We describe a novel filamentous phage, designated VGJ, isolated from strain SG25-1 of Vibrio cholerae O139, which infects all O1 (classical and El Tor) and O139 strains tested. The sequence of the 7,542 nucleotides of the phage genome reveals that VGJ has a distinctive region of 775 nucleotides and a conserved region with an overall genomic organization similar to that of previously characterized filamentous phages, such as CTX of V. cholerae and Ff phages of Escherichia coli. The conserved region carries 10 open reading frames (ORFs) coding for products homologous to previously reported peptides of other filamentous phages, and the distinctive region carries one ORF whose product is not homologous to any known peptide. VGJ, like other filamentous phages, uses a type IV pilus to infect V. cholerae; in this case, the pilus is the mannose-sensitive hemagglutinin. VGJ-infected V. cholerae overexpresses the product of one ORF of the phage (ORF112), which is similar to single-stranded DNA binding proteins of other filamentous phages. Once inside a cell, VGJ is able to integrate its genome into the same chromosomal attB site as CTX, entering into a lysogenic state. Additionally, we found an attP structure in VGJ, which is also conserved in several lysogenic filamentous phages from different bacterial hosts. Finally, since different filamentous phages seem to integrate into the bacterial dif locus by a general mechanism, we propose a model in which repeated integration events with different phages might have contributed to the evolution of the CTX chromosomal region in V. cholerae El Tor.

The main virulence factor of Vibrio cholerae, the cholera toxin, is encoded by the ctxAB operon, which is contained in the genome of the lysogenic filamentous phage CTX. This phage transmits ctxAB genes between V. cholerae bacterial populations that express toxin-coregulated pilus (TCP), the CTX receptor. In investigating new forms of ctxAB transmission, we found that V. cholerae filamentous phage VGJ, which uses the mannose-sensitive hemagglutinin (MSHA) pilus as a receptor, transmits CTX or its satellite phage RS1 by an efficient and highly specific TCP-independent mechanism. This is a novel type of specialized transduction consisting in the site-specific cointegration of VGJ and CTX (or RS1) replicative forms to produce a single hybrid molecule, which generates a single-stranded DNA hybrid genome that is packaged into hybrid viral particles designated HybP (for the VGJ/CTX hybrid) and HybRS (for the VGJ/RS1 hybrid). The hybrid phages replicate by using the VGJ replicating functions and use the VGJ capsid, retaining the ability to infect via MSHA. The hybrid phages infect most tested strains more efficiently than CTX, even under in vitro optimal conditions for TCP expression. Infection and lysogenization with HybP revert the V. cholerae live attenuated vaccine strain 1333 to virulence. Our results reinforce that TCP is not indispensable for the acquisition of CTX. Thus, we discuss an alternative to the current accepted evolutionary model for the emergence of new toxigenic strains of V. cholerae and the importance of our findings for the development of an environmentally safer live attenuated cholera vaccine.The filamentous phage CTX contains the ctxAB genes encoding cholera toxin (CT), the main virulence factor of the pathogenic gram-negative bacterium Vibrio cholerae (49). In toxigenic El Tor and O139 strains of V. cholerae CTX is integrated at the dif site in the bacterial genome arrayed in different tandem structures along with the related satellite phage RS1 (11,39). The genome of RS1 is a short version of the genome of CTX, which contains genes encoding proteins needed for replication (RstA), integration (RstB), and regulation of gene expression (RstR and RstC) but lacks the genes encoding proteins needed for assembling and secretion of viral particles (Psh, Cep, pIII CTX , Ace, and Zot), as well as CT, which is not necessary for phage morphogenesis (11). Thus, satellite phage RS1 can replicate autonomously but depends on its helper phage CTX for assembly and secretion and thereby for transmission of RS1 viral particles (11). Conversely, RS1 encodes the antirepressor RstC, which is not present in CTX (9). This protein promotes transcription of CTX and RS1 genes by counteracting the activity of the phage repressor RstR (9). Thus, RS1 enhances transmission of both CTX and itself by means of RstC antirepressor activity (9).Classical strains of V. cholerae contain nonfunctional CTX prophages, whereas El Tor and O139 strains contain fully active prophages that produce infective CTX viral particles (10). CTX si...

Vibrio cholerae 638 is a living candidate cholera vaccine strain attenuated by deletion of the CTX⌽ prophage from C7258 (O1, El Tor Ogawa) and by insertion of the Clostridium thermocellum endoglucanase A gene into the hemagglutinin/protease coding sequence. This vaccine candidate was previously found to be well tolerated and immunogenic in volunteers. This article reports a randomized, double-blind, placebo-controlled trial conducted to test short-term protection conferred by 638 against subsequent V. cholerae infection and disease in volunteers in Cuba. A total of 45 subjects were enrolled and assigned to receive vaccine or placebo. The vaccine contained 10 9 CFU of freshly harvested 638 buffered with 1.3% NaHCO 3 , while the placebo was buffer alone. After vaccine but not after placebo intake, 96% of volunteers had at least a fourfold increase in vibriocidal antibody titers, and 50% showed a doubling of at least the lipopolysaccharide-specific immunoglobulin A titers in serum. At 1 month after vaccination, five volunteers from the vaccine group and five from the placebo group underwent an exploratory challenge study with 10 9 CFU of ⌬CTX⌽ attenuated mutant strain V. cholerae 81. Only two volunteers from the vaccine group shed strain 81 in their feces, but none of them experienced diarrhea; in the placebo group, all volunteers excreted the challenge strain, and three had reactogenic diarrhea. An additional 12 vaccinees and 9 placebo recipients underwent challenge with 7 ؋ 10 5 CFU of virulent strain V. cholerae 3008 freshly harvested from a brain heart infusion agar plate and buffered with 1.3% NaHCO 3 . Three volunteers (25%) from the vaccine group and all from the placebo group shed the challenge agent in their feces. None of the 12 vaccinees but 7 volunteers from the placebo group had diarrhea, and 2 of the latter exhibited severe cholera (>5,000 g of diarrheal stool). These results indicate that at 1 month after ingestion of a single oral dose (10 9 CFU) of strain 638, volunteers remained protected against cholera infection and disease provoked by the wild-type challenge agent V. cholerae 3008. We recommend that additional vaccine lots of 638 be prepared under good manufacturing practices for further evaluation.

No abstract

No abstract