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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

Amit Sarkar,1 Ranjan K. Nandy,2 G. Balakrish Nair,2 and Asoke C. Ghose1*

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

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.

* 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

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.



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