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 threedimensional 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.bacterial pathogenesis ͉ G5 domain ͉ Aap ͉ chelation ͉ Staphylococcus

Staphylococcal biofilm-forming protein has a contiguous rod-like structure

“…Notwithstanding, in vivo studies demonstrated that zinc is essential for protein-mediated biofilm formation in S. epidermidis (13) and S. aureus (12). Aap G5 domains, which contain native C-terminal histidine residues (not present in SasG), dimerize in a zinc-dependent manner (13). However, there is no experimental evidence that directly links this in vitro dimerization to the Zn 2+ -dependence of proteinmediated biofilm formation in S. epidermidis.…”

“…Previously, we and others showed that SasG and Aap B repeats dimerize in the presence of Zn 2+ ; however, the structural basis of dimerization was not clear (12,13). The crystal structure of a SasG G5 2 dimer (Fig.…”

“…Unlabeled, 15 N-labeled, and 13 C-15 N-labeled proteins were produced in E. coli BL21(DE3) by using Luria-Bertani, 15 N-M9, and 13 C-15 N-M9 medium, respectively. Selenomethionylated E-G5 2 -L17M-L103M was produced in E. coli B834(DE3) (Novagen) by using SeMet-supplemented minimal medium.…”

“…However, recent findings indicated that the full-length protein exposed on the bacterial surface undergoes limited processing within B repeats (12). The current model of proteinmediated intercellular adhesion promoted by Aap and SasG in staphylococci is known as a zinc zipper, in which Zn 2+ -mediated self-association events occur between stretches of B repeats on opposing Aap or SasG molecules (12,13).…”

“…The A domains of SasG and Aap have been implicated in adhesion of bacteria to desquamated epithelial cells (10) and B repeat regions are responsible for cellto-cell accumulation during biofilm formation (11,12). Although annotations in the literature differ (13,14), the Pfam database (15) recognizes a B-repeat sequence as containing a G5 domain (∼80 residues) (14) followed by an approximately 50-residue sequence (herein called E; Fig. 1A).…”