PilZ domain proteins bind cyclic diguanylate and regulate diverse processes in Vibrio cholerae

Abstract

Cyclic diguanylate (c-di-GMP) is an allosteric activator and second messenger implicated in the regulation of a variety of biological processes in diverse bacteria. In Vibrio cholerae, c-di-GMP has been shown to inversely regulate biofilm-specific and virulence gene expression, suggesting that c-di-GMP signaling is important for the transition of V. cholerae from the environment to the host. However, the mechanism behind this regulation remains unknown. Recently, it was proposed that the PilZ protein domain represents a c-di-GMP-binding domain. Here we show that V. cholerae PilZ proteins bind c-di-GMP specifically and are involved in the regulation of biofilm formation, motility, and virulence. These findings confirm a role for PilZ proteins as c-di-GMP-sensing proteins within the c-di-GMP signaling network.

FIGURE 1. Binding of radiolabeled c-di-GMP to purified V. cholerae PilZ domain proteins

A–C, lanes 1–7, PlzD, PlzE, PlzA, PlzB, PlzC, CheY, and mock-purified, respectively. A, after native electrophoretic separation conditions, proteins were transferred to nitrocellulose and probed with [³²P]c-di-GMP. PlzD and PlzC bound c-di-GMP. B, to avoid potential protein denaturation during transfer to nitrocellulose, the PilZ domain proteins and controls were spotted directly onto nitrocellulose and probed. Again, PlzD and PlzC bound c-di-GMP. C, after denaturing electrophoretic separation conditions, proteins were transferred to nitrocellulose, renatured, and probed. Only PlzC bound c-di-GMP. D, to test the specificity of PlzD and PlzC binding to c-di-GMP, proteins spotted to nitrocellulose were probed with [³²P]c-di-GMP alone, [³²P]c-di-GMP plus excess cold c-di-GMP, or [³²P]c-di-GMP plus excess cold GTP, as indicated to the right. Incubation with excess cold c-di-GMP dramatically decreased the amount of binding to [³²P]c-di-GMP, whereas incubation with excess cold GTP did not, indicating that binding of c-di-GMP to PlzD and PlzC is specific. The fractions of [³²P]c-di-GMP bound in the presence of cold competitor compared with that of c-di-GMP alone are shown below each lane.

FIGURE 2. Conserved PilZ domain residues are required for c-di-GMP binding

A, multiple alignment of V. cholerae PilZ domains with the first 39 amino acid residues from the AcsA1 PilZ domain using MAFFT (available on the World Wide Web at www.biophys.kyoto-u.ac.jp/~katoh/programs/align/mafft/). V. cholerae proteins are listed by their locus names, and AcsA1 represents the PilZ domain from cellulose synthase 1 from G. xylinus. Each of the two PilZ domains of PlzC are included and labeled accordingly. Highly conserved residues, those present in six of seven PilZ domains, are marked with an asterisk at the top. Conserved hydrophobic residues are marked with a filled circle. B, ribbon diagram of one monomer of the PlzDdimer showing the locations of highly conserved residues targeted for mutation, Arg¹³⁶, Arg¹⁴⁰, Asp¹⁶², Ser¹⁶⁴, and Gly¹⁶⁷. C, Western blot of purified His₆-tagged wild-type and point mutant PlzD proteins. Protein purification and Western blots were performed as described under “Experimental Procedures.” D, binding of [³²P]c-di-GMP by PlzD point mutants. Binding of 10 µg of each His₆-tagged purified protein (CheY, PlzD, PlzDR136A, PlzDR140A, PlzDD162A, PlzDS164A, and PlzDG167A) to [³²P]c-di-GMP was measured in solution as described under “Experimental Procedures.” The amount of [³²P]c-di-GMP bound is shown relative to that bound by wild-type PlzD. The mean and S.D. values from triplicate assays are shown.

