Cholera stool bacteria repress chemotaxis to increase infectivity

Abstract

Factors that enhance the transmission of pathogens are poorly understood. We show that Vibrio cholerae shed in human 'rice-water' stools have a 10-fold lower oral infectious dose in an animal model than in vitro grown V. cholerae, which may aid in transmission during outbreaks. Furthermore, we identify a bacterial factor contributing to this enhanced infectivity: The achievement of a transient motile but chemotaxis-defective state upon shedding from humans. Rice-water stool V. cholerae have reduced levels of CheW-1, which is essential for chemotaxis, and were consequently shown to have a chemotaxis defect when tested in capillary assays. Through mutational analyses, such a state is known to enhance the infectivity of V. cholerae. This is the first report of a pathogen altering its chemotactic state in response to human infection in order to enhance its transmission.

Fig. 1

Examination of the chemotactic abilities of cheW and cheR paralogue mutants using semisolid agar chemotaxis plates (1% tryptone, 0.5% NaCl, 0.3% agar; 37°C overnight). A. Analysis of the requirements of the cheW paralogues for chemotaxis. B. Analysis of the requirements of the cheR paralogues for chemotaxis.

Fig. 2

Examination of virulence in an infant mouse competition assay. Each data point represents the CI from an individual mouse. The horizontal bars show the geometric means from each experiment. The CI is calculated as the ratio of mutant to the test strain after a 24 h infection corrected for the input ratio. No differences for in vitro growth for any strain were observed compared with the wild-type. A. The cheW-1G69D, cheW-1G69D + pcheW-1, cheW-1G69D + empty vector (pMMB67EH) and ΔcheW-2,3 mutant strains (all LacZ⁻) were each competed against the wild-type strain (LacZ⁺). The cheW-1G69D mutant was also competed against a LacZ⁺ ΔcheY-3 mutant. B. The ΔcheR-1, ΔcheR-2 and ΔcheR-3 mutants (all LacZ⁻) were competed against the wild-type strain (LacZ⁺). The ΔcheR-2 mutant was also competed against a LacZ⁺ ΔcheY-3 mutant.

Fig. 3

Analysis of CheW-1 content and chemotactic ability of rice-water stool V. cholerae. A. Quantitative Western blot analysis of CheW-1 normalized to flagellin in in vitro grown V. cholerae and in rice-water stools from five patients. Wild-type strain AC-V999 was grown for 18 h at 37°C on LB agar (lane 1) or to stationary phase in LB broth (lane 2). A ΔcheW-1 negative control strain was grown in LB broth to stationary phase (lane 3). The amount of CheW-1 in each stool sample was normalized to flagellin, and the fold decrease relative to the amount of CheW-1 in lane 2 (wild-type, stationary phase) is shown below each lane. Stool samples 1, 2 and 3 are from three patients with V. cholerae O1 El Tor serotype Ogawa; samples 4 and 5 are from two patients with V. cholerae O1 El Tor serotype Inaba. The number of V. cholerae loaded in lanes 1−8 was approximately 3, 3, 2, 0.4, 0.3, 0.3, 1 and 1 × 10⁷ cfu. B. Capillary chemotaxis assays using PBS or a mixture of the attractants listed were performed as competitions using a mixture of stool and in vitro grown (LB agar) V. cholerae. The experiments shown include stool samples of both the Ogawa and Inaba serotypes. Each data point represents the geometric mean of the results from eight capillary tubes from an individual experiment using one patient stool sample. The mean of each set of nine experiments (nine different patient stool samples) is shown by the horizontal bar.

Fig. 4

Competition experiments with rice-water stool V. cholerae O1 El Tor serotype Ogawa. Infant mice competition experiments were performed with V. cholerae O1 El Tor serotype Ogawa from human stool (LacZ⁺) competed against either the wild-type strain AC-V984 or the ΔcheY-3 mutant strain AC-V1736 (both LacZ⁻; both grown overnight in LB broth). Each data point represents the CI from an individual mouse. The horizontal bars show the geometric means from each experiment. The CI is calculated as the ratio of stool V. cholerae to the test strain after a 24 h infection corrected for the input ratio. Similar results were also obtained using an independent stool sample from another patient (data not shown).

Fig. 5

Determination of the ID₅₀ for stool and in vitro grown V. cholerae. Groups of five mice were infected with either stool V. cholerae O1 El Tor serotype Ogawa (filled circles) or strain AC-V999 grown overnight in vitro (empty circles). The doses used are shown on the x-axis, and the percentage of mice infected after 24 h is shown on the y-axis. The ID₅₀ value for each was determined graphically and is indicated by the broken lines.