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VGJφ, a Novel Filamentous Phage of Vibrio cholerae, Integrates into the Same Chromosomal Site as CTXφ

We describe a novel filamentous phage, designated VGJφ, 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 VGJφ 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 CTXφ 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. VGJφ, like other filamentous phages, uses a type IV pilus to infect V. cholerae; in this case, the pilus is the mannose-sensitive hemagglutinin. VGJφ-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, VGJφ is able to integrate its genome into the same chromosomal attB site as CTXφ, entering into a lysogenic state. Additionally, we found an attP structure in VGJφ, 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 vaccine candidate Vibrio cholerae 638 is protective against cholera in healthy volunteers.

ABSTRACT Vibrio cholerae 638 is a living candidate cholera vaccine strain attenuated by deletion of the CTXΦ 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 109 CFU of freshly harvested 638 buffered with 1.3% NaHCO3, 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 109 CFU of ΔCTXΦ 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 × 105 CFU of virulent strain V. cholerae 3008 freshly harvested from a brain heart infusion agar plate and buffered with 1.3% NaHCO3. 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 (109 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.

Novel Type of Specialized Transduction for CTXφ or Its Satellite Phage RS1 Mediated by Filamentous Phage VGJφ in Vibrio cholerae

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 phi. This phage transmits ctxAB genes between V. cholerae bacterial populations that express toxin-coregulated pilus (TCP), the CTX phi receptor. In investigating new forms of ctxAB transmission, we found that V. cholerae filamentous phage VGJ phi, which uses the mannose-sensitive hemagglutinin (MSHA) pilus as a receptor, transmits CTX phi 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 phi and CTX phi (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 phi (for the VGJ phi/CTX phi hybrid) and HybRS phi (for the VGJ phi/RS1 hybrid). The hybrid phages replicate by using the VGJ phi replicating functions and use the VGJ phi capsid, retaining the ability to infect via MSHA. The hybrid phages infect most tested strains more efficiently than CTX phi, even under in vitro optimal conditions for TCP expression. Infection and lysogenization with HybP phi revert the V. cholerae live attenuated vaccine strain 1333 to virulence. Our results reinforce that TCP is not indispensable for the acquisition of CTX phi. 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.

Differential Interleukin-8 Response of Intestinal Epithelial Cell Line to Reactogenic and Nonreactogenic Candidate Vaccine Strains of Vibrio cholerae

ABSTRACT In this study, we analyzed whether attachment of Vibrio cholerae vaccine strains to human intestinal epithelial cells can induce an interleukin-8 (IL-8) response. The IL-8 transcripts were detected by PCR amplification of reverse-transcribed mRNA, and the gene product secretion was measured by an enzyme-linked immunosorbent assay. Infection of monolayers of the undifferentiated HT29-18N2 cell line with reactogenic (JBK70 and 81) and nonreactogenic (CVD103HgR and 638) vaccine strains of V. cholerae resulted in markedly higher IL-8 expression by epithelial cells exposed to reactogenic strains than by cells exposed to the nonreactogenic strains. Additionally, epithelial cells produced IL-8 transcripts following stimulation with cholera vaccine strains in a concentration-dependent manner. These results represent a new insight into the inflammatory component of reactogenicity and could be used as a predictive marker of vaccine reactogenicity prior to human testing.

Construction and Characterization of a Nonproliferative El Tor Cholera Vaccine Candidate Derived from Strain 638

ABSTRACT In recent clinical assays, our cholera vaccine candidate strain,Vibrio cholerae 638 El Tor Ogawa, was well tolerated and immunogenic in Cuban volunteers. In this work we describe the construction of 638T, a thymidine auxotrophic version of improved environmental biosafety. In so doing, the thyAgene from V. cholerae was cloned, sequenced, mutated in vitro, and used to replace the wild-type allele. Except for its dependence on thymidine for growth in minimal medium, 638T is essentially indistinguishable from 638 in the rate of growth and morphology in complete medium. The two strains showed equivalent phenotypes with regard to motility, expression of the celAmarker, colonization capacity in the infant mouse cholera model, and immunogenicity in the adult rabbit cholera model. However, the ability of this new strain to survive environmental starvation was limited with respect to that of 638. Taken together, these results suggest that this live, attenuated, but nonproliferative strain is a new, promising cholera vaccine candidate.

Anaerobic growth promotes synthesis of colonization factors encoded at the Vibrio pathogenicity island in Vibrio cholerae El Tor

Pathogenesis of the facultative anaerobe Vibrio cholerae takes place at the gut under low oxygen concentrations. To identify proteins which change their expression level in response to oxygen availability, proteomes of V. cholerae El Tor C7258 grown in aerobiosis, microaerobiosis and anaerobiosis were compared by two-dimensional electrophoresis. Twenty-six differentially expressed proteins were identified which are involved in several processes including iron acquisition, alanine metabolism, purine synthesis, energy metabolism and stress response. Moreover, two proteins implicated in exopolysaccharide synthesis and biofilm formation were produced at higher levels under microaerobiosis and anaerobiosis, which suggests a role of oxygen deprivation in biofilm development in V. cholerae. In addition, six proteins encoded at the Vibrio pathogenicity island attained the highest expression levels under anaerobiosis, and five of them are required for colonization: three correspond to toxin-coregulated pilus biogenesis components, one to soluble colonization factor TcpF and one to accessory colonization factor A. Thus, anaerobiosis promotes synthesis of colonization factors in V. cholerae El Tor, suggesting that it may be a key in vivo signal for early stages of the pathogenic process of V. cholerae.

