The N3 subdomain in a domain of fibronectin-binding protein B isotype I is an independent risk determinant predictive for biofilm formation of Staphylococcus aureus clinical isolates

Fibronectin-binding proteins (FnBP), FnBPA and FnBPB, are purported to be involved in biofilm formation of Staphylococcus aureus. This study was performed to find which of three consecutive N subdomains of the A domain in the FnBP is the key domain in FnBP. A total of 465 clinical isolates of S. aureus were examined for the biofilm forming capacity and the presence of N subdomains of FnBP. In the biofilm-positive strains, N2 and N3 subdomains of FnBPA, and N1 and N3 subdomains of FnBPB were significantly more prevalent. Multivariate logistic regression analysis of 246 biofilm-positive and 123 biofilm-negative strains identified only the FnBPB-N3 subdomain as an independent risk determinant predictive for biofilm-positive strains of S. aureus (Odds ratio [OR], 13.174; P<0.001). We also attempted to delete each of the fnbA-N2 and -N3 and fnbB-N1 and -N3 from S. aureus strain 8325-4 and examined the biofilm forming capacity in the derivative mutants. In agreement with the results of the multivariate regression analysis, deletion of either the fnbA-N2 or ?N3, or fnbB-N1 did not significantly diminish the capacity of strain 8325-4 to develop a biofilm, while deletion of the fnbB-N3 did. Therefore, it is suggested that the FnBPB-N3 subdomain of isotype I may be a key domain in FnBP which is responsible for the causing biofilm formation in S. aureus clinical isolates.     

Evidence for Steric Regulation of Fibrinogen Binding to Staphylococcus aureus Fibronectin-binding Protein A (FnBPA)

The adjacent fibrinogen (Fg)- and fibronectin (Fn)-binding sites on Fn-binding protein A (FnBPA), a cell surface protein from Staphylococcus aureus, are implicated in the initiation and persistence of infection. FnBPA contains a single Fg-binding site (that also binds elastin) and multiple Fn-binding sites. Here, we solved the structure of the N2N3 domains containing the Fg-binding site of FnBPA in the apo form and in complex with a Fg peptide. The Fg binding mechanism is similar to that of homologous bacterial proteins but without the requirement for “latch” strand residues. We show that the Fg-binding sites and the most N-terminal Fn-binding sites are nonoverlapping but in close proximity. Although Fg and a subdomain of Fn can form a ternary complex on an FnBPA protein construct containing a Fg-binding site and single Fn-binding site, binding of intact Fn appears to inhibit Fg binding, suggesting steric regulation. Given the concentrations of Fn and Fg in the plasma, this mechanism might result in targeting of S. aureus to fibrin-rich thrombi or elastin-rich tissues.     

Staphylococcus aureus Host Cell Invasion and Virulence in Sepsis Is Facilitated by the Multiple Repeats within FnBPA

Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to α₅β₁ integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis.     

Sequence diversity in the A domain of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>fibronectin-binding protein A

Background  

Fibronectin-binding protein A (FnBPA) mediates adhesion of Staphylococcus aureus to fibronectin, fibrinogen and elastin. We previously reported that S. aureus strain P1 encodes an FnBPA protein where the fibrinogen/elastin-binding domain (A domain) is substantially divergent in amino acid sequence from the archetypal FnBPA of S. aureus NCTC8325, and that these variations created differences in antigenicity. In this study strains from multilocus sequence types (MLST) that spanned the genetic diversity of S.aureus were examined to determine the extent of FnBPA A domain variation within the S. aureus population and its effect on ligand binding and immuno-crossreactivity.     

