Removal of a Membrane Anchor Reveals the Opposing Regulatory Functions of Vibrio cholerae Glucose-Specific Enzyme IIA in Biofilms and the Mammalian Intestine

Abstract

The Vibrio cholerae phosphoenolpyruvate phosphotransferase system (PTS) is a well-conserved, multicomponent phosphotransfer cascade that coordinates the bacterial response to carbohydrate availability through direct interactions of its components with protein targets. One such component, glucose-specific enzyme IIA (EIIA^Glc), is a master regulator that coordinates bacterial metabolism, nutrient uptake, and behavior by direct interactions with cytoplasmic and membrane-associated protein partners. Here, we show that an amphipathic helix (AH) at the N terminus of V. cholerae EIIA^Glc serves as a membrane association domain that is dispensable for interactions with cytoplasmic partners but essential for regulation of integral membrane protein partners. By deleting this AH, we reveal previously unappreciated opposing regulatory functions for EIIA^Glc at the membrane and in the cytoplasm and show that these opposing functions are active in the laboratory biofilm and the mammalian intestine. Phosphotransfer through the PTS proceeds in the absence of the EIIA^Glc AH, while PTS-dependent sugar transport is blocked. This demonstrates that the AH couples phosphotransfer to sugar transport and refutes the paradigm of EIIA^Glc as a simple phosphotransfer component in PTS-dependent transport. Our findings show that Vibrio cholerae EIIA^Glc, a central regulator of pathogen metabolism, contributes to optimization of bacterial physiology by integrating metabolic cues arising from the cytoplasm with nutritional cues arising from the environment. Because pathogen carbon metabolism alters the intestinal environment, we propose that it may be manipulated to minimize the metabolic cost of intestinal infection.IMPORTANCE The V. cholerae phosphoenolpyruvate phosphotransferase system (PTS) is a well-conserved, multicomponent phosphotransfer cascade that regulates cellular physiology and virulence in response to nutritional signals. Glucose-specific enzyme IIA (EIIA^Glc), a component of the PTS, is a master regulator that coordinates bacterial metabolism, nutrient uptake, and behavior by direct interactions with protein partners. We show that an amphipathic helix (AH) at the N terminus of V. cholerae EIIA^Glc serves as a membrane association domain that is dispensable for interactions with cytoplasmic partners but essential for regulation of integral membrane protein partners. By removing this amphipathic helix, hidden, opposing roles for cytoplasmic partners of EIIA^Glc in both biofilm formation and metabolism within the mammalian intestine are revealed. This study defines a novel paradigm for AH function in integrating opposing regulatory functions in the cytoplasm and at the bacterial cell membrane and highlights the PTS as a target for metabolic modulation of the intestinal environment.

Keywords: Vibrio cholerae; amphipathic helix; biofilms; phosphoenolpyruvate phosphotransferase system; sugar transport.

FIG 1

The N terminus of EIIA^Glc forms an AH that mediates membrane association and is essential for PTS-dependent activation of sugar transport through EIICs. (A and B) Immunoblots of the lysate, cytoplasmic, and membrane fractions of control or ΔEIIBC^Glc ΔmshH Δcya mutant (ΔΔΔ) V. cholerae cells expressing affinity-tagged full-length EIIA^Glc (FL) or Δ16 EIIA^Glc (Δ16). The membrane association index (MAI) with respect to FL is given below. (C) Immunoblots of the lysate, cytoplasmic, and membrane fractions of wild-type V. cholerae expressing neon green alone (AH−) or as a C-terminal fusion to the AH of EIIA^Glc (AH+) encoded on a plasmid. The membrane association index (MAI) with respect to AH− is given below. (D to F) A₆₁₅ measured over time for V. cholerae expressing the indicated EIIA^Glc alleles or deletion (ΔEIIA) and cultured in minimal medium supplemented with pH indicators and sucrose (D), trehalose (E), and N-acetylmuramic acid (F). The decrease in A₆₁₅ indicates medium acidification due to transport and fermentation of the indicated sugar. Results are representative of three independent experiments.

