A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains

Abstract

The bacterial species Vibrio cholerae includes harmless aquatic strains as well as strains capable of causing epidemics and global pandemics of cholera. While investigating the relationship between pathogenic and nonpathogenic strains, we identified a chromosomal pathogenicity island (PAI) that is present in epidemic and pandemic strains but absent from nonpathogenic strains. Initially, two ToxR-regulated genes (aldA and tagA) were studied and were found to be associated with epidemic and pandemic strains but absent in nontoxigenic strains. The region containing aldA and tagA comprises 13 kb of previously unidentified DNA and is part of a PAI that contains a regulator of virulence genes (ToxT) and a gene cluster encoding an essential colonization factor and the cholera toxin phage receptor (toxin-coregulated pilus; TCP). The PAI is 39.5 kb in size, has low %G+C (35%), contains putative integrase and transposase genes, is flanked by att sites, and inserts near a 10Sa RNA gene (ssrA), suggesting it may be of bacteriophage origin. We found this PAI in two clinical non-O1/non-O139 cholera toxin-positive strains, suggesting that it can be transferred within V. cholerae. The sequence within this PAI includes an ORF with homology to a gene associated with the type IV pilus gene cluster of enteropathogenic Escherichia coli, a transposase from Vibrio anguillarum, and several ORFs with no known homology. As the PAI contains the CTXPhi receptor, it may represent the initial genetic factor required for the emergence of epidemic and pandemic cholera. We propose to call this island VPI (V. cholerae pathogenicity island).

Figure 1

The VPI. Arrows above genes show PCR primers used and are identified by the number of the KAR primer series, whereas arrows within genes show direction of transcription. ▪, Probes used; ▧, common chromosomal flanking DNA; ☒ at each end of VPI denotes att sites; and ░⃞ within the left end of the VPI indicates the defective transposase. The + and − indicate the presence and absence, respectively, of the region in epidemic and pandemic strains and nontoxigenic strains.

Figure 2

PCR analysis of the left and right junctions of the VPI. (A) Similar sized fragments containing the left junction for all strains with primers KAR94/KAR97. (B) Similar sized fragments containing the right junction for all strains with KAR23/KAR87. Lanes: 1, 6th pandemic strain 395; 2, 7th pandemic strain N16961; 3, O139 Bengal strain AI1837; 4, U.S. Gulf coast strain E506; 5, 1937 Sulawesi strain 66–2; 6, non-O1/non-O139 CT-positive strain ATCC25872 from Czechoslovakia; 7, non-O1/non-O139 CT-positive strain S-21 from Sudan; center lane, 1-kb marker.

Figure 3

Sequence of the left and right junctions of the VPI and its insertion site into the V. cholerae chromosome. (A) The left and right junctions of VPI. (B) att site at the 3′ end of the ssrA gene in three VPI-negative strains. Bold text indicates att sites. Note that the absence of the adenine in the att site at the left junction of VPI-positive strains (A) is also found in the VPI-negative CT-positive strain E9120 (B). Differences in sequence flanking the att site in the VPI-negative strain DK63 are underlined (B).