Genomic analysis of the Mozambique strain of Vibrio cholerae O1 reveals the origin of El Tor strains carrying classical CTX prophage

Abstract

Cholera outbreaks in subSaharan African countries are caused by strains of the El Tor biotype of toxigenic Vibrio cholerae O1. The El Tor biotype is the causative agent of the current seventh cholera pandemic, whereas the classical biotype, which was associated with the sixth pandemic, is now extinct. Besides other genetic differences the CTX prophages encoding cholera toxin in the two biotypes of V. cholerae O1 have distinct repressor (rstR) genes. However, recent incidences of cholera in Mozambique were caused by an El Tor biotype V. cholerae O1 strain that, unusually, carries a classical type (CTX class ) prophage. We conducted genomic analysis of the Mozambique strain and its CTX prophage together with chromosomal phage integration sites to understand the origin of this atypical strain and its evolutionary relationship with the true seventh pandemic strain. These analyses showed that the Mozambique strain carries two copies of CTX class prophage located on the small chromosome in a tandem array that allows excision of the prophage, but the excised phage genome was deficient in replication and did not produce CTX class virion. Comparative genomic microarray analysis revealed that the strain shares most of its genes with the typical El Tor strain N16961 but did not carry the TLC gene cluster, and RS1 sequence, adjacent to the CTX prophage. Our data are consistent with the Mozambique strain's having evolved from a progenitor similar to the seventh pandemic strain, involving multiple recombination events and suggest a model for origination of El Tor strains carrying the classical CTX prophage. cholera ͉ CTX phage ͉ TLC element ͉ evolution C holera caused by toxigenic Vibrio cholerae is a major public health problem confronting many developing countries, where outbreaks occur frequently and are particularly associated with poverty and poor sanitation (1, 2). The occurrence of seven distinct pandemics of cholera have been recorded since the beginning of the first pandemic in 1817. The current seventh pandemic, which originated in Indonesia in 1961, is the most extensive in geographic spread and duration, and the causative agent is V. cholerae O1 of the El Tor biotype. The sixth pandemic and presumably the earlier pandemics were caused by V. cholerae O1 of the classical biotype. These two biotypes of V. cholerae O1 differ in certain phenotypic and genetic characteristics (2). In toxigenic V. cholerae, the genes encoding cholera toxin (ctxAB) are part of the CTX prophage (3). Besides other genetic differences between the two biotypes of V. cholerae O1, the CTX prophages in these two biotypes are also distinct in their sequence of the repressor gene (rstR), although most of the phage genomes are similar in the two biotypes (4, 5). The factors that led to the extinction of the classical biotype and its replacement by the El Tor biotype of V. cholerae O1 as a causative agent of cholera are not clearly known.The seventh pandemic of cholera reached sub-Saharan West Africa in the early 1970s and caused explosive outbreaks...

Drug response and genetic properties of Vibrio cholerae associated with endemic cholera in north-eastern Thailand, 2003–2011

“…Interestingly, a sharp genetic transition of V. cholerae from the ET Mozambique variant to the Asian altered ET (possessing ctxB of the CL biotype) was observed in north-east Thailand in 2007, with all of the tested V. cholerae strains acquiring RS1 and TLC and switching the repressor rstR allele from rstR CL (CL) to rstR ET (ET), as in the altered ET reported from Bangladesh (Nair et al, 2006) and India (Raychoudhuri et al, 2008). Our data on the temporal absence of virulence and related genetic elements and their acquisitions clearly indicate horizontal gene transfer, which has been proposed to provide V. cholerae strains with improved evolutionary fitness (Faruque et al, 2007).…”

“…However, the tcpA gene of V. cholerae O1 ET strains isolated in 2007 and thereafter had a mutation at amino acid position 64 (NAS). Although the change is subtle and it is not clear whether such genetic switching of the tcpA gene can provide V. cholerae O1 strains with increased environmental and/or epidemiological fitness (Faruque et al, 2007), a similar change of amino acid at position 64 of TcpA was found in ctx 2 V. cholerae O1 strains ZJ65 (2006) isolated in China (GenBank accession no. EU622532) and V. cholerae strain 2010EL1786 (GenBank accession no.…”

“…The data also show that, unlike the altered ET strains reported from Bangladesh (Nair et al, 2006) and India (Raychoudhuri et al, 2008), V. cholerae O1 ET strains isolated between 2003 and 2004, and one strain isolated in 2007 from a cholera outbreak in Khon Kaen, Thailand, did not carry rstC, a repressor gene sequence of the RS1 genetic element (Safa et al, 2010) or the TLC gene cluster, which encodes a filamentous phage replicase and is proposed to be a satellite phage. Besides, all of these strains contained the repressor rstR gene (allele) of CL biotype, and thus appeared indistinguishable from the Mozambique variant of ET carrying CL CTX prophage (Ansaruzzaman et al, 2004;Faruque et al, 2007). Interestingly, a sharp genetic transition of V. cholerae from the ET Mozambique variant to the Asian altered ET (possessing ctxB of the CL biotype) was observed in north-east Thailand in 2007, with all of the tested V. cholerae strains acquiring RS1 and TLC and switching the repressor rstR allele from rstR CL (CL) to rstR ET (ET), as in the altered ET reported from Bangladesh (Nair et al, 2006) and India (Raychoudhuri et al, 2008).…”

Molecular keys of the tropism of integration of the cholera toxin phage

“…Studies of the mechanism of integration of the classical variant of CTXϕ were complicated because no phage production was observed in any of the strains harboring it (3,11). To circumvent this difficulty, we used conjugation to deliver circular DNA molecules carrying the replication and integration region of the phage found in the 569B classical strain, hereafter referred to as "RS Cl ," directly into V. cholerae cells.…”

“…1C). Nevertheless, the classical variant of CTXϕ was found integrated at dif2 in classical strains (3) and in recent El Tor isolates (4,(10)(11)(12)(13)(14). The mode of dissemination of this variant is of particular interest, because it allows the production of an elevated amount of cholera toxin (15,16) that seems to be implicated in a high proportion of severe infections associated with classical strains (17).…”