The factors that enhance the transmission of pathogens during epidemic spread are ill defined. Water-borne spread of the diarrhoeal disease cholera occurs rapidly in nature, whereas infection of human volunteers with bacteria grown in vitro is difficult in the absence of stomach acid buffering. It is unclear, however, whether stomach acidity is a principal factor contributing to epidemic spread. Here we report that characterization of Vibrio cholerae from human stools supports a model whereby human colonization creates a hyperinfectious bacterial state that is maintained after dissemination and that may contribute to epidemic spread of cholera. Transcriptional profiling of V. cholerae from stool samples revealed a unique physiological and behavioural state characterized by high expression levels of genes required for nutrient acquisition and motility, and low expression levels of genes required for bacterial chemotaxis.

The role of chemotaxis in the virulence of gastrointestinal pathogens is ill defined. Counterintuitively, nonchemotactic mutants of the polarly flagellated pathogen Vibrio cholerae greatly outcompete the wild-type strain during infection of the small intestine. We show that the out-competition phenotype is dependent on the direction of flagellar rotation and independent of Toxin Co-regulated Pilus function. Specifically, the out-competition associated with the loss of chemotaxis required the presence of counterclockwise-biased flagellar rotation and smooth straight runs by the bacteria. In contrast, a nonchemotactic strain with clockwise-biased flagellar rotation was confined to small-scale net movement and was attenuated for infection. The significance of the out-competition phenotype was examined and was shown to correlate with a true increase in infectivity. Counterclockwisebiased mutants are aberrantly distributed throughout the infant mouse small intestine and we find that the expression of virulence factors occurs normally in all segments. Thus, alteration of the chemotactic properties of V. cholerae allows it to exploit additional niches in the host intestine.T he Gram-negative bacterium Vibrio cholerae is the causative agent of the epidemic disease cholera. Cholera patients are afflicted with a profuse watery diarrhea resulting from the action of the ADP-ribosylating cholera toxin (CT). This organism is highly motile by means of a single polar sheathed flagellum and is believed to use the processes of motility and chemotaxis to travel from the lumen of the small intestine to its preferred intestinal niche on the intestinal epithelium. In this niche V. cholerae expresses a number of virulence factors including CT and the toxin co-regulated type IV pilus (TCP). The latter has been shown to be essential for colonization of humans (1), as well as in the infant mouse model of infection (2).Within the V. cholerae genome are multiple paralogues of chemotaxis genes (3). Despite the presence of three chemotaxis operons, only one of these operons is required for chemotaxis (4). The function of the remaining chemotaxis operons remains unknown. Chemotaxis in many organisms is achieved by modulating change in the direction of flagellar rotation from the default direction of counterclockwise (CCW) to clockwise (CW) rotation. In the peritrichously flagellated bacterium Escherichia coli, CCW rotation results in the bacterium swimming smoothly in a mostly straight line, whereas CW rotation causes the cell to turn abruptly in a process known as tumbling (5). Because of the presence of a single polar flagellum, V. cholerae does not tumble per se but instead reverses direction brief ly, thereby allowing the bacterium to randomly reorient itself and swim in a new direction.Although motility and chemotaxis are believed to guide V. cholerae to its preferred colonization site within the small intestine, the precise role of each of these processes during infection has not been clearly established. Previous work by Freter et al. ...

We devised a noninvasive genetic selection strategy to identify positive regulators of bacterial virulence genes during actual infection of an intact animal host. This strategy combines random mutagenesis with a switch-like reporter of transcription that confers antibiotic resistance in the off state and sensitivity in the on state. Application of this technology to the human intestinal pathogen Vibrio cholerae identified several regulators of cholera toxin and a central virulence gene regulator that are operative during infection. These regulators function in chemotaxis, signaling pathways, transport across the cell envelope, biosynthesis, and adherence. We show that phenotypes that appear genetically independent in cell culture become interrelated in the host milieu. Vibrio cholerae, the causative agent of the epidemic disease cholera, requires the coordinated expression of multiple virulence factors. Two of the most well studied factors are cholera toxin (CT) and toxin-coregulated pilus (TCP). CT is an ADP-ribosylating toxin largely responsible for eliciting the profuse diarrhea characteristic of this disease, and TCP is a type IV bundle-forming pilus that is essential for intestinal colonization in humans and in animal models of cholera (1, 2). Regulation of these factors involves the concerted actions of three proteins, ToxR, TcpP, and ToxT, which together form the V. cholerae virulence gene regulatory cascade (3-5). ToxR and TcpP are inner-membrane-associated transcriptional regulators that act cooperatively to induce the expression of ToxT in response to particular environmental signals. ToxT is a cytoplasmic transcriptional activator responsible for directly activating the transcription of the structural genes for CT, TCP, and other virulence factors (3). Investigation of the regulatory networks coordinating expression of these genes has been predominately limited to in vitro culture conditions largely because of the inability to monitor regulatory processes during infection of an intact host. Therefore, the true nature of the in vivo regulation of these virulence factors within a host remains unclear.To extend our knowledge of virulence gene regulation during infection, we developed a noninvasive genetic selection that incorporates the use of the recombination-based in vivo expression technology (RIVET) and transposon mutagenesis to identify positive regulators of virulence genes during infection. RIVET relies on the construction of transcriptional fusions to a promoterless reporter gene, tnpR, encoding a site-specific DNA recombinase. After it is produced, TnpR functions in trans to permanently excise from the bacterial genome a tetracycline resistance (Tc R ) marker (tet) that is flanked by recombinase recognition sequences (res). The subsequent conversion to tetracycline (Tc) sensitivity serves as an ex post facto indicator of increased transcription of the gene fusion. RIVET has been used as a promoter-trap method to identify bacterial and fungal genes induced during infection (6)(7)(8) and as a too...

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