FIGURE 3. PilZ protein PlzB regulates motility in V. cholerae

V. cholerae strains were inoculated into chemotaxis plates and incubated at 30 °C for 16 h. A (tryptone-based chemotaxis plate), wild type (wt), AC61; vieAE170A, AC1596; ΔflaA, AC1539; ΔplzB, AC2342; ΔplzB (pPlzB) (pAC2140), AC2348; ΔplzD, AC2344; ΔplzC, AC2346; vieAE170A ΔplzB, AC2343; vieAE170A ΔplzD, AC2345; vieAE170A ΔplzC, AC2347. B (tryptone-based chemotaxis plate), wt, AC61; ΔplzB, AC2342; plzBR96A, AC2458; plzBR96A (pPlzB) (pAC2140), AC2459. Magnification is X 3. C (M9 + NRES chemotaxis plate), wt, AC61; vieAE170A, AC1596; ΔplzB, AC2342; ΔplzB (pPlzB) (pAC2140), AC2348; ΔplzD, AC2344; ΔplzC, AC2346; ΔplzC (pPlzC) (pAC2141), AC2347. D (M9 + NRES chemotaxis plate plus 1 mm isopropyl-β-d-thiogalactopyranoside), wt (pMMB67EH), AC1109; vieAE170A (pMMB67EH), wt (pPlzA) (pAC2138), AC2144; wt (pPlzB) (pAC2140), AC2146; wt (pPlzC) (pAC2141), AC2147; wt (pPlzD) (pAC2137), AC2143; wt (pPlzE) (pAC2139), AC2145.

FIGURE 4. Biofilm formation is regulated by V. choleraePilZ protein PlzB

V. cholerae strains were grown in LB broth standing cultures at room temperature for 16 h, and adherent biofilms were stained with crystal violet. Staining was quantified by solubilizing bound crystal violet in ethanol and measuring the absorbance at 570 nm. The mean and S.D. values from triplicate cultures are shown. wt, AC61; vieAE170A, AC1596; vpsR∷pGP704, AC1869; ΔplzB, AC2342; plzBR96A, AC2458; ΔplzB (pPlzB) (pAC2140), AC2348; plzBR96A (pPlzB) (pAC2140), AC2459; vieAE170A ΔplzB, AC2342; vieAE170A ΔplzB (pPlzB) (pAC2140), AC2349; ΔplzC, AC2346, vieAE170A ΔplzC, AC2347; vieAE170A ΔplzC (pPlzC) (pAC2141), AC2347; ΔplzD, AC2344; vieAE170A ΔplzD, AC2345.

FIGURE 5. V. cholerae ΔplzB and plzBR96A strains are attenuated for virulence

Competition assays were performed using the infant mouse model of infection. A, strains ΔplzB (AC2342), plzBR96A (AC2458), were competed against wild-type O395 (AC50). Strains ΔplzB (pPlzB (pAC2140)) (AC2348) and plzBR96A (pPlzB (pAC2140)) (AC2459) were competed against O395 (pMMB67EH) (AC1109), which contained the empty vector. B, strains ΔplzC (AC2356), ΔplzD (AC2344), ΔplzC ΔplzD (AC2550), and ΔplzC plzDR140A (AC2553) were competed against wild-type O395 (AC50). Strains ΔplzC ΔplzD (pPlzC (pAC2141)) (AC2551), ΔplzC ΔplzD (pPlzD (pAC2137)) (AC2552), and ΔplzC plzDR140A (pPlzD (pAC2137)) (AC2554) were competed against O395 (pMMB67EH) (AC1109). The competitive index is the ratio of mutant to wild-type bacteria recovered from the small intestine corrected for the input ratio. Each data point represents the competitive index from an individual mouse; the gray bar represents the geometric mean. The ΔplzB, plzBR96A, ΔplzC ΔplzD, and ΔplzC plzDR140A strains are significantly attenuated (p < 0.01) by Student’s two-tailed t test.