Microbial adhesins recognizing extracellular matrix macromolecules.

Microorganisms express a family of cell-surface adhesins that specifically recognize and bind components of the extracellular matrix. Adhesion of microorganisms to host tissues represents a critical phase in the development of many types of infections. Recent studies have focused on the mechanisms of microbial attachment at a molecular level, including the identification of ligand-binding domains in several cell-surface adhesins from Gram-positive bacteria and the construction of adhesin-deficient isogenic mutants.

Vaccination with a recombinant fragment of collagen adhesin provides protection against Staphylococcus aureus-mediated septic death.

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. Morbidity and mortality due to infections such as sepsis, osteomyelitis, septic arthritis, and invasive endocarditis remain high despite the use of antibiotics. The emergence of antibiotic resistant super bugs mandates that alternative strategies for the prevention and treatment of S. aureus infections are developed. We investigated the ability of vaccination with a recombinant fragment of the S. aureus collagen adhesin to protect mice against sepsis-induced death. Actively immunized NMRI mice were intravenously inoculated with the S. aureus clinical isolate strain Phillips. 14 d after inoculation, mortality in the collagen adhesin-vaccinated group was only 13%, compared with 87% in the control antigen immunized group (P < 0.001). To determine if the protective effect was antibody mediated, we passively immunized naive mice with collagen adhesin-specific antibodies. Similar to the active immunization strategy, passive transfer of collagen adhesin-specific antibodies protected mice against sepsis-induced death. In vitro experiments indicated that S. aureus opsonized with sera from collagen adhesin immunized mice promoted phagocytic uptake and enhanced intracellular killing compared with bacteria opsonized with sera from control animals. These results indicate that the collagen adhesin is a viable target in the development of immunotherapeutics against S. aureus.

Vaccines and immunotherapy for staphylococcal infections.

Nosocomial or hospital-acquired infections are associated with prolonged hospitalizations and increased healthcare costs. Infections associated with surgical implants are becoming more difficult and more costly to manage, as they require repeated surgical procedures and a longer period of time to treat patients. Continued advances in the use of medical devices, an increase in the number of immunocompromised patients, and a steady rise in the prevalence of antibiotic-resistant organisms has renewed interest in the development of novel therapies that can be used to prevent and treat nosocomial infections. This review provides an overview of bacterial adhesins and focuses on novel immunological therapies developed to treat staphylococcal infections.

Isolation and characterization of pcp, a gene encoding a pyrrolidone carboxyl peptidase in Staphylococcus aureus.

The pcp gene, encoding a pyrrolidone carboxyl peptidase (PYRase), was cloned from a lambda GT11 genomic library prepared from Staphylococcus aureus FDA 574 and sequenced. The pcp gene is located 740 bp downstream from cna, a gene that encodes a collagen-binding adhesin in S. aureus. S. aureus pcp encodes a 212-amino-acid (aa) polypeptide. The pcp gene was overexpressed in Escherichia coli and the PYRase purified to homogeneity. The recombinant enzyme exhibited biological activity, as determined using the chromogenic substrate L-pyroglutamyl-beta-napthylamide. Biochemical analysis of the PYRase using thiol-blocking chemicals suggested that the enzyme belongs to the cysteine peptidase family. Moreover, multiple sequence alignment revealed a high degree of similarity to previously described bacterial PYRases. This family of peptidases has been used to selectively remove the N-terminal pyrrolidone carboxylic acid residue found on certain blocked proteins and peptides prior to aa sequencing. However, the exact biological role of PYRases has yet to be elucidated.

The Staphylococcus aureus collagen adhesin-encoding gene (cna) is within a discrete genetic element.

Although the gene (cna) encoding the Staphylococcus aureus (Sa) collagen adhesin is not present in all strains, the DNA both upstream and downstream of cna is present in all Sa strains. Using oligo primers corresponding to the conserved nt flanking cna and template DNA from Sa strains that do not encode cna, we amplified a 372-bp fragment. These results illustrate that the conserved regions upstream and downstream of cna are contiguous in strains that do not encode cna. Using primers corresponding to the conserved flanking DNA together with primers corresponding to the 5' and 3' ends of cna, we also amplified DNA fragments containing the junctions between the cna genetic element and the conserved flanking sequences. Sequence comparisons of the amplification products from four cna negative and four cna positive strains revealed that cna is within a discrete genetic element that extends 202 bp upstream from the cna start codon and 100 bp downstream of the cna stop codon. Sequence analysis of the ends of the cna element did not reveal any of the repeats characteristic of transposable elements. These results suggest that cna may be part of a larger element (e.g., a phage) that may or may not contain cna. Alternatively, cna may be a subject to a precise excision event resulting in its deletion from the chromosome. Based on sequence analysis of the flanking DNA amplified from strains that do not encode cna, the presence of a cna genetic element does not disrupt an ORF.