Fibronectin-binding protein A of Staphylococcus aureus has multiple, substituting, binding regions that mediate adherence to fibronectin and invasion of endothelial cells

Invasive Staphylococcus aureus infection frequently involves bacterial seeding from the bloodstream to other body tissues, a process necessarily involving interactions between circulating bacteria and vascular endothelial cells. Staphylococcus aureus fibronectin‐binding protein is central to the invasion of endothelium, fibronectin forming a bridge between bacterial fibronectin‐binding proteins and host cell receptors. To dissect further the mechanisms of invasion of endothelial cells by S. aureus, a series of truncated FnBPA proteins that lacked one or more of the A, B, C or D regions were expressed on the surface of S. aureus and tested in fibronectin adhesion, endothelial cell adhesion and invasion assays. We found that this protein has multiple, substituting, fibronectin‐binding regions, each capable of conferring both adherence to fibronectin and endothelial cells, and endothelial cell invasion. By expressing S. aureus FnBPA on the surface of the non‐invasive Gram‐positive organism Lactococcus lactis, we have found that no other bacterial factor is required for invasion. Furthermore, we have demonstrated that, as with other cell types, invasion of endothelial cells is mediated by integrin α₅β₁. These findings may be of relevance to the development of preventive measures against systemic infection, and bacterial spread in the bacteraemic patient.     

SdrC induces staphylococcal biofilm formation through a homophilic interaction

The molecular pathogenesis of many Staphylococcus aureus infections involves growth of bacteria as biofilm. In addition to polysaccharide intercellular adhesin (PIA) and extracellular DNA, surface proteins appear to mediate the transition of bacteria from planktonic growth to sessile lifestyle as well as biofilm growth, and can enable these processes even in the absence of PIA expression. However, the molecular mechanisms by which surface proteins contribute to biofilm formation are incompletely understood. Here we demonstrate that self‐association of the serine‐aspartate repeat protein SdrC promotes both bacterial adherence to surfaces and biofilm formation. However, this homophilic interaction is not required for the attachment of bacteria to abiotic surfaces. We identified the subdomain that mediates SdrC dimerization and subsequent cell‐cell interactions. In addition, we determined that two adjacently located amino acid sequences within this subdomain are required for the SdrC homophilic interaction. Comparative amino acid sequence analysis indicated that these binding sites are conserved. In summary, our study identifies SdrC as a novel molecular determinant in staphylococcal biofilm formation and describes the mechanism responsible for intercellular interactions. Furthermore, these findings contribute to a growing body of evidence suggesting that homophilic interactions between surface proteins present on neighbouring bacteria induce biofilm growth.     

Identification of the Immunodominant Regions of Staphylococcus aureus Fibronectin-Binding Protein A

Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a diverse spectrum of human diseases and a leading cause of nosocomial and community-acquired infections. Development of a vaccine against this pathogen is an important goal. The fibronectin binding protein A (FnBPA) of S. aureus is one of multifunctional ‘microbial surface components recognizing adhesive matrix molecules'' (MSCRAMMs). It is one of the most important adhesin molecules involved in the initial adhesion steps of S. aureus infection. It has been studied as potential vaccine candidates. However, FnBPA is a high-molecular-weight protein of 106 kDa and difficulties in achieving its high-level expression in vitro limit its vaccine application in S. aureus infection diseases control. Therefore, mapping the immunodominant regions of FnBPA is important for developing polyvalent subunit fusion vaccines against S. aureus infections. In the present study, we cloned and expressed the N-terminal and C-terminal of FnBPA. We evaluated the immunogenicity of the two sections of FnBPA and the protective efficacy of the two truncated fragments vaccines in a murine model of systemic S. aureus infection. The results showed recombinant truncated fragment F1_30-500 had a strong immunogenicity property and survival rates significantly increased in the group of mice immunized with F1_30-500 than the control group. We futher identified the immunodominant regions of FnBPA. The mouse antisera reactions suggest that the region covering residues 110 to 263 (F1B_110-263) is highly immunogenic and is the immunodominant regions of FnBPA. Moreover, vaccination with F1B_110-263 can generate partial protection against lethal challenge with two different S. aureus strains and reduced bacterial burdens against non-lethal challenge as well as that immunization with F1_30-500. This information will be important for further developing anti- S. aureus polyvalent subunit fusion vaccines.     

