Calcium inhibits bap-dependent multicellular behavior in Staphylococcus aureus

Abstract

Bap (biofilm-associated protein) is a 254-kDa staphylococcal surface protein implicated in formation of biofilms by staphylococci isolated from chronic mastitis infections. The presence of potential EF-hand motifs in the amino acid sequence of Bap prompted us to investigate the effect of calcium on the multicellular behavior of Bap-expressing staphylococci. We found that addition of millimolar amounts of calcium to the growth media inhibited intercellular adhesion of and biofilm formation by Bap-positive strain V329. Addition of manganese, but not addition of magnesium, also inhibited biofilm formation, whereas bacterial aggregation in liquid media was greatly enhanced by metal-chelating agents. In contrast, calcium or chelating agents had virtually no effect on the aggregation of Bap-deficient strain M556. The biofilm elicited by insertion of bap into the chromosome of a biofilm-negative strain exhibited a similar dependence on the calcium concentration, indicating that the observed calcium inhibition was an inherent property of the Bap-mediated biofilms. Site-directed mutagenesis of two of the putative EF-hand domains resulted in a mutant strain that was capable of forming a biofilm but whose biofilm was not inhibited by calcium. Our results indicate that Bap binds Ca2+ with low affinity and that Ca2+ binding renders the protein noncompetent for biofilm formation and for intercellular adhesion. The fact that calcium inhibition of Bap-mediated multicellular behavior takes place in vitro at concentrations similar to those found in milk serum supports the possibility that this inhibition is relevant to the pathogenesis and/or epidemiology of the bacteria in the mastitis process.

FIG. 1.

EF-hand-like domains in the Bap sequence. (A) Positions of the potential EF hands, shown in a diagram representing the structural organization of Bap. S, signal peptide; A, region A; B, region B; C, C-repeat region; D, region rich in serine-aspartate (SD) repeats; WM, cell wall anchor, membrane-spanning region, and positively charged tail. The positions of the potential EF-hand-like motifs, designated EF1 to EF4, are indicated by vertical grey lines. (B) Sequences of the four potential EF-hand motifs in Bap and degrees of similarity to the consensus sequence. Residues that do not conform to the EF-hand loop consensus sequence are in lowercase letters. The positions that participate in coordinating Ca²⁺ are underlined. aa, amino acid.

FIG. 2.

Effects of divalent cations and chelating agents on V329 biofilm formation. Bacteria were inoculated onto microtiter plates containing TSB-glucose supplemented with Ca²⁺, Mg²⁺, Mn²⁺, EGTA, or EDTA at the final concentrations indicated. After 24 h of incubation the microplates were washed and stained with crystal violet. (A) Effect of Ca²⁺ addition on the ability of S. aureus strains V329, M556, 15981, and 12313 to form biofilms. (B) Effect of Ca²⁺, Mg²⁺, and Mn²⁺ on the ability of V329 to form a biofilm. (C) Effect of EGTA addition on the biofilm formed by V329 in TSB-glucose. (D) Effect of EDTA addition on the biofilm formed by V329 in TSB-glucose. The results of a representative experiment are shown.

FIG. 3.

Ca²⁺ inhibits V329 bacterial clumping in liquid culture tubes. (A) Culture tubes containing TSB-glucose were inoculated with V329 or M556, and Ca²⁺, EGTA, and EDTA were added as indicated to final concentrations of 10, 1.25, and 1.25 mM, respectively. After 16 h of incubation at 37°C, the tubes were photographed. (B) Percentages of suspended cells in V329 and M556 liquid cultures grown in TSB-glucose supplemented with different amounts of Ca²⁺. The results of a representative experiment are shown. (C) Same as panel B, except that EDTA was added to the culture tubes. Symbols: ▪, V329; ▴, M556.

FIG. 4.

Expression of Bap changes the multicellular behavior of the Newman strain. (A) Biofilm formation by strains Newman and Newman_Bap on microtiter plates containing TSB-glucose supplemented with different amounts of Ca²⁺. Newman cells did not form a biofilm after 24 h of incubation, whereas the Newman_Bap mutant that contained a chromosomal copy of bap formed a biofilm and this biofilm was inhibited by millimolar amounts of added Ca²⁺. (B) Liquid culture tubes containing Newman and Newman_Bap grown overnight in TSB-glucose or TSB-glucose supplemented with 10 mM Ca²⁺. Liquid cultures of Newman_Bap exhibited a greater degree of bacterial clumping than Newman cultures, and the clumping was inhibited by addition of 10 mM Ca²⁺ to the growth medium. The results of a representative experiment are shown. (C) Colony morphology on Congo red agar plates incubated at 37°C for 24 h for strains V329, M556, Newman, and Newman_Bap.

FIG. 5.

Mutation of the potential EF-hand motifs EF2 and EF3. (A) Amino acid substitutions introduced into the chromosomal copy of bap in V329 to configure mutant V329_EF23. The positions at which residues were replaced are underlined, and the arrows indicate the residues introduced into the mutant. (B) Formation of biofilms on microplates by V329 and mutant V329_EF23 grown overnight in TSB-glucose supplemented with different amounts of Ca²⁺. (C) Liquid culture tubes containing V329_EF23 grown overnight in TSB-glucose or TSB-glucose supplemented with 10 mM Ca²⁺. The degree of bacterial clumping of the mutant was not affected by Ca²⁺ addition. The results of a representative experiment are shown.

FIG. 6.

Analysis of surface protein preparations. (A) SDS-PAGE of surface protein preparations obtained under isosmotic conditions. Lane M, Bio-Rad prestained broad-range M_r standards; lane 1, V329 grown in TSB-glucose; lane 2, V329 grown in TSB-glucose supplemented with 10 mM Ca²⁺; lane 3, mutant V329_EF23 grown in TSB-glucose; lane 4, mutant V329_EF23 grown in TSB-glucose supplemented with 10 mM Ca²⁺; lane 5, negative control preparation obtained from Bap-negative S. aureus strain CH142 grown in TSB-glucose. (B) Western blot analysis with anti-Bap serum. For lane contents see above. The amounts of each preparation loaded for SDS-PAGE and Western analysis were 10 and 0.2 μl, respectively. The arrow indicates the position of full-size Bap.

FIG. 7.

Model illustrating the effect of Ca²⁺ on Bap conformation and biofilm formation. Metal-free Bap participates in the series of interactions that lead to cell-to-cell adhesion and biofilm formation. Occupation of the inhibitory site(s) by Ca²⁺ induces a conformational change in Bap and renders the protein noncompetent for biofilm formation. The ligand for Bap on neighboring cells is illustrated as other Bap molecules, but it could be a different type of molecule. Only at high Ca²⁺ concentrations would Bap be saturated with the metal, whereas chelating agents would favor the metal-free form.