Role of Staphylococcus aureus surface adhesins in orthopaedic device infections: are results model-dependent?

Bacterial colonisation of prosthetic material can lead to clinical infection or implant failure, or both, often requiring removal of the device. Adherence of Staphylococcus aureus to bioprosthetic materials is mediated by adhesins belonging to the MSCRAMM (microbial surface components recognising adhesive matrix molecules) family of microbial cell surface proteins. The objective of this study was to compare the virulence of a mutant strain of S. aureus Newman that possesses all three fibrinogen-, fibronectin- and collagen-binding MSCRAMMs (MSCRAMM-positive strain) with that of a mutant strain that lacks all three types of MSCRAMMs (MSCRAMM-negative strain) in a rabbit model of orthopaedic device-related infection. After a hole was drilled into the knee joint of each animal, a group of 10 rabbits was inoculated with the MSCRAMM-positive strain and another group of 10 rabbits received the MSCRAMM-negative strain. A stainless steel screw was then placed into the drilled hole. Two weeks later, the rabbits were killed and serum samples, bone tissue and implants were harvested for bacteriological and histopathological evaluation. No significant difference in infection rates was demonstrated between the two groups. The ability to delineate the role of S. aureus surface adhesins in causing orthopaedic device-related infection could be model-dependent.

Identification and biochemical characterization of the ligand binding domain of the collagen adhesin from Staphylococcus aureus.

We have recently shown that the expression of a collagen adhesin is both necessary and sufficient to mediate the attachment of Staphylococcus aureus to cartilage, a complex collagen-containing substrate [Switalski, L. M., Patti, J. M., Butcher, W., Gristina, A. G., Speziale, P., & Höök, M. (1993) Mol. Microbiol. 7, 99-107]. We now report on the localization of the ligand binding site within the 135-kDa S. aureus collagen adhesin. Using deletion mutagenesis in combination with Western ligand blot and direct binding assays, the collagen binding domain (CBD) was localized to a 168 amino acid long segment [CBD(151-318)] within the N-terminal portion of the adhesin. Using biospecific interaction analysis, pepsin-digested bovine type II collagen was found to contain eight binding sites for CBD(151-318); two binding sites were of "high" affinity (Kd = 3 microM) and six sites were of low affinity (Kd = 30 microM). Short truncations in the terminal flanking regions of CBD(151-318) resulted in two CBDs (180-318 and 151-297) that lacked collagen binding activity. Analysis by circular dichroism of the recombinant CBDs in the far UV revealed similar secondary structures, predominantly beta-sheet, whereas the near-UV spectra indicated dramatic changes in the degree of intermolecular packing (tertiary structure). The deduced amino acid sequence of the ligand binding domain of the collagen adhesin is presented.

Transcriptional regulation of the Staphylococcus aureus collagen adhesion gene, cna.

We demonstrate that transcription of the Staphylococcus aureus collagen adhesin gene (cna) is temporally regulated, with expression being highest in exponentially growing cultures and falling to almost undetectable levels as cultures enter the post-exponential-growth phase. The temporal regulation of cna transcription was not affected by mutation of agr. We also demonstrate that the collagen adhesin is expressed by both agr+ and agr-negative S. aureus cells growing in bone.

The biosynthetic incorporation of selenomethionine and telluromethionine into pyrrolidone carboxyl peptidase (PYRase) from S. aureus.

Heavy-atom derivatives of PYRase proteins prepared in the past have been unsuitable for x-ray diffraction analysis. Thus, we propose utilizing unnatural metalloid-containing amino acids as an alternative to heavy-atom derivatization. Selenomethionine-containing proteins analyzed by multiwavelength anomalous diffraction provides a facile means of addressing the phase problem, whose solution is necessary to determine protein structures by X-ray Crystallography [Hendrickson, et al., 1991 and references therein]. Telluromethionine-containing proteins offer the same investigational potential, and additionally allow further simplification of the data collection technique by requiring only traditional methods of phase analysis [Boles et al., 1995 and references therein]. We sought to introduce the required Se and Te atoms into Staphylococcus aureus Pyrrolidone Carboxyl Peptidase (PYRase) via selenomethionine (SeMet) and telluromethionine (TeMet). Complete incorporation of SeMet into S. aureus PYRase was succeeded with little change in enzymatic properties. Incomplete incorporation (75%) of TeMet was accomplished in preparing TeMet-PYRase, however, representing the highest incorporation to date of a tellurium-containing amino acid. Enzymatic properties remained unchanged when TeMet was incorporated. We report herein the biosynthetic substitution and expression, protein purification and comparative biochemistry of SeMet-PYRase and TeMet-PYRase.

