Variance in fibronectin binding and fnb locus polymorphisms in Staphylococcus aureus: identification of antigenic variation in a fibronectin binding protein adhesin of the epidemic CMRSA-1 strain of methicillin-resistant S. aureus.

The fnbA and fnbB genes of Staphylococcus aureus 8325-4 encode fibronectin (Fn) binding proteins FnBPA and FnBPB, which promote adherence to host tissues. Each adhesin contains three copies of a repeated D motif that binds Fn and is a target for vaccine development. In this study, we assess variability within the Fn-binding domain of the FnBP adhesins and evaluate factors that promote variance in Fn binding among clinical isolates. Based on variation in the number of fnb genes or the number of D motifs, we identified five polymorphism groups. S. aureus 8325-4 and 91% of methicillin-resistant S. aureus (MRSA) isolates belong to polymorphism group I, with two fnb genes and three copies of the D motif. Polymorphism group II contained one fnb gene with only two D motifs and was associated with the epidemic CMRSA-4 strain, which exhibited high protease activity and low Fn binding. Polymorphism group III was unique to the epidemic CMRSA-1 strain, defined by the presence of a fourth D motif that exhibited antigenic variation within a conserved sequence that is essential for Fn binding. However, the sequence of the D motifs was otherwise highly conserved among the other polymorphism groups. Variation in Fn binding among MRSA isolates was inversely related to protease activity but not to the number of fnb genes or the number of D motifs. Therefore, the fnb locus is polymorphic in a small number of strains, but this does not contribute to variation in Fn binding. The antigenic variation that was observed only in the epidemic CMRSA-1 strain may have evolved in response to a host immune response encountered during successive cycles of colonization, transmission, and infection in the nosocomial environment.

Description of staphylococcus serine protease (ssp) operon in Staphylococcus aureus and nonpolar inactivation of sspA-encoded serine protease.

Signature tagged mutagenesis has recently revealed that the Ssp serine protease (V8 protease) contributes to in vivo growth and survival of Staphylococcus aureus in different infection models, and our previous work indicated that Ssp could play a role in controlling microbial adhesion. In this study, we describe an operon structure within the ssp locus of S. aureus RN6390. The ssp gene encoding V8 protease is designated as sspA, and is followed by sspB, which encodes a 40.6-kDa cysteine protease, and sspC, which encodes a 12.9-kDa protein of unknown function. S. aureus SP6391 is an isogenic derivative of RN6390, in which specific loss of SspA function was achieved through a nonpolar allelic replacement mutation. In addition to losing SspA, the culture supernatant of SP6391 showed a loss of 22- to 23-kDa proteins and the appearance of a 40-kDa protein corresponding to SspB. Although the 40-kDa SspB protein could degrade denatured collagen, our data establish that this is a precursor form which is normally processed by SspA to form a mature cysteine protease. Culture supernatant of SP6391 also showed a new 42-kDa glucosaminidase and enhanced glucosaminidase activity in the 29 to 32 kDa range. Although nonpolar inactivation of sspA exerted a pleiotropic effect, S. aureus SP6391 exhibited enhanced virulence in a tissue abscess infection model relative to RN6390. Therefore, we conclude that SspA is required for maturation of SspB and plays a role in controlling autolytic activity but does not by itself exert a significant contribution to the development of tissue abscess infections.

Molecular analysis of the accessory gene regulator (agr) locus and balance of virulence factor expression in epidemic methicillin-resistant Staphylococcus aureus.

Potential relationships between virulence factor expression and transmissibility were assessed in epidemic methicillin-resistant Staphylococcus aureus (MRSA) clones CMRSA-1 and CMRSA-3. A major subtype of CMRSA-1 exhibited normal transcription of RNAIII, which facilitates the induction of secreted virulence factors and repression of colonization factor expression at high cell density. However, these isolates characteristically did not express alpha-toxin or protease and displayed a limited profile of secreted proteins. CMRSA-1 also expressed a novel cell surface glycoprotein and exhibited a unique polymorphism within the accessory gene regulator (agr) locus. CMRSA-3 displayed attenuated activation of RNAIII transcription, which was consistent with its higher fibronectin-binding and coagulase activity relative to sporadic MRSA or CMRSA-1 (P=.05), low protease activity, and limited profile of secreted proteins. Thus, the balance of virulence factor expression in CMRSA-1 and CMRSA-3 favors the colonization phase of infection, and CMRSA-1 possesses unique genotypic and phenotypic traits.

