Response of Vibrio cholerae to Low-Temperature Shifts: CspV Regulation of Type VI Secretion, Biofilm Formation, and Association with Zooplankton

Abstract

The ability to sense and adapt to temperature fluctuation is critical to the aquatic survival, transmission, and infectivity of Vibrio cholerae, the causative agent of the disease cholera. Little information is available on the physiological changes that occur when V. cholerae experiences temperature shifts. The genome-wide transcriptional profile of V. cholerae upon a shift in human body temperature (37°C) to lower temperatures, 15°C and 25°C, which mimic those found in the aquatic environment, was determined. Differentially expressed genes included those involved in the cold shock response, biofilm formation, type VI secretion, and virulence. Analysis of a mutant lacking the cold shock gene cspV, which was upregulated >50-fold upon a low-temperature shift, revealed that it regulates genes involved in biofilm formation and type VI secretion. CspV controls biofilm formation through modulation of the second messenger cyclic diguanylate and regulates type VI-mediated interspecies killing in a temperature-dependent manner. Furthermore, a strain lacking cspV had significant defects for attachment and type VI-mediated killing on the surface of the aquatic crustacean Daphnia magna Collectively, these studies reveal that cspV is a major regulator of the temperature downshift response and plays an important role in controlling cellular processes crucial to the infectious cycle of V. cholerae

Importance: Little is known about how human pathogens respond and adapt to ever-changing parameters of natural habitats outside the human host and how environmental adaptation alters dissemination. Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, experiences fluctuations in temperature in its natural aquatic habitats and during the infection process. Furthermore, temperature is a critical environmental signal governing the occurrence of V. cholerae and cholera outbreaks. In this study, we showed that V. cholerae reprograms its transcriptome in response to fluctuations in temperature, which results in changes to biofilm formation and type VI secretion system activation. These processes in turn impact environmental survival and the virulence potential of this pathogen.

FIG 1

The V. cholerae transcriptome is altered by temperature downshifts. Expression profiles of a selected set of genes in V. cholerae cells grown at 37°C compared to those shifted to 15°C or 25°C for 1 h. Induced expression is represented in yellow, and repressed expression is represented in blue. Differential expression of genes involved in virulence (I), biofilm formation (II), T6SS (III), and cold shock (IV) was observed upon a cold shift. The color scale is shown at the bottom. The details of the expression data are provided in Table S1 in the supplemental material.

FIG 2

Biofilm formation is modulated by temperature. (A) Analysis of biofilm gene expression in response to a low-temperature shift by qRT-PCR. Error bars indicate standard deviations of the results from three biological replicates. All genes exhibited a statistically significant difference between 15°C and 37°C (P < 0.05). (B) Three-dimensional biofilm structures of wild-type V. cholerae and the ΔaphA, ΔvpsR, and ΔvpsT mutants formed at 37°C, 25°C, and 15°C after 24 h of incubation. Images shown are from one representative experiment of three independent experiments. The scale bars represent 40 μm.

FIG 3

The T6SS is modulated by temperature. (A) Analysis of hcp gene expression by qRT-PCR. Error bars indicate standard deviations of the results from three biological replicates (P < 0.05). (B) Hcp production levels by Western blot analysis before (at 37°C) and 1 h after (15°C or 25°C) a low-temperature shift. BSA, bovine serum albumin control; α, anti. (C) T6SS-mediated killing of E. coli at 37°C, 25°C, and 15°C. Error bars indicate standard deviations of the results from three biological replicates. *, P < 0.05; n.s., not significantly different (P > 0.05).

FIG 4

Expression of three cold shock genes is differentially regulated by temperature. Analysis of cold shock gene expression in response to a shift from 37°C to 15°C (A) or 25°C (B) by qRT-PCR. Error bars indicate standard deviations of the results from three biological replicates. *, P < 0.05; **, P < 0.005; n.s., not significantly different (P > 0.05).

FIG 5

cspV regulates biofilm formation. (A) Biofilm gene expression in the ΔcspV mutant compared with wild-type V. cholerae 1 h after a shift from 37°C to 15°C by qRT-PCR. All genes exhibited a statistically significant difference between the ΔcspV mutant and the wild type at 15°C (*,P < 0.05). Error bars indicate standard deviations of the results from four biological replicates. (B) Three-dimensional biofilm structures of wild-type V. cholerae and the strain lacking cspV formed at 25 and 15°C after 24 h of incubation. The images shown are from one representative experiment of three independent experiments. Scale bars represent 40 μm. (C) c-di-GMP levels in the ΔcspV mutant and wild-type V. cholerae grown at 37°C and then shifted to 15°C for 1 h. (A and C) Error bars indicate standard deviations of the results from four biological replicates. *, P < 0.05; n.s., not significantly different (P > 0.05).

FIG 6

CspV impacts the T6SS. Analysis of hcp expression by qRT-PCR in the ΔcspV mutant and wild-type V. cholerae upon a shift from 37°C to 15°C (A) or to 25°C (B). Error bars indicate standard deviations of the results from four biological replicates. (C) Hcp production levels by Western blot analysis in the ΔcspV mutant at 25°C. (D) T6SS-mediated killing of E. coli wild-type and mutant strains at 37°C, 25°C, and 15°C. Error bars indicate standard deviations of the results from three biological replicates. *, P < 0.05; n.s., not significantly different (P > 0.05).

FIG 7

CspV impacts attachment to live D. magna. (A) SEM images showing the wild type and ΔcspV mutant attached to D. magna. Scale bars represent 5 μM. (B and C) Analysis of the abilities of wild-type V. cholerae, the ΔmshA mutant, and the ΔcspV mutant to colonize D. magna (B) and survive in ADaM (C). Error bars indicate standard deviations of results from eight biological replicates. *, P < 0.05; n.s., P > 0.05.

FIG 8

CspV contributes to interspecies killing on the surface of live D. magna. T6SS-mediated killing of Aeromonas sp. in vitro (A) and on the surface of D. magna (B). Error bars indicate standard deviations of the results from eight biological replicates. *, P < 0.05; n.s., not significantly different (P > 0.05).