Vibrio cholerae, the causative agent of cholera, requires two coordinately regulated factors for full virulence: cholera toxin (CT), a potent enterotoxin, and toxin-coregulated pili (TCP), surface organelles required for intestinal colonization. The structural genes for CT are shown here to be encoded by a filamentous bacteriophage (designated CTXphi), which is related to coliphage M13. The CTXphi genome chromosomally integrated or replicated as a plasmid. CTXphi used TCP as its receptor and infected V. cholerae cells within the gastrointestinal tracts of mice more efficiently than under laboratory conditions. Thus, the emergence of toxigenic V. cholerae involves horizontal gene transfer that may depend on in vivo gene expression.

Shiga toxin-producing Escherichia coli (STEC) cause significant disease; treatment is supportive and antibiotic use is controversial. Ciprofloxacin but not fosfomycin causes Shiga toxin-encoding bacteriophage induction and enhanced Shiga toxin (Stx) production from E. coli O157:H7 in vitro. The potential clinical relevance of this was examined in mice colonized with E. coli O157:H7 and given either ciprofloxacin or fosfomycin. Both antibiotics caused a reduction in fecal STEC. However, animals treated with ciprofloxacin had a marked increase in free fecal Stx, associated with death in two-thirds of the mice, whereas fosfomycin did not. Experiments that used a kanamycin-marked Stx2 prophage demonstrated that ciprofloxacin, but not fosfomycin, caused enhanced intraintestinal transfer of Stx2 prophage from one E. coli to another. These observations suggest that treatment of human STEC infection with bacteriophage-inducing antibiotics, such as fluoroquinolones, may have significant adverse clinical consequences and that fluoroquinolone antibiotics may enhance the movement of virulence factors in vivo.

The mammalian gastrointestinal (GI) tract provides a complex and competitive environment for the microbiota1. Successful colonization by pathogens depends on scavenging nutrients, sensing chemical signals, competing with the resident bacteria, and precisely regulating expression of virulence genes2. The GI pathogen enterohemorrhagic E.coli (EHEC) relies on inter-kingdom chemical sensing systems to regulate virulence gene expression3–4. Here we show that these systems control the expression of a novel two-component signal transduction system, named FusKR, where FusK is the histidine sensor kinase (HK), and FusR the response regulator (RR). FusK senses fucose and controls expression of virulence and metabolic genes. This fucose-sensing system is required for robust EHEC colonization of the mammalian intestine. Fucose is highly abundant in the intestine5. Bacteroides thetaiotaomicron (B.theta) produces multiple fucosidases that cleave fucose from host glycans, resulting in high fucose availability in the gut lumen6. During growth in mucin, B.theta contributes to EHEC virulence by cleaving fucose from mucin, thereby activating the FusKR signaling cascade, modulating EHEC’s virulence gene expression. Our findings suggest that EHEC uses fucose, a host-derived signal made available by the microbiota, to modulate EHEC pathogenicity and metabolism.

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Vibrio cholerae O139 is the first non-O1 serogroup of V. cholerae to give rise to epidemic cholera. Apparently, this new serogroup arose from an El Tor O1 strain of V. cholerae, but V. cholerae O139 is distinguishable from V. cholerae El Tor O1 by virtue of its novel antigenic structure and also its characteristic pattern of resistances to the antibiotics sulfamethoxazole, trimethoprim, streptomycin, and furazolidone. We found that the first three of these antibiotic resistances are carried on an approximately 62-kb self-transmissible, chromosomally integrating genetic element which we have termed the SXT element. This novel conjugative transposon-like element could be conjugally transferred from V. cholerae O139 to V. cholerae O1 and Escherichia coli strains, where it integrated into the recipient chromosomes in a site-specific manner independent of recA. To study the potential virulence properties of the SXT element as well as to improve upon the live attenuated O139 vaccine strain Bengal-2, a large internal deletion in the SXT element was crossed on to the Bengal-2 chromosome. The resulting strain, Bengal-2.SXT s , is sensitive to sulfamethoxazole and trimethoprim and colonizes the intestines of suckling mice as well as wild-type strains do, suggesting that the SXT element does not encode a colonization factor. Derivatives of Bengal-2.SXT s are predicted to be safe, antibiotic-sensitive, live attenuated vaccines for cholera due to the O139 serogroup.Cholera is a severe and sometimes lethal diarrheal disease caused by the gram-negative bacterium Vibrio cholerae. Historically, only the O1 serogroup of V. cholerae has been associated with epidemic cholera. However, in early 1993 in India and Bangladesh, a major cholera epidemic was caused by a novel non-O1 serogroup of V. cholerae named V. cholerae O139 (6). Strains belonging to this newly emerged V. cholerae serogroup replaced the endemic El Tor O1 strains of V. cholerae to become the principal clinical and environmental isolate of V. cholerae on the Indian subcontinent (6). In the past year and a half, however, El Tor O1 strains have returned to India and Bangladesh and are the cause of the majority of cholera cases in the region (5).The initial microbiologic characterization of V. cholerae O139 revealed that this serogroup was closely related to the El Tor biotype of V. cholerae O1. The shared properties of V. cholerae O139 and El Tor O1 strains include (i) the agglutination of chicken erythrocytes (6), (ii) resistance to polymyxin B (6), (iii) in vitro growth conditions for the expression of virulence factors (42), (iv) identical-sized restriction fragments for genes which have known polymorphisms (4, 42), (v) identical electrophoretic types by multilocus enzyme electrophoresis analysis (31), (vi) tandem duplications of the CTX genetic element (41), and (vi) identical chromosomal location of the CTX genetic element (41). These findings support the hypothesis that V. cholerae O139 is a derivative of an El Tor O1 strain of V. cholerae. DNA sequence analysis of...