Construction and characterisation of O139 cholera vaccine candidates

The hemagglutinin/protease (HA/P) seems to be an attractive locus for the insertion of heterologous tags in live cholera vaccine strains. A deltaCTXphi spontaneous mutant derived from a pathogenic strain of O139 Vibrio cholerae was sequentially manipulated to obtain hapA Colon, two colons celA derivatives which were later improved in their environmental safety by means of a thyA mutation. All the strains here obtained showed similar phenotypes in traits known to be remarkable for live cholera vaccines irrespective of their motility phenotypes, although the hapA mutants had a 10-fold decrease in their colonisation capacity compared with their parental strains in the infant mouse cholera model. However, the subsequent thyA mutation did not affect their colonisation properties in the same model. These preliminary results pave the way for further clinical assays to confirm the possibilities of these vaccine prototypes as safe and effective tools for the prevention of O139 cholera.

TLP01, an mshA mutant of Vibrio cholerae O139 as vaccine candidate against cholera

No commercially live vaccine against cholera caused by Vibrio cholerae O139 serogroup is available and it is currently needed. Virulent O139 strain CRC266 was genetically modified by firstly deleting multiple copies of the filamentous phage CTXφ, further tagging by insertion of the endoglucanase A coding gene from Clostridium thermocellum into the hemagglutinin/protease gene and finally deleting the mshA gene, just to improve the vaccine biosafety. One of the derived strains designated as TLP01 showed full attenuation and good colonizing capacity in the infant mouse cholera model, as well as highly immunogenic properties in the adult rabbit and rat models. Since TLP01 lacks MSHA fimbriae, it is refractory to infection with another filamentous phage VGJφ and therefore protected of acquiring CTXφ from a recombinant hybrid VGJφ/CTXφ. This strategy could reduce the possibilities of stable reversion to virulence out of the human gut. Furthermore, this vaccine strain was impaired to produce biofilms under certain culture conditions, which might have implications for the strain survival in natural settings contributing to vaccine biosafety as well. The above results has encouraged us to consider TLP01 as a live attenuated vaccine strain having an adequate performance in animal models, in terms of attenuation and immunogenicity, so that it fulfills the requirements to be evaluated in human volunteers.

Periplasmic proteins encoded by VCA0261-0260 and VC2216 genes together with copA and cueR products are required for copper tolerance but not for virulence in Vibrio cholerae.

The bacterial pathogen Vibrio cholerae requires colonizination of the human small intestine to cause cholera. The anaerobic and slightly acidic conditions predominating there enhance toxicity of low copper concentrations and create a selective environment for bacteria with evolved detoxifying mechanisms. We reported previously that the VCA0260, VCA0261 and VC2216 gene products were synthesized only in V. cholerae grown in microaerobiosis or anaerobiosis. Here we show that ORFs VCA0261 and VCA0260 are actually combined into a single gene encoding a 18.7 kDa protein. Bioinformatic analyses linked this protein and the VC2216 gene product to copper tolerance. Following the approach of predict-mutate and test, we describe for the first time, to our knowledge, the copper tolerance systems operating in V. cholerae. Copper susceptibility analyses of mutants in VCA0261-0260, VC2216 or in the putative copper-tolerance-related VC2215 (copA ATPase) and VC0974 (cueR), under aerobic and anaerobic growth, revealed that CopA represents the main tolerance system under both conditions. The VC2216-encoded periplasmic protein contributes to resistance only under anaerobiosis in a CopA-functional background. The locus tag VCA0261-0260 encodes a copper-inducible, CueR-dependent, periplasmic protein, which mediates tolerance in aerobiosis, but under anaerobiosis its role is only evident in CopA knock-out mutants. None of the genes involved in copper homeostasis were required for V. cholerae virulence or colonization in the mouse model. We conclude that copper tolerance in V. cholerae, which lacks orthologues of the periplasmic copper tolerance proteins CueO, CusCFBA and CueP, involves CopA and CueR proteins along with the periplasmic Cot (VCA0261-0260) and CopG (VC2216) V. cholerae homologues.

El Tor and Calcutta CTXΦ precursors coexisting with intact CTXΦ copies in Vibrio cholerae O139 isolates

Restriction fragment length polymorphism analyses of the array of CTXPhi prophages in strains CRC262 and CRC266 of Vibrio cholerae O139 revealed the presence of copies of complete CTXPhi and pre-CTXPhi prophages coexisting at a single chromosomal locus in each strain. Restriction pattern and comparative nucleotide sequence analysis revealed pre-CTXPhi precursors of both the El Tor and Calcutta lineages. Thus, we hypothesize that two precursor variants independently acquired cholera toxin genes and gave rise to the current El Tor and Calcutta CTXPhi prophages. We discuss the implications of these results in terms of the evolution and origin of the current diversity of CTXPhi prophages.