A MAM7 Peptide-Based Inhibitor of Staphylococcus aureus Adhesion Does Not Interfere with In Vitro Host Cell Function

Adhesion inhibitors that block the attachment of pathogens to host tissues may be used synergistically with or as an alternative to antibiotics. The wide-spread bacterial adhesin Multivalent Adhesion Molecule (MAM) 7 has recently emerged as a candidate molecule for a broad-spectrum adhesion inhibitor which may be used to prevent bacterial colonization of wounds. Here we have tested if the antibacterial properties of a MAM-based inhibitor could be used to competitively inhibit adhesion of methicillin-resistant Staphylococcus aureus (MRSA) to host cells. Additionally, we analyzed its effect on host cellular functions linked to the host receptor fibronectin, such as migration, adhesion and matrix formation in vitro, to evaluate potential side effects prior to advancing our studies to in vivo infection models. As controls, we used inhibitors based on well-characterized bacterial adhesin-derived peptides from F1 and FnBPA, which are known to affect host cellular functions. Inhibitors based on F1 or FnBPA blocked MRSA attachment but at the same time abrogated important cellular functions. A MAM7-based inhibitor did not interfere with host cell function while showing good efficacy against MRSA adhesion in a tissue culture model. These observations provide a possible candidate for a bacterial adhesion inhibitor that does not cause adverse effects on host cells while preventing bacterial infection.     

Fibronectin-binding protein B variation in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Background  

Fibronectin binding proteins A and B (FnBPA and FnBPB) mediate adhesion of S. aureus to fibrinogen, elastin and fibronectin. We previously identified seven different isotypes of FnBPA based on divergence in the fibrinogen- and elastin-binding A domains. The variation created differences in antigenicity while ligand binding functions were retained. Here, FnBPB variation was examined in both human and bovine isolates and compared to that of FnBPA.     

Tissue Plasminogen Activator Coating on Implant Surfaces Reduces Staphylococcus aureus Biofilm Formation

Staphylococcus aureus biofilm infections of indwelling medical devices are a major medical challenge because of their high prevalence and antibiotic resistance. As fibrin plays an important role in S. aureus biofilm formation, we hypothesize that coating of the implant surface with fibrinolytic agents can be used as a new method of antibiofilm prophylaxis. The effect of tissue plasminogen activator (tPA) coating on S. aureus biofilm formation was tested with in vitro microplate biofilm assays and an in vivo mouse model of biofilm infection. tPA coating efficiently inhibited biofilm formation by various S. aureus strains. The effect was dependent on plasminogen activation by tPA, leading to subsequent local fibrin cleavage. A tPA coating on implant surfaces prevented both early adhesion and later biomass accumulation. Furthermore, tPA coating increased the susceptibility of biofilm infections to antibiotics. In vivo, significantly fewer bacteria were detected on the surfaces of implants coated with tPA than on control implants from mice treated with cloxacillin. Fibrinolytic coatings (e.g., with tPA) reduce S. aureus biofilm formation both in vitro and in vivo, suggesting a novel way to prevent bacterial biofilm infections of indwelling medical devices.     

Surface proteins and the formation of biofilms by Staphylococcus aureus

Staphylococcus aureus biofilms pose a serious clinical threat as reservoirs for persistent infections. Despite this clinical significance, the composition and mechanism of formation of S. aureus biofilms are unknown. To address these problems, we used solid-state NMR to examine S. aureus (SA113), a strong biofilm-forming strain. We labeled whole cells and cell walls of planktonic cells, young biofilms formed for 12–24 h after stationary phase, and more mature biofilms formed for up to 60 h after stationary phase. All samples were labeled either by (i) [¹⁵N]glycine and l-[1-¹³C]threonine, or in separate experiments, by (ii) l-[2-¹³C,¹⁵N]leucine. We then measured ¹³C-¹⁵N direct bonds by C{N} rotational-echo double resonance (REDOR). The increase in peptidoglycan stems that have bridges connected to a surface protein was determined directly by a cell-wall double difference (biofilm REDOR difference minus planktonic REDOR difference). This procedure eliminates errors arising from differences in ¹⁵N isotopic enrichments and from the routing of ¹³C label from threonine degradation to glycine. For both planktonic cells and the mature biofilm, 20% of pentaglycyl bridges are not cross-linked and are potential surface-protein attachment sites. None of these sites has a surface protein attached in the planktonic cells, but one-fourth have a surface protein attached in the mature biofilm. Moreover, the leucine-label shows that the concentration of β-strands in leucine-rich regions doubles in the mature biofilm. Thus, a primary event in establishing a S. aureus biofilm is extensive decoration of the cell surface with surface proteins that are linked covalently to the cell wall and promote cell-cell adhesion.     