Synthetic peptide immunogens elicit polyclonal and monoclonal antibodies specific for linear epitopes in the D motifs of Staphylococcus aureus fibronectin-binding protein, which are composed of amino acids that are essential for fibronectin binding.

A fibronectin (Fn)-binding adhesin of Staphylococcus aureus contains three tandem 37- or 38-amino-acid motifs (D1, D2, and D3), which function to bind Fn. Plasma from patients with S. aureus infections contain antibodies that preferentially recognize ligand induced binding sites in the D motifs and do not inhibit Fn binding (F. Casolini, L. Visai, D. Joh, P. G. Conaldi, A. Toniolo, M. Höök, and P. Speziale, Infect. Immun. 66:5433-5442, 1998). To eliminate the influence of Fn binding on antibody development, we used synthetic peptide immunogens D1(21-34) and D3(20-33), which each contain a conserved pattern of amino acids that is essential for Fn binding but which cannot bind Fn without N- or C-terminal extensions. The D3(20-33) immunogen promoted the production of polyclonal antibodies that were 10-fold more effective as inhibitors of Fn-binding to the D3 motif than antibodies obtained by immunizing with an extended peptide D3(16-36), which exhibits functional Fn binding. The D3(20-33) immunogen also facilitated the production of a monoclonal antibody, 9C3, which was highly specific for the epitope SVDFEED, and abolished Fn binding by the D3 motif. When mixed with polyclonal anti-D1(21-34) immunoglobulin G, 70% inhibition of Fn binding to the three tandem D motifs was achieved compared to no more than 30% inhibition with either antibody preparation alone. Therefore, by immunizing with short synthetic peptides that are unable to bind Fn, we have effectively stimulated the production of antibodies specific for epitopes comprised of amino acids that are essential for Fn binding. Although these epitopes occur within a conserved pattern of amino acids that is required for Fn binding, the antibodies recognized specific linear epitope sequences and not a conserved structure common to all repeated motifs.

Modification of the Staphylococcus aureus fibronectin binding phenotype by V8 protease.

The amount of cell surface fibronectin (Fn)-binding protein (FnBP) adhesin expressed by Staphylococcus aureus is maximal during exponential growth but disappears rapidly as the culture progresses into stationary phase. To identify factors responsible for the loss of cell surface FnBP, a culture of S. aureus L170, which shows high levels of Fn binding, was supplemented at the time of inoculation with concentrated stationary-phase supernatant from S. aureus L530, a strain which binds Fn poorly. The resulting exponential-phase cells were devoid of FnBP. The factor responsible for this activity was purified from the culture supernatant and identified as V8 protease. When cultured with 375 ng of exogenous V8 protease ml(-1), exponential-phase cells of S. aureus L170 were devoid of cell surface FnBP, and concentrations as low as 23 ng x ml(-1) resulted in reduced amounts of FnBP. Addition of the protease inhibitor alpha2-macroglobulin to the culture medium prevented the growth-phase-dependent loss of cell surface FnBP, whereas growth with exogenous V8 protease resulted in reduced adherence to the solid-phase N-terminal fragment of Fn and to the extracellular matrix synthesized by fetal rabbit lung fibroblasts. Although FnBP was extremely sensitive to V8 protease, exogenous protease did not exert a significant influence on the amount of cell surface protein A. However, a limited number of other high-molecular-weight cell surface proteins were also sensitive to V8 protease. Therefore, both the adhesive phenotype and cell surface protein profile of S. aureus can be modified by V8 protease activity.

Identification of D motif epitopes in Staphylococcus aureus fibronectin-binding protein for the production of antibody inhibitors of fibronectin binding.

A fibronectin-binding protein (FnBP) adhesin of Staphylococcus aureus possesses three 37- or 38-amino-acid motifs (D1, D2, and D3) that can each bind fibronectin (Fn) with low affinity and that in tandem comprise D1-3, a high-affinity Fn-binding domain. To identify epitopes for the generation of adhesion-blocking antibodies, rabbits were immunized with recombinant D1-3 or with a glutathione S-transferase fusion protein, GSTD1-3. Affinity-purified antibodies from the D1-3 immunization were poor inhibitors of Fn binding to S. aureus and recognized several different epitopes, with a preference for clusters of acidic amino acids that do not contribute to Fn binding. Antibodies generated with GSTD1-3 as an immunogen were more effective inhibitors, but concentrations in excess of 20 microg x ml-1 did not promote more than 50% inhibition. These antibodies were highly specific for amino acids 21 to 34 of D1 (D1(21-34)), which contain a sequence that is essential for Fn binding and are identical to D2 at 12 of 14 residues. Neither antibody preparation recognized D3(20-33) of the D3 motif, where the only homology to D1(21-34) and D2(21-34) comprises a sequence motif, GG(X3,4)(I/V)DF, that is critical to Fn binding. However, antibodies specific for both D1(21-34) and D3(20-33) could be obtained by using synthetic peptides corresponding to these sequences as immunogens. F(ab')2 fragments derived from these antibodies each caused 40 to 50% inhibition of Fn binding to S. aureus, and their ability to bind to purified FnBP was eliminated by competing Fn. However, mixtures of the two F(ab')2 preparations did not provide additive or synergistic inhibition of Fn binding. Therefore, inhibition of Fn binding to S. aureus requires antibodies specific for D1(21-34) and D3(20-33), but a mixture of antibodies specific for both sequences did not provide complete inhibition.

