Alkaline pH Increases Swimming Speed and Facilitates Mucus Penetration for Vibrio cholerae

Abstract

Intestinal mucus is the first line of defense against intestinal pathogens. It acts as a physical barrier between epithelial tissues and the lumen that enteropathogens must overcome to establish a successful infection. We investigated the motile behavior of two Vibrio cholerae strains (El Tor C6706 and Classical O395) in mucus using single-cell tracking in unprocessed porcine intestinal mucus. We determined that V. cholerae can penetrate mucus using flagellar motility and that alkaline pH increases swimming speed and, consequently, improves mucus penetration. Microrheological measurements indicate that changes in pH between 6 and 8 (the physiological range for the human small intestine) had little effect on the viscoelastic properties of mucus. Finally, we determined that acidic pH promotes surface attachment by activating the mannose-sensitive hemagglutinin (MshA) pilus in V. cholerae El Tor C6706 without a measurable change in the total cellular concentration of the secondary messenger cyclic dimeric GMP (c-di-GMP). Overall, our results support the hypothesis that pH is an important factor affecting the motile behavior of V. cholerae and its ability to penetrate mucus. Therefore, changes in pH along the human small intestine may play a role in determining the preferred site for V. cholerae during infection.IMPORTANCE The diarrheal disease cholera is still a burden for populations in developing countries with poor sanitation. To develop effective vaccines and prevention strategies against Vibrio cholerae, we must understand the initial steps of infection leading to the colonization of the small intestine. To infect the host and deliver the cholera toxin, V. cholerae has to penetrate the mucus layer protecting the intestinal tissues. However, the interaction of V. cholerae with intestinal mucus has not been extensively investigated. In this report, we demonstrated using single-cell tracking that V. cholerae can penetrate intestinal mucus using flagellar motility. In addition, we observed that alkaline pH improves the ability of V. cholerae to penetrate mucus. This finding has important implications for understanding the dynamics of infection, because pH varies significantly along the small intestine, between individuals, and between species. Blocking mucus penetration by interfering with flagellar motility in V. cholerae, reinforcing the mucosa, controlling intestinal pH, or manipulating the intestinal microbiome will offer new strategies to fight cholera.

Keywords: Vibrio cholerae; cell tracking; flagellar motility; intestinal mucus; microrheology; pH.

FIG 1

V. cholerae Classical O395 flagellar motility through unprocessed porcine intestinal mucus (PIM). (A) Mucus scraped from the medial part of the small intestine of an adult pig. (B) Representative epifluorescence image at ×40 magnification of V. cholerae Classical O395 (expressing the green fluorescent protein) swimming in unprocessed pig intestinal mucus between two glass coverslips. (C) Motile cells can be distinguished from nonmotile by comparing the trajectories of effective diffusion coefficients. (D) Distributions of diffusion coefficients from individual trajectories in liquid and PIM. Motile wild-type V. cholerae O395 (WT) was compared to a nonmotile mutant (flrA) in PIM. Each distribution represents 3 to 12 replicates combining between 500 and 6,000 individual trajectories (between 250 and 1,700 min of cumulative time).

FIG 2

Effects of pH on the motility of V. cholerae through porcine intestinal mucus. (A) Distributions of diffusion coefficient from individual trajectories in mucus buffered at different pH. Cells with a diffusion coefficient of <10^−0.5 μm²/s were categorized as nonmotile or trapped and were excluded from the following analyses. (B) Distributions of swimming speed from the motile cell populations. (C) Distributions of directional persistence time scales from the motile cell populations. Each distribution represents 8 to 12 replicates combining between 6,000 and 19,000 individual trajectories (between 1,000 and 2,600 min of cumulative time). Circles indicate means for the motile populations.

FIG 3

Passive microrheology of porcine intestinal mucus. (A) Mean-squared displacement (mean sq. disp.) of PEG-coated 1-μm polystyrene beads with respect to time at different pHs (represented by different colors) and after incubation with V. cholerae. The data points (circles) are the average trajectories from 4 to 6 replicates (10 to 25 individual trajectories). A polynomial fit to the data was used to calculate the storage and loss moduli using the generalized Stokes-Einstein relation. (B) Storage moduli (elasticity) of porcine intestinal mucus at different pHs. (C) Loss moduli (viscosity) of porcine intestinal mucus at different pHs. (D) Distributions of the diffusion coefficient of nonmotile V. cholerae (flrA) after incubation in mucus at pH 8. Each distribution represents 6 replicates combining between 1,000 and 2,000 individual trajectories (∼150 min of cumulative time). Circles indicate means.

FIG 4

Effects of pH on the spreading of V. cholerae colonies in soft agar. (A) Representative colonies from the Classical O395 and El Tor C6706 (wild type and mshA) at different pHs (bar is 10 mm). (B) Measured colony diameters at different pHs for all experimental replicates. (C) Measured growth rates in batch cultures at different pHs for all experimental replicates.

FIG 5

Effects of pH on V. cholerae flagellar motility. (A) Distributions of diffusion coefficient of Classical O395 from single-cell trajectories in spent medium (Spent) or in fresh medium at different pHs. Trajectories below 10 μm²/s were categorized as nonmotile and excluded from the remaining analyses. (B) Distributions of swimming speed from the motile cell populations. (C) Distributions of trajectory directional persistence from the motile cell populations. (D) Distributions of diffusion coefficients of El Tor C6706 from single-cell trajectories in spent medium (Spent) or in fresh medium at different pHs. An mshA mutant was also tracked. (E) Distributions of swimming speed from the motile cell populations. (F) Distributions of trajectory directional persistence from the motile cell populations. Each distribution represents 3 replicates combining between 2,000 and 10,000 individual trajectories (between 100 and 500 min of cumulative time). Circles indicate means for the motile populations.

FIG 6

Effects of inhibiting the Na⁺-NQR pump on flagellar motility in V. cholerae. (A) Distributions of diffusion coefficient in liquid and 0.3% (wt/vol) agarose buffered at pH 8 and with the addition of 100 μM HQNO. Each distribution represents 6 replicates combining between 1,000 and 3,000 individual trajectories (∼1,000 min of cumulative time). (B) Distributions of swimming speed at different pHs as a function of HQNO concentration. Each distribution represents at least 6 replicates combining between 2,000 and 6,000 individual trajectories (between 500 and 1,000 min of cumulative time). Circles indicate means for the motile populations.