Model for studying bacterial adherence to skin wounds.

The adherence of bacterial pathogens to wounded skin is probably the first step in wound infection. This report describes the development of a bioassay to simulate the adherence of bacteria to wounds. The adherence of bacteria was examined by exposing wounds to known quantities of pathogens, washing the wounds with distilled water, and quantitating the number of adherent bacteria per cm2 of tissue. Our studies focused on the effects of naturally occurring mediators of bacterial adherence, such as wound fluid, serum, and fibronectin. Bacterial adherence was shown to be challenge dependent. Addition of wound fluid, serum, and heat-inactivated serum was shown to reduce the adherence of Pseudomonas aeruginosa to the wound surface compared with that of a saline control. Additional fibronectin treatment of the wound had no effect on the adherence of Staphylococcus aureus. The ability to identify what affects the binding of bacteria to wounded skin can lead to a better understanding of wound infection.

Protection against experimental Staphylococcus aureus arthritis by vaccination with clumping factor A, a novel virulence determinant.

The importance of the fibrinogen-binding adhesin clumping factor A (ClfA) in the pathogenesis of Staphylococcus aureus septic arthritis was examined in an animal model. The protective effect of active and passive immunization with ClfA also was investigated in S. aureus infection models. The severity of arthritis was markedly reduced in mice challenged intravenously with a clfA mutant, compared with mice infected with the wild-type strain. Mice immunized with recombinant ClfA and challenged with S. aureus developed less-severe arthritis than did mice immunized with a control antigen. Passive immunization of mice with rat and rabbit anti-ClfA antibodies protected against S. aureus arthritis and sepsis-induced death, indicating that the protection by active immunization is antibody mediated. Taken together, these data strongly suggest that ClfA is a crucial virulence determinant for septic arthritis and an excellent target for the generation of immune therapies directed against S. aureus.

Inhibition of Staphylococcus aureus adherence to collagen under dynamic conditions.

Staphylococcus aureus is the most common etiological agent of bacterial arthritis and acute osteomyelitis and has been shown to bind to type II collagen under static and dynamic conditions. We have previously reported the effect of shear on the adhesion of S. aureus Phillips to collagen and found that this process is shear dependent (Z. Li, M. Höök, J. M. Patti, and J. M. Ross, Ann. Biomed. Eng. 24[Suppl. 1]:S-55). In this study, we used recombinant collagen adhesin fragments as well as polyclonal antibodies generated against adhesin fragments in attempts to inhibit bacterial adhesion. A parallel-plate flow chamber was used in a dynamic adhesion assay, and quantification of adhesion was accomplished by phase contrast video microscopy coupled with digital image processing. We report that both recombinant fragments studied, M19 and M55, and both polyclonal antibodies studied, alpha-M17 and alpha-M55, inhibit adhesion to varying degrees and that these processes are shear dependent. The M55 peptide and alpha-M55 cause much higher levels of inhibition than M19 and alpha-M17, respectively, at all wall shear rates studied. Our results demonstrate the importance of using a dynamic system in the assessment of inhibitory strategies and suggest the possible use of M55 and alpha-M55 in clinical applications to prevent infections caused by S. aureus adhesion to collagen.

MSCRAMM-mediated adherence of microorganisms to host tissues.

Microbial adhesion to host tissue is the initial critical event in the pathogenesis of most infections and, as such, is an attractive target for the development of new antimicrobial therapeutics. Specific microbial components (adhesins) mediate adherence to host tissues by participating in amazingly sophisticated interactions with host molecules. This review focuses on a class of cell surface adhesins that specifically interact with extracellular matrix components and which we have designated MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). MSCRAMMs recognizing fibronectin-, fibrinogen-, collagen-, and heparin-related polysaccharides are discussed in terms of structural organization, ligand-binding structures, importance in host tissue colonization and invasion, and role as virulence factors.

Fibronectin receptors from gram-positive bacteria: comparison of active sites.