Fibrinogen and elastin bind to the same region within the A domain of fibronectin binding protein A, an MSCRAMM of Staphylococcus aureus

The fibronectin binding protein, FnBPA, is a multifunctional microbial surface component recognizing adhesive matrix molecule (MSCRAMM) that promotes bacterial adherence to immobilized fibrinogen and elastin via the N-terminal A domain. The binding site for fibrinogen and elastin was localized to subdomains N2N3. A three-dimensional structural model of FnBPA was created based on the known crystal structure of the domains N2N3 of clumping factor A (ClfA). The role of individual residues in the putative ligand binding trench was examined by testing the affinity of mutants for fibrinogen and elastin. Two residues (N304 and F306) were crucial for binding both ligands and are in the equivalent positions to residues known to be important for fibrinogen binding by ClfA. A peptide comprising the C-terminus of the gamma-chain of fibrinogen and a monoclonal anti-rAFnBPA antibody were potent inhibitors of the FnBPA-elastin interaction. This suggests that FnBPA binds to fibrinogen and elastin in a similar manner. Amino acid sequence divergence of 26.5% occurred between the A domains of FnBPA from strains 8325-4 and P1. Most variant residues were predicted to be located on the surface of domains N2N3 while few occurred in the putative ligand binding trench and the latching peptide explaining limited immunocross reactivity while ligand binding activity is conserved.     

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.     

Structural organization of the fibrinogen-binding region of the clumping factor B MSCRAMM of Staphylococcus aureus

The clumping factor B (ClfB) of Staphylococcus aureus is a surface protein that binds to fibrinogen (Ni Eidhin, D., Perkins, S., Francois, P., Vaudaux, P., Hook, M., and Foster, T. J., 1998 Mol. Microbiol. 30, 245-257). The ligand-binding activity is located in the approximately 500-residue A-region (residues 44-542), which represents the N-terminal half of the MSCRAMM protein. We now hypothesize that the ClfB A-region is composed of three subdomains, which we have named N1, N2, and N3, respectively. To examine this hypothesis, we expressed recombinant forms of the individual putative subdomains, the tandem motifs N12 and N23, and the full-length A-region N123. Far UV circular dichroism spectra showed that each subdomain is composed mainly of beta-sheets with little or no discernible alpha-helices. Heat-induced unfolding of individual subdomains occurred with a single state transition and was reversible, indicating that the subdomains can fold as discreet units. Gel permeation chromatography indicated that N2, N3, and N23 are globular. In contrast, domain N1 appeared to be elongated and conferred a somewhat elongated structure on segments containing this subdomain (i.e. N12 or N123). N123, N12, and N23 all bound to fibrinogen, but N23 had a higher affinity for fibrinogen than that observed for the full-length A-region; N123 or for N12. However, an extended N terminus of N23 was required for ligand binding. A form of N23 that was generated by proteolytic processing and lacked the N-terminal extension was unable to bind fibrinogen. Recombinant forms of individual subdomains did not bind fibrinogen. The addition of recombinant N23 effectively inhibited ClfB-mediated bacterial adherence to fibrinogen, and N123 caused some reduction in bacterial attachment, whereas N12 was essentially inactive. Antibodies raised against the central N2 domain of the A-region were the most effective at inhibiting bacterial adhesion to immobilized fibrinogen, although anti-N3 or anti-N1 antibodies also caused some reduction in ClfB-mediated adherence to fibrinogen.     

