A zinc-dependent adhesion module is responsible for intercellular adhesion in staphylococcal biofilms

Abstract

Hospital-acquired bacterial infections are an increasingly important cause of morbidity and mortality worldwide. Staphylococcal species are responsible for the majority of hospital-acquired infections, which are often complicated by the ability of staphylococci to grow as biofilms. Biofilm formation by Staphylococcus epidermidis and Staphylococcus aureus requires cell-surface proteins (Aap and SasG) containing sequence repeats known as G5 domains; however, the precise role of these proteins in biofilm formation is unclear. We show here, using analytical ultracentrifugation (AUC) and circular dichroism (CD), that G5 domains from Aap are zinc (Zn(2+))-dependent adhesion modules analogous to mammalian cadherin domains. The G5 domain dimerizes in the presence of Zn(2+), incorporating 2-3 Zn(2+) ions in the dimer interface. Tandem G5 domains associate in a modular fashion, suggesting a "zinc zipper" mechanism for G5 domain-based intercellular adhesion in staphylococcal biofilms. We demonstrate, using a biofilm plate assay, that Zn(2+) chelation specifically prevents biofilm formation by S. epidermidis and methicillin-resistant S. aureus (MRSA). Furthermore, individual soluble G5 domains inhibit biofilm formation in a dose-dependent manner. Thus, the complex three-dimensional architecture of staphylococcal biofilms results from the self-association of a single type of protein domain. Surface proteins with tandem G5 domains are also found in other bacterial species, suggesting that this mechanism for intercellular adhesion in biofilms may be conserved among staphylococci and other Gram-positive bacteria. Zn(2+) chelation represents a potential therapeutic approach for combating biofilm growth in a wide range of bacterial biofilm-related infections.

Fig. 1.

Characterization of the G5 domain from Aap. (A) The five regions of Aap are illustrated: the A-repeat region, the putative globular domain (α/β), the B-repeat region containing 5–17 tandem G5 domains, the collagen-like proline/glycine-rich region and a cell wall anchoring motif (LPXTG). The proteolysis site is illustrated with scissors. The domain boundaries of the Brpt1.0, Brpt1.5, and Brpt2.5 constructs are illustrated. (B) Sequence alignment of the terminal G5 domain and C-terminal half-repeat motif from Aap and SasG. Identical amino acids are indicated by dashes (–); histidines are highlighted with arrowheads. Blast alignment (41) shows 80% conservation and 65% identity. (C) Far-UV circular dichroism spectrum of Brpt1.5 in 20 mM Tris pH 7.4, 50 mM NaF. Deconvolution of the data reveals predominantly β-sheet and coil secondary structure elements (Table S1). (D) Sedimentation coefficient distribution plot for Brpt1.5 at varying concentrations. Molecular weight estimation indicates Brpt1.5 is monomeric. (E) Representative sedimentation equilibrium data for Brpt1.5, confirming a monomeric state (Table S3). Black lines show the global fits; residuals are shown above.

Fig. 2.

Characterization of Zn²⁺-dependent G5 dimerization. (A) Sedimentation velocity analyses of Brpt1.5 in the presence of divalent cations revealed a Zn²⁺-specific dimerization event. The dashed line label (––) represents cation-free Brpt1.5. (B) Far-UV CD spectra of Brpt1.5 in the presence (squares) and absence (triangles) of Zn. (C) Sedimentation equilibrium analysis of Zn²⁺-mediated dimerization of Brpt1.5 (triangles) and Brpt2.5 (squares). Brpt2.5 dimerization requires lower [Zn²⁺] (EC₅₀ = 3.7 mM) compared to Brpt1.5 (EC₅₀ = 5.4 mM) and shows a steeper slope for the monomer–dimer transition, suggesting enhanced cooperativity of Zn²⁺ binding with increasing numbers of tandem G5 domains. Error bars show 95% confidence intervals. (D) Linked equilibrium plot of Zn binding by Brpt1.5. Linear regression analysis of the slope indicates that the number of Zn²⁺ ions taken up upon Brpt1.5 dimerization (i.e., ΔZn²⁺) is approximately three. (E) Linked equilibrium plot of Zn binding by Brpt2.5. Linear regression analysis of the slope indicates that approximately five Zn²⁺ ions are bound upon Brpt2.5 dimerization.

Fig. 3.

Zn²⁺ chelation inhibits biofilm formation by S. epidermidis and S. aureus. (A) S. epidermidis RP62A biofilms formed on polystyrene and were visualized with crystal violet; addition of the Zn²⁺ chelator DTPA (≥30 μM) inhibits biofilm formation. Representative wells have been scanned (Bottom). Untreated (“RP62A”) and vehicle (“HCl”) controls are shown. Error bars show standard deviation (*, P < 0.05 relative to untreated control; n = 3). (B) Methicillin-resistant S. aureus USA300 biofilms form on fibronectin-coated plates but are inhibited by addition of DTPA (≥30 μM). (*, P < 0.05; n = 4). (C) Addition of 5–20 μM ZnCl₂ at a minimal inhibitory dose of DTPA (30 μM) rescues biofilm formation by RP62A. (#, P < 0.05 relative to the 0 μM ZnCl₂/30 μM DTPA control; ^, data are statistically indistinguishable from untreated control RP62A; n = 3). (D) Addition of 15–20 μM ZnCl₂ at a minimal inhibitory dose of DTPA (30 μM) rescues biofilm formation by USA300. (#, P < 0.05 compared to 0 μM ZnCl₂/30 μM DTPA; ^, statistically indistinguishable from untreated control; n = 4).

Fig. 4.

Soluble G5 domain inhibits biofilm formation in a dose-dependent manner. (A) Addition of soluble MBP–Brpt1.5 inhibits RP62A biofilms in a dose-dependent manner in the presence of 0.75–1 mM ZnCl₂. MBP alone was statistically indistinguishable from untreated control at all concentrations tested. (*, P < 0.05 relative to RP62A control at the relevant Zn²⁺ concentration; n = 3). (B) Dose−response of biofilm inhibition by MBP–Brpt1.5 at a fixed 1 mM ZnCl₂ concentration. (*, P < 0.0005; n = 3). (C) The zinc zipper model for intercellular adhesion in staphylococcal biofilms mediated by zinc-dependent self-association of G5 domains.