Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System Control of Carbohydrate Transport, Biofilm Formation, and Colonization of the Germfree Mouse Intestine

PTS sugars activate PTS gene transcription. EI, EIIA^Glc, HPr, and FPr transcript levels in wild-type V. cholerae grown in MM alone or MM supplemented with the indicated carbon sources were analyzed by quantitative RT-PCR. Three experimental replicates were performed. The data were analyzed using the ΔΔCT method for comparison to measurements from bacteria grown in MM alone. clpX was used as a standard.

HPr and FPr repress biofilm-associated growth and vps gene transcription. The total growth and biofilm-associated growth of and vps transcription in wild-type (WT) V. cholerae and various PTS mutants were quantified. (A) Biofilm-associated and total growth in MM supplemented with glucose. (B) Biofilm-associated and total growth in MM supplemented with pyruvate. (C) β-Galactosidase activities of strains carrying a chromosomal vps-lacZ fusion at the lacZ site in MM supplemented with pyruvate. Error bars indicate the standard deviations of results from at least three experimental replicates. Measurements for the indicated V. cholerae mutants were compared to those for wild-type V. cholerae by using the t test of statistical significance. Biofilm measurements that are significantly different from wild-type biofilm measurements are marked with an asterisk (P < 0.0005). β-Galactosidase measurements for all mutants were significantly different from that for the wild-type strain (P < 0.01). Furthermore, the β-galactosidase activity of the ΔEI mutant was significantly different from that of the ΔHPr ΔFPr mutant (P = 0.002).

HPr and FPr must be phosphorylated to repress biofilm-associated growth. (A) Quantification of total and biofilm-associated growth of wild-type (WT) V. cholerae harboring a pBAD expression vector carrying a control sequence (pCTL) or of a ΔHPr ΔFPr mutant harboring a pBAD expression vector carrying either a control sequence (pCTL), the wild-type HPr gene (pHPr), a sequence encoding an unphosphorylatable form of HPr (pHPr{H15A}), the wild-type FPr gene (pFPr), a sequence encoding an unphosphorylatable form of FPr (FPr{H324A}), or a sequence encoding the C-terminal HPr-like domain of FPr including residues 309 to 401 (pFPr^trunc) in MM supplemented with pyruvate. Protein expression was induced with 0.04% l-arabinose. Schematic representations of the rescue constructs provided in trans are illustrated above the data. Error bars indicate the standard deviations of results from three experimental replicates. Asterisks indicate measurements significantly different from the measurement for the ΔHPr ΔFPr (pCTL) strain (P < 0.0014). (B) Western blots demonstrating expression of the relevant protein from the rescue plasmid. A V5 epitope tag was used for visualization.

HPr and FPr are downstream of EI in the pathways regulating biofilm growth. (A) Quantification of total and biofilm-associated growth of a ΔPTS mutant and a ΔPTS ΔFPr mutant harboring a pBAD expression vector carrying either a control sequence (pCTL) or the wild-type gene encoding EI (pEI) in MM supplemented with pyruvate. Protein expression was induced with 0.04% l-arabinose. Error bars indicate the standard deviations of results from three experimental replicates. The asterisk indicates a measurement significantly different from that for the ΔPTS (pCTL) mutant (P = 0.0019). (B) Western blot demonstrating expression of the relevant protein from the rescue plasmid. A V5 epitope tag was used for visualization. WT, wild type.

V. cholerae persists in the distal portion of the adult germfree mouse intestine for 1 month. (A) Enumeration of V. cholerae CFU in stool pellets over time. Mice were infected by having free access to V. cholerae-inoculated saline solution for 24 h. At each indicated time point, two stool pellets were collected from each mouse. Stool pellets from cohoused mice were pooled. The samples were weighed and homogenized in 1 ml of PBS. Serial dilutions of this suspension were spread onto LB agar plates, and the resulting colonies were enumerated. Data were normalized with respect to stool weight. This experiment included 10 mice housed 2 to a cage. Cage 1 mice were monitored for 6 days, mice in cages 2 and 3 were monitored for 13 days, and cage 4 and 5 mice were monitored for 23 days. (B) Quantification of V. cholerae bacteria in the proximal small intestine (1), the middle small intestine (2), the distal small intestine (3), the cecum (4), and the large intestine (5) at 6 and 36 days postinoculation. Measurements at 6 days are shown in blue, while measurements at 36 days are shown in red. Bars represent the geometric means of measurements. Numbers of CFU in all segments of the small intestine were statistically significantly different from those in the large intestine (P = 0.0286). (C) Micrographs of different hematoxylin- and eosin-stained portions of the small intestine of a V. cholerae-infected, previously germfree mouse. The intestine was fixed in neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Bar, 10 μm.

V. cholerae is found primarily in the lumen of the large intestine rather than on the epithelial surface. Micrographs of Gram-stained sections of the small intestine of a previously germfree, V. cholerae-infected mouse are shown. Bacteria are indicated by arrows. Bar, 10 μm.