Interaction of N-terminal fragments of fibronectin with synthetic and recombinant D motifs from its binding protein on Staphylococcus aureus studied using fluorescence anisotropy.

The N-terminal 29-kDa fragment of fibronectin (Fn29K) contains five type I "finger" modules. It binds to heparin, fibrin, and bacteria and is involved in fibronectin (Fn) matrix assembly. Binding to Staphylococcus aureus involves a cell wall-associated protein that contains approximately three repeats of a 38-residue D motif (Signäs, C., Raucci, G., Jönsson, K., Lindgren, P.-E., Anantharamaiah, G.M., Höök, M., and Lindberg, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 699-703). Synthetic peptides representing D1, D2, and D3, when labeled with fluorescein isothiocyanate (FITC), exhibited increases in fluorescence anisotropy upon addition of Fn29K but not other Fn fragments. The response could be reversed by titration with unlabeled peptides to yield inhibition constants that agreed with the dissociation constants obtained by fitting the initial response. Values of Kd ranged between 2 and 12 microM, with D3 having the highest affinity. Specificity of D3 for Fn29K was further illustrated by the fact that its C-terminal half (D3b, Lys801 to Lys821), when immobilized, selectively adsorbed Fn29K from a thermolysin digest of fibronectin. The binding site in Fn was further localized within Fn29K by analyzing smaller proteolytic or recombinant subfragments. Those containing fingers, F3-5 and F4-5, were purified on D3b-Sepharose and bound FITC-D3b with Kd values of 4-6 microM. Subfragments containing pairs of fingers 1-2, 2-3, or single fingers 1, 4, or 5 were inactive. Whole D1-3, expressed in Escherichia coli and labeled with fluorescein, bound 1.9 mol/mol of Fn29K with Kd = 1.5 nM. F4-5 and F2-3 bound with respective Kd values of 0.35 and 4.4 microM. These and other results indicate that binding of the individual D region peptides is mediated through their C-terminal halves, primarily to fingers 4 and 5 of fibronectin. The possible basis of the much higher affinity of D1-3 is discussed.

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 Streptococcus dysgalactiae and Staphylococcus aureus. Involvement of conserved residues in ligand binding.

The nucleotide sequence of two genes encoding fibronectin (Fn) receptors FnBA and FnBB of Streptococcus dysgalactiae S2 revealed the presence of repeated motifs (called RA1-A3 and RB1-B3, respectively) which encode Fn binding activity (Lindgren, P.-E., McGavin, M. J., Signäs, C., Guss, B., Gurusiddappa, S., Höök, M., and Lindberg, M. (1993) Eur. J. Biochem. 214, 819-827). Synthetic peptides of 32-37 amino acids, corresponding to individual repeated motifs, were assayed for the ability to inhibit Fn binding to cells of S. dysgalactiae. Within the RA motifs, peptide A2 was 10-fold more active than either A1 or A3, while in the RB motifs, only B3 was active. The same level of activity is observed when these synthetic peptides were assayed for inhibition of Fn binding to cells of Staphylococcus aureus. Likewise, synthetic peptides corresponding to the RD1-D3 motifs, which comprise a ligand binding domain in a Fn receptor from S. aureus, inhibit binding of Fn to both S. aureus and S. dysgalactiae. Assays of chemically modified peptides and peptide fragments derived from chemical or proteolytic cleavage suggest that a conserved core sequence, defined as ED(T/S) (X9,10)GG(X3,4)(I/V)DF, within a 30-amino acid-long segment is present in the active RA and RD motifs. Analyses of the importance of individual residues of this core sequence indicate that the ED(T/S) motif is nonessential, whereas the GG and the (I/V)DF together with additional acidic residues in the C-terminal half of the peptide are required for activity.