Protein A-Mediated Multicellular Behavior in Staphylococcus aureus

The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.Staphylococcus aureus is a gram-positive bacterium that lives as part of the normal microflora on the skin and mucous membranes of humans and animals. If S. aureus passes through the epithelial barrier and reaches internal organs, it can cause a variety of diseases, ranging from minor skin infections, such as furuncles or boils, to severe infections, such as bacteremia, pneumonia, osteomyelitis, or endocarditis. Despite the progress with antibiotics in the treatment of bacterial infections over the last 2 decades, the number of infections due to S. aureus has increased (11, 30). The infection rate has been correlated with an increase in the use of prosthetic and indwelling devices in modern medical practices (24, 26). S. aureus, as well as other coagulase-negative staphylococci, displays a strong capacity to irreversibly attach to the surface of implanted medical devices and forms multilayered communities of bacteria, known as biofilms, that grow embedded in a self-produced extracellular matrix (23). The biofilm formation process occurs in two steps: first, bacterial cells irreversibly attach to a surface, and second, they interact with each other and accumulate in multilayered cell clusters embedded in a self-produced extracellular matrix. Primary attachment is mediated by physico-chemical cell surface properties as well as specific factors that mediate the attachment to the host-derived extracellular matrix components that rapidly coat the biomaterial following insertion into the patient. Numerous proteins from the MSCRAMMs family (microbial surface components recognizing adhesive matrix molecules) are involved in the first step of S. aureus biofilm formation, such as clumping factors ClfA (37) and ClfB (41) and fibrinogen and fibronectin binding proteins (FnBPA and FnBPB) (25, 31). Once bacteria accumulate in multilayered cell clusters, most have no direct contact with the surface, and thus cell-to-cell interactions become essential for biofilm development and maintenance. An extracellular polysaccharide intercellular adhesin (PIA, or PNAG), produced by icaADBC operon-encoded enzymes, is currently the best-characterized element mediating intercellular interactions in vitro (8, 23, 34, 35, 38). Alternatively, a number of surface proteins can replace PIA/PNAG exopolysaccharide in promoting intercellular adhesion and biofilm development, including the surface protein Bap (9). All the tested staphylococcal isolates harboring the bap gene were shown to be strong biofilm producers, and inactivation of the icaADBC operon in bap-positive strains had no effect on in vitro biofilm formation (57). Remarkably, proteins homologous to Bap are involved in the biofilm formation process in diverse bacterial species (33). A second surface protein, SasG, as well as its homologous protein in Staphylococcus epidermidis, Aap, also mediates intercellular interactions and biofilm development in the absence of the ica operon (7, 51). More recently, two independent laboratories have shown that fibronectin binding proteins A and B (FnBPA and FnBPB) induce biofilm development of clinical isolates of S. aureus (45, 55). Finally, there is growing evidence that extracellular DNA, despite not being sufficient to replace PIA/PNAG exopolysaccharide, is an important S. aureus biofilm matrix component (50).During the course of a systematic mutagenesis study of the 17 two-component systems of S. aureus that aimed to identify biofilm-negative regulators, we found that S. aureus agr arlRS double mutants developed an alternative, ica-independent biofilm in a chemically defined medium, Hussain-Hastings-White (HHW) medium (56). This study focused on the identification of the proteinaceous compound responsible for the biofilm developed by S. aureus agr arlRS mutants. Here, we show that S. aureus protein A is responsible for the aggregative phenotype and capacity for biofilm formation displayed by this strain. Furthermore, overproduction of protein A in wild-type S. aureus strains or addition of soluble protein A to bacterial growth medium induced aggregation and biofilm development, suggesting that protein A does not need to be covalently linked to the cell wall to promote multicellular behavior. Moreover, deletion of the spa gene significantly decreased the capacity of S. aureus to colonize subcutaneously implanted catheters. Our findings support a novel role for protein A in promoting multicellular behavior and suggest that protein A-mediated biofilm development may have a critical function during the infection process of S. aureus.     