Many parasitic bacteria express fibronectin binding proteins that are located on the cell surface. These proteins may act as adhesins and mediate the adherence of the microorganisms to fibronectin-containing host tissues. The ligand binding sites in the fibronectin receptor proteins from Gram-positive bacteria are composed of unique 37-48 amino acid long motifs that are repeated 3-4 times. We have now expressed the ligand binding sites of fibronectin receptors from Staphylococcus aureus, Streptococcus dysgalactiae (two receptors), and Streptococcus pyogenes as recombinant proteins. The purified recombinant proteins have the expected molecular weights as indicated by electrospray mass spectroscopy although they migrate abnormally on SDS-PAGE. Each recombinant protein effectively inhibited the binding of 125I-labeled intact fibronectin or the N-terminal fibronectin domain to Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus pyogenes. The relative inhibitory potency of the different recombinant proteins was similar for all target bacteria and is reflected in their relative affinities for fibronectin. Synthetic peptides corresponding to the repeat units of the ligand binding site of the fibronectin receptor proteins were shown to inhibit the binding of the N-terminal fibronectin fragment to Streptococcus pyogenes cells. Together with amino acid sequence comparison, these data demonstrate that the repeat motif of the fibronectin receptor of Streptococcus pyogenes conforms to the consensus sequence previously reported for the Staphylococcus aureus receptor and to one of the Streptococcus dysgalactiae receptors (McGavin et al., 1993).

Staphylococcus aureus expresses a major histocompatibility complex class II analog.

Staphylococcus aureus expresses various surface proteins which specifically recognize and bind to different host molecules. We have previously identified a bacterial protein that exhibits a broad specificity and binds to several mammalian extracellular proteins. The gene encoding this bacterial component has now been cloned and sequenced. The deduced protein consists predominantly of six repeated domains of 110 residues. Each of the repeated domains contain a subdomain of 31 residues that share striking sequence homology with a segment in the peptide binding groove of the beta chain of the major histocompatibility complex (MHC) class II proteins from different mammalian species. The purified recombinant bacterial protein bound several mammalian proteins, including recombinant osteopontin, suggesting a protein-protein interaction and also specifically recognized a 15-amino acid residue synthetic peptide. Taken together, these results suggest that the bacterial protein resembles mammalian MHC class II molecules with respect to both sequence similarities and peptide binding capabilities.

Critical residues in the ligand-binding site of the Staphylococcus aureus collagen-binding adhesin (MSCRAMM).

We have identified a discrete collagen-binding site within the Staphylococcus aureus collagen adhesin that is located in a region between amino acids Asp209 and Tyr233. Polyclonal antibodies raised against a recombinant form of the collagen adhesin inhibited the binding of collagen type II to S. aureus. When overlapping synthetic peptides mimicking segments of the adhesin fragment were tested for their ability to neutralize the inhibitory activity of the antibody only one peptide, CBD4 was found to be active. CBD4 bound directly to collagen and at high concentrations inhibited the binding of collagen to S. aureus. A synthetic peptide derivative of CBD4 lacking 2 carboxyl-terminal residues (Asn232, Tyr233) had no inhibitory activity. The importance of these residues for collagen binding was confirmed by biospecific interaction analysis. Mutant adhesin proteins N232-->A and Y233-->A exhibited dramatic changes in collagen binding activity. The dominant dissociation rate for the binding of mutant adhesin protein N232-->A to immobilized collagen II decreased almost 10-fold, while the Y233-->A and the double mutant exhibited even more significant decreases in affinity and apparent binding ratio when compared to the wild type protein.

Molecular characterization and expression of a gene encoding a Staphylococcus aureus collagen adhesin.

Some strains of Staphylococcus aureus bind collagen with a high degree of specificity and affinity. This interaction can represent a mechanism of substrate adhesion and may be an important step in the pathogenesis of osteomyelitis and infectious arthritis. We now report on the cloning, sequencing, and expression of a gene name cna, encoding a S. aureus collagen adhesin. The cna gene was isolated from a lambda GT11 S. aureus genomic library and encodes an 1185 amino acid polypeptide. The deduced amino acid sequence reveals several structural characteristics similar to previously described Gram-positive bacterial cell surface proteins. Antibodies raised against the native collagen adhesin from S. aureus recognize the recombinant collagen adhesin. Collagen binding activity can be detected in a lysate obtained from Escherichia coli cells, which harbor the cloned cna gene on an expression plasmid. Collagen-binding proteins can be detected in the lysate when analyzed by a Western blot type assay in which the membrane-transferred proteins are probed with radioactively labeled collagen. Finally, the bacterial lysate containing the recombinant adhesin can effectively inhibit the binding of soluble collagen to cells of S. aureus.

Staphylococcal fibronectin binding protein interacts with heat shock protein 60 and integrins: role in internalization by epithelial cells.