The temporal expression patterns of the critical Vibrio cholerae virulence genes, tcpA and ctxA, were determined during infection using a recombinase reporter. TcpA was induced biphasically in two temporally and spatially separable events in the small intestine, whereas ctxA was induced monophasically only after, and remarkably, dependent upon, tcpA expression; however, this dependence was not observed during in vitro growth. The requirements of the virulence regulators, ToxR, TcpP, and ToxT, for expression of tcpA and ctxA were determined and were found to differ significantly during infection versus during growth in vitro. These results illustrate the importance of examining virulence gene expression in the context of bona fide host-pathogen interactions.

SummaryVibrio cholerae O139, the ®rst non-O1 serogroup of V. cholerae to give rise to epidemic cholera, is characteristically resistant to the antibiotics sulphamethoxazole, trimethoprim, chloramphenicol and streptomycin. Resistances to these antibiotics are encoded by a 62 kb self-transmissible, conjugative, chromosomally integrating element designated the`SXT element'. We found that the SXT element integrates site speci®cally into both V. cholerae and Escherichia coli K-12 into the 58 end of prfC, the gene encoding peptide chain release factor 3. Integration of the SXT element interrupts the chromosomal prfC gene, but the element encodes a new 58 end of prfC that restores the reading frame of this gene. The recombinant prfC allele created upon element integration is functional. The integration and excision mechanism of the SXT element shares many features with site-speci®c recombination found in lambdoid phages. First, like l, the SXT element forms a circular extrachromosomal intermediate through speci®c recombination of the left and right ends of the integrated element. Second, chromosomal integration of the element occurs via site-speci®c recombination in a 17 bp sequence found in the circular form of the SXT element and a similar 17 bp sequence in prfC. Third, both chromosomal integration and excision of the SXT element were found to require an element-encoded int gene with strong similarities to the l integrase family. Based on the properties of the SXT element, we propose to classify this element as a CONSTIN, an acronym for a conjugative, self-transmissible, integrating element.

SummaryCTX is a filamentous phage that encodes cholera toxin, one of the principal virulence factors of Vibrio cholerae. CTX is unusual among filamentous phages because it can either replicate as a plasmid or integrate into the V. cholerae chromosome at a specific site. The CTX genome has two regions, the 'core' and RS2. Integrated CTX is frequently flanked by an element known as RS1 which is related to RS2. The nucleotide sequences of RS2 and RS1 were determined. These related elements contain three nearly identical open reading frames (ORFs), which in RS2 were designated rstR, rstA2 and rstB2. RS1 contains an additional ORF designated rstC. Functional analyses indicate that rstA2 is required for CTX replication and rstB2 is required for CTX integration. The amino terminus of RstR is similar to the amino termini of other phageencoded repressors, and RstR represses the expression of rstA2. Although genes with related functions are clustered in the genome of CTX in a way similar to those for other filamentous phages, the CTX RS2-encoded gene products mediating replication, integration and repression appear to be novel.