In this study, we show that human-shed V. cholerae have a transiently reduced chemotactic state. This is, to our knowledge, the first report demonstrating that a pathogen alters its chemotactic state in response to human infection. In addition, these findings provide the first explanation for the enhanced virulence previously observed for stool V. cholerae. Consistent with the presence of defective chemotaxis, rice-water stool V. cholerae are an order of magnitude more infectious than when grown in vitro. If infection of infant mice mimics that in humans, then it appears that pathogenic V. cholerae have evolved to repress chemotaxis upon exiting the human host in order to enhance transmissibility. This lower infectious dose would be predicted to aid in the spread of V. cholerae during localized outbreaks of cholera.
Although all three cheW
and three cheR
paralogues appeared to be transcriptionally downregulated in rice-water stool V. cholerae
compared with V. cholerae
grown in vitro
, we have determined that only the cheW-1
paralogues are required for chemotaxis. We also showed that the level of CheW-1 protein is decreased, which would suggest the presence of smooth swimming by stool V. cholerae
. We have not confirmed the downregulation of CheR-2 by Western blot analysis; however, decreased levels of CheW-1 would be sufficient to cause a chemotaxis defect. If the remaining cheW
paralogues are also downregulated at the protein level, the significance of that finding would currently be unclear because no function has yet been ascribed to the chemotaxis genes located outside of the dominant chemotaxis cluster (with the exception of cheR-2
). As mentioned previously, alternative chemotaxis clusters often regulate processes like biofilm formation and flagellar-independent motility such as twitching motility. However, V. cholerae
do not appear to exhibit flagellar-independent surface motility (Watnick et al., 1999
), and we have been thus far unable to detect any defects in biofilm formation for any of the cheW
paralogue mutants (S. Butler and A. Camilli, unpubl. data). Therefore the significance of any potential downregulation of these proteins may not become evident until such time as their function becomes known.
No functional analysis of the chemotactic state of rice-water stool V. cholerae has been reported previously. We therefore examined the chemotactic ability of rice-water stool V. cholerae using a capillary assay and found that these bacteria have an approximately threefold defect in chemotaxis compared with V. cholerae that are wild-type for chemotaxis. Furthermore this chemotaxis defect was observed with two independent O1 El Tor serotypes (Ogawa and Inaba). These data suggest that this defect in chemotaxis is a genuine characteristic V. cholerae O1 El Tor exiting the human host.
We have previously shown that smooth-swimming non-chemotactic mutants of V. cholerae
have a lower infectious dose than the wild-type strain. This increase in infectivity is likely a combination of the increased area of the small intestine colonized by these mutants and the potential avoidance of an antimicrobial response as proposed earlier. We hypothesize that by temporarily downregulating chemotaxis, rice-water stool V. cholerae
can initially colonize the upper small intestine. If so, this property would reduce the infectious dose required for infection because more of these bacteria will initially colonize the intestine than if they exhibited functional chemotaxis. Presumably, after a period of time these bacteria may reinitiate and use chemotaxis to penetrate deeper in the intervillous spaces and to reach the distal small intestine. This is likely to be important because it has been previously shown that spontaneous non-chemotactic mutants exhibit an approximately threefold decrease in penetration of the infant mouse intestinal mucosa (Allweiss et al., 1977
), which is remarkably similar in magnitude to the threefold decrease in chemotaxis we observe for stool V. cholerae
. An additional role for the downmodulation of chemotaxis may be to facilitate dissemination from the host. If the V. cholerae
were fully chemotactic they might respond to chemoattractants present within the host and be more likely to remain in the intestinal crypts, where they are known to colonize (Freter et al., 1981
). A temporary decrease in chemotaxis may therefore facilitate improved entry into the lumen and subsequent exit from the host.
A number of questions remain unanswered regarding the temporary defect in chemotaxis that is observed. First, we do not know whether the chemotaxis defect observed is due to an equivalent defect among the population as a whole, or rather results from a subpopulation of V. cholerae that are impaired for chemotaxis, as the capillary assays do not distinguish between these two possibilities. Regardless, a defect in chemotaxis in any proportion of the population may have important implications for transmission of V. cholerae following dissemination from a host. Second, it is unknown whether it is the environment of the rice-water stool itself that causes the decrease in CheW-1 levels and the resulting increase in infectivity, or whether they have undergone these changes prior to entering into the intestinal lumen. Finally, it is unclear how long the chemotaxis defect and subsequent increase in infectivity persists in the environment after exiting the human host. We know from previous studies that the hypervirulent phenotype survives incubation in pond water for 5 h; however, the chemotactic ability and infectivity of such bacteria has not been tested.
A recent paper has described the use of mouse-passaged V. cholerae
as an equivalent model for the increase in infectivity that is observed with human-passaged V. cholerae
(Alam et al., 2005
). Although V. cholerae
in mid-exponential phase have a competitive advantage over stationary-phase V. cholerae
during infection, the authors showed that the increase in infectivity observed when V. cholerae
are first passaged through mice is not solely due to differences in growth phase or priming for virulence gene expression. It is therefore also likely that the increase in infectivity observed with rice-water stool V. cholerae
is not due to differences in growth phase alone. Unfortunately, this model is not suitable for an analysis of the chemotactic ability of these mouse-passaged V. cholerae
because it would be extremely difficult to remove the bacteria from the small intestine homogenate material. Therefore, the use of rice-water V. cholerae
is crucial in this instance for examining the chemotactic ability of stool V. cholerae
We do not know whether non-chemotactic smooth-swimming mutants or rice-water stool V. cholerae
are more infectious in humans. However, the infant mouse model has been a good predictor of factors important for V. cholerae
colonization and virulence in past studies (Klose, 2000
). In addition, although we have determined the infectivity of human-passaged V. cholerae
in infant mice, the infectivity of pathogenic V. cholerae
in the environment prior to human passage is unknown. Determination of this infectivity would be difficult because V. cholerae
cannot be directly cultured in large numbers from the environment, but require an enrichment step to enable culturing (Faruque et al., 2004
). The increased infectivities of natural forms of pathogens compared with experimental infection using in vitro
grown bacteria have been demonstrated in pathogens such as Citrobacter rodentium
and entero-haemorrhagic E. coli
(Cornick and Helgerson, 2004
; Wiles et al., 2005
). Interestingly, all toxigenic V. cholerae
isolated from local water sources during a 2004 outbreak in Dhaka, Bangladesh were of a single clone, which was the same clone present in clinical isolates from that outbreak (Faruque et al., 2005
). It is conceivable that this widespread outbreak was caused by a single clone that was initially passaged by one or a few susceptible individuals, and with the subsequent increased numbers of shed bacteria combined with a lower infectious dose, was able to be amplified within the human population and thus surrounding bodies of water.
We have shown in this study that V. cholerae shed from humans are an order of magnitude more infectious than V. cholerae grown in vitro for infection of infant mice. Furthermore, these human-shed V. cholerae have a chemotaxis defect compared with in vitro grown V. cholerae. This downmodulation of chemotactic ability by V. cholerae shed from humans is likely to be one reason for the hyperinfectivity observed and provides us with an insight into the multiple strategies that V. cholerae may employ during its life cycle to take advantage of its human host.