Pyrrolomycins as antimicrobial agents. Microwave-assisted organic synthesis and insights into their antimicrobial mechanism of action

New compounds able to counteract staphylococcal biofilm formation are needed. In this study we investigate the mechanism of action of pyrrolomycins, whose potential as antimicrobial agents has been demonstrated. We performed a new efficient and easy method to use microwave organic synthesis suitable for obtaining pyrrolomycins in good yields and in suitable amount for their in vitro in-depth investigation. We evaluate the inhibitory activity towards Sortase A (SrtA), a transpeptidase responsible for covalent anchoring in Gram-positive peptidoglycan of many surface proteins involved in adhesion and in biofilm formation. All compounds show a good inhibitory activity toward SrtA, having IC₅₀ values ranging from 130 to 300?µM comparable to berberine hydrochloride. Of note compound 1d shows a good affinity in docking experiment to SrtA and exhibits the highest capability to interfere with biofilm formation of S. aureus showing an IC₅₀ of 3.4?nM. This compound is also effective in altering S. aureus murein hydrolase activity that is known to be responsible for degradation, turnover, and maturation of bacterial peptidoglycan and involved in the initial stages of S. aureus biofilm formation.     

A Modified Chronic Infection Model for Testing Treatment of Staphylococcus aureus Biofilms on Implants

Bacterial biofilms causing implant-associated osteomyelitis is a severe complication with limited antimicrobial therapy options. We designed an animal model, in which implant associated osteomyelitis arise from a Staphylococcus aureus biofilm on a tibia implant. Two bioluminescently engineered (luxA-E transformed), strains of S. aureus were utilized, Xen29 and Xen31. Biofilm formation was assessed with epifluorescence microscopy. Quantitative measurements were performed day 4, 6, 8, 11 and 15 post-surgery. Bacteria were extracted from the biofilm by sonication and the bacterial load quantified by culturing. Biofilm formation was evident from day 6 post-implantation. Mean bacterial load from implants was ∼1×10⁴ CFU/implant, while mean bacterial load from infected tibias were 1×10⁶ CFU/bone. Bioluminesence imaging revealed decreasing activity throughout the 15-day observation period, with signal intensity for both strains reaching that of the negative control by day 15 while there was no significant reduction in bacterial load. The model is suitable for testing antimicrobial treatment options for implant associated OM, as treatment efficacy on both biofilm and viable counts can be assessed.     

The A domain of fibronectin-binding protein B of Staphylococcus aureus contains a novel fibronectin binding site

The fibronectin-binding proteins FnBPA and FnBPB are multifunctional adhesins than can also bind to fibrinogen and elastin. In this study, the N2N3 subdomains of region A of FnBPB were shown to bind fibrinogen with a similar affinity to those of FnBPA (2 μM). The binding site for FnBPB in fibrinogen was localized to the C-terminus of the γ-chain. Like clumping factor A, region A of FnBPB bound to the γ-chain of fibrinogen in a Ca(2+)-inhibitable manner. The deletion of 17 residues from the C-terminus of domain N3 and the substitution of two residues in equivalent positions for crucial residues for fibrinogen binding in clumping factor A and FnBPA eliminated fibrinogen binding by FnBPB. This indicates that FnBPB binds fibrinogen by the dock-lock-latch mechanism. In contrast, the A domain of FnBPB bound fibronectin with K(D) = 2.5 μM despite lacking any of the known fibronectin-binding tandem repeats. A truncate lacking the C-terminal 17 residues (latching peptide) bound fibronectin with the same affinity, suggesting that the FnBPB A domain binds fibronectin by a novel mechanism. The substitution of the two residues required for fibrinogen binding also resulted in a loss of fibronectin binding. This, combined with the observation that purified subdomain N3 bound fibronectin with a measurable, but reduced, K(D) of 20 μM, indicates that the type I modules of fibronectin bind to both the N2 and N3 subdomains. The fibronectin-binding ability of the FnBPB A domain was also functional when the protein was expressed on and anchored to the surface of staphylococcal cells, showing that it is not an artifact of recombinant protein expression.