, annealing at 55°C for 50 s, and extension at 72°C for 50 s.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Schematic representation of V. cholerae VPI with its right (RJ) and left (LJ) junctions. Amplicons generated with strain 10259 by using various primer pairs are shown with their respective sizes. Arrows represent forward and reverse primer pairs.

The CTX genetic element of the strain 10259 was also probed with the primers for ctxA, ctxB, zot, ace, orfU, and cep genes, as listed in Table 2. All of these primers produced amplicons of the desired sizes, thereby documenting the presence of these genes in this non-O1 non-O139 strain. Amplicons generated with ctxA and orfU primers were subjected to sequencing analysis, and data were compared with those of O1 strains. The ctxA of 10259 showed only minimum changes from those of classical (0 bp) and El Tor (2 bp) strains. On the other hand, the 10259 orfU sequence diverged significantly from that of the classical strain by 56 bp (9.1% with 14 synonymous and 42 nonsynonymous changes), although it differed from the El Tor orfU by only 8 bp (1.3% with 6 synonymous and 2 nonsynonymous changes).

Recently, orfU has been proposed to be involved in the interaction of CTX to its receptor TCP on the V. cholerae surface (5). A significant difference between the orfU sequences of classical and El Tor biotype strains was postulated to be responsible for the specific recognition of CTXs by biotype-specific TcpA proteins, the major structural unit of TCP. Therefore, the fact that the orfU sequence of 10259 shows close similarity to that of El Tor (but not of classical orfU), appears to be somewhat at variance to this concept, since 10259 TcpA was predicted to differ equally from both classical and El Tor TcpA proteins (Table 3). Our results are, however, in agreement with those obtained with certain other non-O1 non-O139 strains, the orfU sequences of which were shown to be more similar to those of El Tor than to those of classical strains (5). At least, two of these strains, 158 and 208, possessed a new variant of TcpA with significant differences from classical, El Tor, as well as 10259 TcpA (35). All of these considerations would suggest that the drift in the TcpA sequence in non-O1 non-O139 V. cholerae strains may not necessarily be related to the need to bind to different OrfU proteins for the acquisition of new type of CTX. As a matter of fact, TcpA-independent acquisition of CTX by V. cholerae under selective conditions has been documented recently (4, 13).

Documentation of the presence of VPI and CTX elements in strain 10259 and partial characterization of its virulence-associated genes have prompted us to address the question of their expression in relation to pathogenesis. Therefore, the expression of toxR, tcpP/H, toxT, ctxA, and tcpA genes was studied by reverse transcription-PCR (RT-PCR) with the set of primers listed in Table 1. Briefly, bacterial RNA was extracted with TRIZOL reagent (GIBCO-BRL) from cells grown under the appropriate culture conditions, and the extracted material was treated with RNase-free DNase (Ambion). Purified RNA was used to obtain cDNA. For this, 1 µg of RNA was mixed with 0.1 M dithiothreitol (DTT), 2 pmol of each primer of the primer pair, 10 mM deoxynucleotide triphosphate (dNTP), and 5x first-strand buffer in 9.5 µl of reaction volume, and the mixture was incubated at 42°C for 2 min followed by immediate cooling. Next, 100 U of the enzyme reverse transcriptase (GIBCO-BRL) was added to this mixture, which was incubated at 42°C for 50 min. The reaction was terminated by incubating the mixture at 70°C for 15 min. The cDNA preparation thus obtained was amplified by PCR by the methodology described earlier.

The results obtained in the RT-PCR experiments demonstrate that strain 10259, when grown in LB at pH 6.5 for 16 h at 30°C under mild shaking conditions, expressed transcripts for toxR, tcpP/H, toxT, and tcpA, but not for ctxA (Fig. 2A). Similar results were obtained when the organism was grown in the same LB medium under static conditions or in colonization factor antigen (CFA) agar plates (data not shown). In all of these experiments, the V. cholerae O1 classical strain (O395) was used as the positive control to document the expression of virulence gene-associated transcripts as mentioned above (Fig. 2A). The expression of these genes was also tested by growing strain 10259 in modified AKI medium (52), which is known to favor the expression of ctxAB in El Tor biotype strains. While the control El Tor strain AD48 expressed toxR, tcpP/H, toxT, ctxA, and tcpA when grown in AKI medium at 37°C at pH 7.8 for 4 h under static conditions followed by 2 h of shaking conditions (24), strain 10259 showed expression of toxR with only weakly detectable transcripts of toxT and ctxA, but none for tcpP/H or tcpA (Fig. 2B). Culture of strain 10259 for a longer period (4 h of static conditions plus 6 h of shaking conditions) also failed to produce elevated levels of transcripts for these genes (data not shown).

View larger version (35K): [in this window] [in a new window]   FIG. 2. Detection of toxR, tcpP/H, toxT, tcpA, and ctxA transcripts by RT-PCR in V. cholerae strains O395, AD48, 10259, and 10259R. Organisms were grown in LB medium at pH 6.5 at 30°C for 16 h under mild shaking conditions (A) and AKI medium at pH 7.8 at 37°C for 4 h under static conditions followed by 2 h of shaking conditions (B). Columns in the right-hand panel show the relative intensity of the bands (left-hand panel) determined by the densitometric analysis with the Molecular Analyst (version 1.5) software package.