We reported previously that internalization of Staphylococcus aureus by nonprofessional phagocytes involves an interaction between fibronectin (Fn) binding protein (FnBP) and the host cell, resulting in signal transduction, tyrosine kinase activity, and cytoskeletal rearrangement (K. Dziewanowska, J. M. Patti, C. F. Deobald, K. W. Bayles, W. R. Trumble, and G. A. Bohach, Infect. Immun. 67:4673-4678, 1999). The goal of the present study was to identify the host molecules responsible for uptake of the organism through an interaction with FnBP. First, Fn was required for internalization. Addition of small amounts of exogenous Fn stimulated the uptake of S. aureus by HEp-2 cells, which are deficient in Fn synthesis. Fn antibodies blocked internalization of the organism by MAC-T cell monolayers, a bovine epithelial cell line which expresses Fn. Second, a monoclonal antibody (MAb) specific for beta(1) integrins dramatically reduced S. aureus invasion, suggesting that the formation of a Fn bridge linking the host cell beta(1) integrin and FnBP precedes internalization. However, ligand blotting of cell membrane proteins with a functional fragment of FnBP consistently identified an additional approximately 55-kDa receptor on both human and bovine epithelial cells. This protein was purified and identified by N-terminal microsequencing as heat shock protein 60 (Hsp60). The interaction between FnBP and Hsp60 also occurred when the whole cells were used. Cell membrane localization of Hsp60 was confirmed by biotinylation with an agent nonpermeable to the cell membrane. Pretreatment of epithelial cells with a MAb specific for eukaryotic Hsp60 significantly reduced internalization of S. aureus. Combined, these results suggest that the FnBP binds directly to both Hsp60 and Fn and is linked to beta(1) integrins through a Fn bridge. The simultaneous involvement of Fn and two host cell ligands, beta(1) integrins and Hsp60, suggests that FnBP is a multifunctional adhesin that mediates internalization in a manner similar to that proposed for OpaA, the Neisseria gonorrhoeae FnBP homolog (J. P. M. van Putten, T. D. Duensing, and R. L. Cole, Mol. Microbiol. 29:369-379, 1998).

Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells.

Staphylococcus aureus expresses several surface proteins that promote adherence to host cell extracellular matrix proteins, including fibronectin (Fn). Since this organism has recently been shown to be internalized by nonprofessional phagocytes, a process that typically requires high-affinity binding to host cell receptors, we investigated the role of its Fn binding proteins (FnBPs) and other surface proteins in internalization by the bovine mammary gland epithelial cell line (MAC-T). Efficient internalization of S. aureus 8325-4 required expression of FnBPs; an isogenic mutant (DU5883), not expressing FnBPs, was reduced by more than 95% in its ability to invade MAC-T cells. Moreover, D3, a synthetic peptide derived from the ligand binding domain of FnBP, inhibited the internalization of the 8325-4 strain in a dose-dependent fashion and the efficiency of staphylococcal internalization was partially correlated with Fn binding ability. Interestingly, Fn also inhibited the internalization and adherence of S. aureus 8325-4 in a dose-dependent manner. In contrast to internalization, adherence of DU5883 to MAC-T was reduced by only approximately 40%, suggesting that surface binding proteins, other than FnBPs, can mediate bacterial adherence to cells. Adherence via these proteins, however, does not necessarily result in internalization of the staphylococci. An inhibitor of protein tyrosine kinase, genistein, reduced MAC-T internalization of S. aureus by 95%, indicating a requirement for a host signal transduction system in this process. Taken together, these results indicate that S. aureus invades nonprofessional phagocytes by a mechanism requiring interaction between FnBP and the host cell, leading to signal transduction and subsequent rearrangement of the host cell cytoskeleton.

A collagen receptor on Staphylococcus aureus strains isolated from patients with septic arthritis mediates adhesion to cartilage.

Staphylococcus aureus strains isolated from patients with septic arthritis or osteomyelitis possess a collagen receptor present in two forms, which contains either two or three copies of a 187-amino-acid repeat motif. Collagen receptor-positive strains adhered to both collagen substrata and cartilage in a time-dependent process. Collagen receptor-specific antibodies blocked bacterial adherence, as did preincubation of the substrate with a recombinant form of the receptor protein. Furthermore, polystyrene beads coated with the collagen receptor bound collagen and attached to cartilage. Taken together, these results suggest that the collagen receptor is both necessary and sufficient to mediate bacterial adherence to cartilage in a process that constitutes an important part of the pathogenic mechanism in septic arthritis.