Role of Surface Protein SasG in Biofilm Formation by Staphylococcus aureus

The SasG surface protein of Staphylococcus aureus has been shown to promote the formation of biofilm. SasG comprises an N-terminal A domain and repeated B domains. Here we demonstrate that SasG is involved in the accumulation phase of biofilm, a process that requires a physiological concentration of Zn²⁺. The B domains, but not the A domain, are required. Purified recombinant B domain protein can form dimers in vitro in a Zn²⁺-dependent fashion. Furthermore, the protein can bind to cells that have B domains anchored to their surface and block biofilm formation. The full-length SasG protein exposed on the cell surface is processed within the B domains to a limited degree, resulting in cleaved proteins of various lengths being released into the supernatant. Some of the released molecules associate with the surface-exposed B domains that remain attached to the cell. Studies using inhibitors and mutants failed to identify any protease that could cause the observed cleavage within the B domains. Extensively purified recombinant B domain protein is very labile, and we propose that cleavage occurs spontaneously at labile peptide bonds and that this is necessary for biofilm formation.Staphylococcus aureus is a commensal bacterium that is carried persistently in the anterior nares of about 20% of the human population. The organism can cause superficial skin infections, such as abscesses and impetigo, and more dangerous and potentially life-threatening invasive infections, such as endocarditis, osteomyelitis, and septic arthritis (26). Staphylococcus epidermidis and S. aureus are the major causes of infections associated with indwelling medical devices, such as central venous catheters, cardiovascular devices, and artificial joints (34, 54). The ability to form a biofilm is crucial to the microbes'' success in device-related infections. Bacteria in the biofilm matrix are in a semidormant state, are difficult to inhibit with antibiotics, and are impervious to host neutrophils and macrophages (36, 43, 44, 51). Until recently biofilm formation by staphylococci was attributed to the ability to synthesize an extracellular polysaccharide called polysaccharide intercellular adhesin (PIA), which is composed of partially deacetylated poly-N-acetylglucosamine (15, 28, 50). Attachment of bacteria to biomedical devices is mediated by adhesion to the naked plastic or metal surface by a surface component such as the major autolysin Atl (2, 14). Alternatively, adhesion to surfaces that have been conditioned by fibronectin and fibrinogen from host plasma is mediated by surface proteins such as clumping factor A (ClfA) and fibronectin binding proteins (FnBPA/B) of S. aureus or SdrG/Fbe of S. epidermidis (17, 46, 47).Several surface proteins of staphylococci can also promote the accumulation phase of biofilm: (i) the biofilm-associated protein Bap, which is only expressed by bovine strains of S. aureus (8); (ii) the SasC surface protein of S. aureus (41); (iii) fibronectin binding proteins FnBPA and FnBPB, which are particularly associated with biofilm formation by some types of methicillin-resistant S. aureus (MRSA) (35, 48); (iv) the multifactorial virulence factor protein A, which promotes cell accumulation when expressed at high levels, for example,in mutants defective in the accessory gene regulator Agr (31); (v) the extracellular matrix binding protein (Embp) of S. epidermidis (4); (vi) the accumulation-associated protein (Aap) of S. epidermidis and the related protein SasG from S. aureus (7, 19, 40).Aap and SasG are typical LPXTG-anchored multidomain cell wall-associated proteins (see Fig. ​Fig.1A,1A, below). A signal sequence is removed from the N terminus during secretion across the cytoplasmic membrane. The C-terminal domains comprise a sorting signal (LPXTG) and hydrophobic membrane-spanning domain and positively charged residues that are required for covalent attachment of the proteins to cell wall peptidoglycan by sortase A. The N termini of the mature proteins (A domains) comprise related amino acid sequences that have been implicated in adhesion of bacteria to desquamated epithelial cells and could be involved in colonization of the nares and skin (7, 27, 39). The archetypal Aap protein of S. epidermidis RP62a has 12 repeats of almost identical sequences of 128 residues followed by a partial repeat of 68 residues (region B), while SasG from S. aureus strain 8325-4 and strain Newman has seven 128-residue repeats and one partial repeat. The B subunits of Aap and SasG are 64% identical.Open in a separate windowFIG. 1.(A) Schematic representation of SasG domain organization. The positions of the signal sequence (S), A domain, B region (B1 to -8), and the wall/membrane-spanning regions (W/M) are indicated. The LPKTG motif is recognized by the sortase A enzyme, which covalently anchors the protein to the cell wall peptidoglycan. (B) Whole-cell immunoblot validating expression of A domain and B regions of SasG variants. Serial dilutions of SH1000(pALC2073:sasG⁺) (row 1); SH1000(pALC2073sasG⁺ A⁺B⁻) (row 2); SH1000(pALC2073sasG⁺ A⁻B⁺) (row 3), and SH1000(pALC2073sasG⁺ A⁻B⁺) induced with tetracycline (90 ng/ml) (row 4) were applied to a nitrocellulose membrane and probed with anti-SasG A domain and anti-SasG B domain antibodies. (C) Biofilm formation by SH1000 constructs expressing SasG variants. Biofilm was allowed to form for 24 h at 37°C under static conditions in microtiter dishes. Biofilm was stained with crystal violet, and the absorbance was measured at 570 nm.The formation of biofilm by Aap in S. epidermidis is promoted by the removal of the A domain by cleavage by an as-yet-unidentified bacterial protease, an event that can also be precipitated by host proteases (40). The ability of the exposed Aap B domains of different bacterial cells to form homophilic interactions through a Zn²⁺-dependent zipper mechanism was proposed when it was shown that purified B domains formed dimers in vitro that were dependent on the presence of Zn²⁺ (6). Purified recombinant B domain protein, but not the A domain, inhibited biofilm formation, as did antibodies that specifically bound to the B domains (40). The Zn²⁺ chelator diethylenetriaminepentaacetic acid (DTPA) inhibited biofilm formation both by S. epidermidis RP62a (presumed to be due to Aap) and by community-associated MRSA (presumed to be due to SasG) (6).This study set out to investigate the molecular basis of biofilm accumulation promoted by the SasG protein of S. aureus. We demonstrate that processing of SasG occurs during growth and biofilm formation in a manner that is different from that reported for Aap, and we have investigated the mechanism.