View larger version (33K): [in this window] [in a new window]   FIG. 3. Partial nucleotide sequence comparison of the ctxAB upstream regions of V. cholerae strains O395 and 10259. The sequences are aligned to show differences in the numbers of heptanucleotide repeats (in boxes).

Following characterization, the recombinant strain 10259R was tested for expression of virulence genes by RT-PCR as described earlier. When grown in LB at pH 6.5 at 30°C, the organism was shown to express ctxA in addition to tcpP/H, toxR, toxT, and tcpA (Fig. 2A). However, transcription of tcpP/H, toxT, and ctxA was detectable at low levels only in AKI medium-grown cells of strain 10259R (Fig. 2B). The RT-PCR data were extended through determination of the translated products CT and TcpA. Production of CT was estimated by GM1 enzyme-linked immunosorbent assay (ELISA) (17) with culture supernatants of cells grown under the appropriate culture conditions. While the wild-type strain 10259 produced a barely detectable level of CT in the LB-grown culture supernatant, the recombinant strain 10259R produced a significantly (about 1,000-fold) larger amount of CT in cultures grown under the same conditions (Table 4). In fact, CT produced by 10259R was quite comparable to that produced by the classical strain O395 in LB medium. Production of TcpA was also tested by immunoblotting experiments with a rabbit polyclonal antiserum to the protein. Both the wild-type and recombinant strains expressed TcpA when grown in LB medium, and the level of expression was quite comparable to that of the classical strain O395 grown under the same culture conditions (Fig. 4).

View larger version (48K): [in this window] [in a new window]   FIG. 4. Expression of TcpA by V. cholerae strains grown in LB medium (pH 6.5 at 30°C for 16 h) as determined by immunoblotting experiments with anti-TcpA serum. Whole-cell lysates of O395 (lane 1), 10259 (lane 2), 10259R (lane 3), S7 (lane 4), V2 (lane 5), and a tcpA strain, V1228 (lane 6), were used as antigens. An arrowhead indicates 20-kDa TcpA bands.

View larger version (56K): [in this window] [in a new window]   FIG. 5. Detection of toxR, tcpP/H, toxT, tcpA, and ctxA transcripts by RT-PCR in V. cholerae strains V2 and S7. Organisms were grown in LB medium at pH 6.5 at 30°C for 16 h (a) or 6 h (b) under mild shaking conditions. Columns in the right-hand panel show the relative intensity of the bands (left-hand panel) determined by densitometric analysis.

Expression of ctxA in absence of any detectable transcripts of tcpA by strain 10259R in AKI medium raises an interesting question regarding the relative efficiency with which these two genes can be transcriptionally activated in V. cholerae. In a recent study, Yu and DiRita (55) have demonstrated that, although ToxT is the direct activator of both ctxA and tcpA, ctxA transcription regulation is much more complex than that of tcpA. It is also suggested that the ctxA is under the control of a more efficient ToxT-dependent promoter compared to that of tcpA. Thus, under ToxT-limiting conditions, as is the case for strain 10259R grown in AKI medium, expression of ctxA may be achieved even in the absence of a detectable level of tcpA transcripts.

Coordinately regulated expression of CT and TcpA is an essential (although perhaps not sufficient) feature of epidemic-causing strains of V. cholerae, which so far has differentiated them from strains that are not associated with epidemics. Evidently, the genetic background of these strains plays a crucial role in this effect. The emergence of the O139 strain with epidemic potential from an O1 El Tor strain demonstrates that this feature may be retained by a non-O1 strain as well, provided it has a genetic makeup otherwise similar to that of O1 strains (50). Coordinate expression of CT and TcpA in the non-O1 non-O139 V. cholerae strain S7 described here also supports this concept, since this strain was likely to be derived from an O1 classical strain (3) and known to be involved in an epidemic in Sudan in 1968. Results obtained with the genetically engineered strain 10259R, however, demonstrate that coordinate expression of high levels of CT and TcpA is also achievable with a non-O1 non-O139 strain that may not be directly related to an epidemic strain as its origin. Although strain 10259R has shown its pathogenic potential in in vitro experiments, preliminary data generated in our laboratory have demonstrated considerable enhancement of its ability to produce toxin in vivo.

	ACKNOWLEDGMENTS

 
This work was supported by a grant [37(1008)/99-EMR-II] from the Council of Scientific and Industrial Research, Government of India.

The helpful technical assistance of Prabal Gupta and Debashis Mazumder is acknowledged.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Microbiology, Bose Institute, P-1/12 CIT Scheme VIIM, Calcutta-700 054, India. Phone: 033-337-9416/9544. Fax: 91-33-334-3886. E-mail: acghosh{at}boseinst.ernet.in.

Editor: V. J. DiRita

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