Two different genes coding for fibronectin-binding proteins from Streptococcus dysgalactiae. The complete nucleotide sequences and characterization of the binding domains.

The binding of Streptococcus dysgalactiae to fibronectin involves fibronectin-binding protein(s) present on the bacterial surface. Previously, we reported the cloning of two different genes coding for cell-wall-associated fibronectin-binding proteins from S. dysgalactiae strain S2 [Lindgren, P.-E., Speziale, P., McGavin, M. J., Monstein, H.-J., Höök, M., Visai, L., Kostiainen, T., Bozzini, S. & Lindberg, M. (1992) J. Biol. Chem. 267, 1924-1931]. The two genes, fnbA and fnbB, have now been sequenced and the primary amino acid sequences of the two fibronectin-binding proteins, FnBA and FnBB, have been deduced. The two proteins have predicted molecular masses of 117 kDa and 122 kDa, respectively, and are organized in a similar way. The fibronectin-binding activities are localized in repeated motifs, 32-37 amino acids long, in the COOH-terminal regions of the proteins. The two fibronectin-binding proteins have heterologous amino acid sequences, except for the COOH-terminal ends which include the fibronectin-binding repeats. The fibronectin-binding regions of the genes have been fused to IgG-binding domains of protein A, utilizing the IgG-binding capacity of the resulting fusion proteins, to facilitate isolation of the fibronectin-binding domains.

Fibronectin binding determinants of the Staphylococcus aureus fibronectin receptor.

Synthetic peptide analogs mimicking a repeated motif within the Staphylococcus aureus fibronectin receptor inhibit binding of the bacteria to fibronectin (Signäs, C., Raucci, G., Jonsson, K., Lindgren, P. E., Anantharamaiah, G. M., Höök, M., and Lindberg, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 699-703). In this study, we have further localized the fibronectin-binding determinant within the 37 amino acid D3 peptide. Chemical modification of the carboxyl side chains of the glutamic and aspartic residues in D3 abolished fibronectin-binding activity, whereas modifications of lysine or tyrosine residues had little effect. An active peptide encompassing residues 15-36 was isolated from a trypsin digest of D3, and a synthetic peptide S16-36 had activity comparable with that of intact D3. Scrambling the amino acid sequence of S16-36 or replacing the aspartic and glutamic residues with asparagine and glutamine resulted in loss of activity. Therefore, one or more of the acidic residues are essential for activity. However, additional sequence is required. Reduction in the size of S16-36 from either the N- or C-terminal end resulted in peptides with greatly diminished activity. These data suggest that the amino acids essential for binding fibronectin are contained within residues 21-33 of the D3 peptide and that the flanking N- and C-terminal amino acids are necessary for the peptide to acquire a conformation that is favorable for fibronectin binding.

Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3.

The cel-3 gene cloned from Fibrobacter succinogenes into Escherichia coli coded for the enzyme EG3, which exhibited both endoglucanase and cellobiosidase activities. The gene had an open reading frame of 1,974 base pairs, coding for a protein of 73.4 kilodaltons (kDa). However, the enzyme purified from the osmotic shock fluid of E. coli was 43 kDa. The amino terminus of the 43-kDa protein matched amino acid residue 266 of the protein coded for by the open reading frame, indicating proteolysis in E. coli. In addition to the 43-kDa protein, Western immunoblotting revealed a 94-kDa membranous form of the enzyme in E. coli and a single protein of 118 kDa in F. succinogenes. Thus, the purified protein appears to be a proteolytic degradation product of a native protein which was 94 kDa in E. coli and 118 kDa in F. succinogenes. The discrepancy between the molecular weight expected on the basis of the DNA sequence and the in vivo form may be due to anomalous migration during electrophoresis, to glycosylation of the native enzyme, or to fatty acyl substitution at the N terminus. One of two putative signal peptide cleavage sites bore a strong resemblance to known lipoprotein leader sequences. The purified 43-kDa peptide exhibited a high Km (53 mg/ml) for carboxymethyl cellulose but a low Km (3 to 4 mg/ml) for lichenan and barley beta-glucan. The enzyme hydrolyzed amorphous cellulose, and cellobiose and cellotriose were the major products of hydrolysis. Cellotriose, but not cellobiose, was cleaved by the enzyme. EG3 exhibited significant amino acid sequence homology with endoglucanase CelC from Clostridium thermocellum, and as with both CelA and CelC of C. thermocellum, it had a putative active site which could be aligned with the active site of hen egg white lysozyme at the highly conserved amino acid residues Asn-44 and Asp-52.