FIG 2

Repression of alternative glucose transport and biofilm formation is mediated by phospho-EI and relieved by EIIA^Glc AH-independent phosphotransfer to EIIB. (A and B) Phos-tag acrylamide gel electrophoresis and anti-V5 antibody immunoblotting of the affinity-tagged full-length EIIA^Glc (FL), full-length EIIA^Glc with histidine 91 mutated to alanine (FL^H91A), Δ16 EIIA^Glc (Δ16), Δ16 EIIA^Glc with histidine 91 mutated to alanine (Δ16^H91A), or EIIA^Glc in a strain with histidine 189 of EI mutated to alanine (EI^H189A) extracted from cells grown in LB alone or supplemented with glucose. (C and D) A₆₁₅ measured over time for V. cholerae expressing the indicated EIIA^Glc alleles or deletion (ΔEIIA) and cultured in minimal medium supplemented with pH indicators and glucose. (E and F) Quantification of biofilm formation by the indicated V. cholerae strains cultured in MM supplemented with glucose. Data represent the means from three biological replicates, and error bars represent the standard deviation. Statistical significance was calculated using a one-way analysis of variance followed by Tukey’s multiple-comparison test. ns, not significant. (G) A₆₁₅ measured over time in cultures of the indicated V. cholerae control or mutant strains. Minimal medium supplemented with pH indicators and glucose was used. Results are representative of three independent experiments. (H) Quantification of biofilm formation by the indicated V. cholerae strains cultured in MM supplemented with glucose. Data represent the means from three biological replicates, and error bars represent the standard deviation. Statistical significance was calculated using a one-way analysis of variance followed by Tukey’s multiple-comparison test.

FIG 3

EIIA^Glc regulation of integral membrane protein partners is AH dependent. (A to F) Medium acidification (A and B) and growth curves (C to F) carried out in minimal medium supplemented with pH indicators and maltose alone (maltose) or supplemented with α-methylglucoside (maltose + α-MG). (G and H) Northern blot assay of the sRNA csrB in LB-cultured, mid-log-phase cells of the indicated genotype. Samples were harvested at the noted times after addition of rifampin to arrest transcript elongation.

FIG 4

AH swapping establishes that the function of the V. cholerae EIIA^Glc AH is not amino acid sequence dependent. (A) Anti-V5 antibody immunoblot assays of the lysate (L), cytoplasmic (C), and membrane (M) fractions of V. cholerae expressing the indicated affinity-tagged EIIA^Glc constructs. The membrane association index (MAI) with respect to FL is given at the right. (B) Phos-tag acrylamide gel electrophoresis and anti-V5 antibody immunoblot assay of V. cholerae expressing the indicated EIIA^Glc alleles. A₆₁₅ measured over time in cultures of V. cholerae expressing FL-EIIA^Glc (FL), K6P-EIIA^Glc (K6P), 1X-MreB EIIA^Glc (1XMreB), 2X-MreB EIIA^Glc (2XMreB), EIIA^Glc-MinD (MinD), and ΔEIIA^Glc (ΔEIIA). (C to F) Minimal medium supplemented with pH indicators and sucrose (C and D) or glucose (E and F) was used. Results are representative of three independent experiments. (G) Quantification of biofilm formation by strains as indicated in minimal medium supplemented with glucose. Bars represent the mean from biological triplicates. Error bars indicate the standard deviation. Statistical significance was calculated using a one-way analysis of variance followed by Tukey’s multiple-comparison test. (H) Northern blot of the sRNA csrB in LB-cultured, mid-log-phase V. cholerae cells that express the indicated EIIA^Glc alleles. Samples were harvested at the noted times after addition of rifampin to arrest transcription elongation. (I and J) Immunoblots of adenylate cyclase (AC) (I) and MshH (J) in cell lysates and the bound fractions that coimmunoprecipitated with FL EIIA^Glc or Δ16 EIIA^Glc. The partner association index (PAI) with respect to FL is given at the right.

FIG 5

The metabolic environments created by V. cholerae Δ16 EIIA^Glc and ΔEIIA^Glc strains in cecal fluid are distinct and independent of classical virulence factors. (A and B) Colonization levels of indicated V. cholerae strains in the ileum (A) and colon (B). (C) qRT-PCR quantification of tcpA in ileal tissue. (D) Cecal fluid volume. (E) qRT-PCR quantification of ctxA in ileal tissue. (F and G) Edema (F) and congestion (G) score according to rubric shown in Data Set S1 and Fig. S4. A one-way analysis of variance followed by Dunnett’s multiple-comparison test was used to calculate statistical significance. (H and I) Cluster analysis of the 25 most significantly different metabolites in the supernatants of cecal fluid harvested from infant rabbits (H) or spent LB broth supernatants (I) inoculated with the indicated V. cholerae strains. Statistical significance was calculated by a one-way analysis of variance followed by a Fisher least significant difference test. Hierarchical clustering was performed using a Euclidean distance algorithm within Morpheus software from the Broad Institute (54). Intensities were mapped to colors using the minimum and maximum of each row independently with red representing values higher than the mean and blue representing values lower than the mean.