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PubMed2002

Identification of a fibronectin-binding protein from Staphylococcus epidermidis.

Staphylococcus epidermidis has been reported to bind to a number of host cell extracellular matrix proteins, including fibronectin. Here we report the identification of a fibronectin-binding protein from S. epidermidis. A phage display library of S. epidermidis genomic DNA was constructed and panned against immobilized fibronectin. A number of phagemid clones containing overlapping inserts were identified, and one of these clones, pSE109FN, contained a 1.4-kb insert. Phage pSE109FN was found to bind to fibronectin but not to collagen, fibrinogen, laminin, or vitronectin. However, pSE109FN also bound to heparin, hyaluronate, and plasminogen, although to a lesser extent than it bound to fibronectin. Analysis of The Institute for Genomic Research S. epidermidis genome sequence database revealed a 1.85-kb region within a putative 30.5-kb open reading frame, to which the overlapping DNA inserts contained within the fibronectin-binding phagemids mapped. We have designated the gene encoding the fibronectin-binding domain embp. A recombinant protein, Embp32, which encompassed the fibronectin-binding domain of Embp, blocked the binding of S. epidermidis, but not the binding of Staphylococcus aureus, to fibronectin. In contrast, a recombinant protein, FnBPB[D1-D4], spanning the fibronectin-binding domain of the S. aureus fibronectin-binding protein FnBPB, blocked binding of S. aureus to fibronectin but had a negligible effect on the binding of S. epidermidis.

Adhesins, Bacterial↗

PubMed2002

Genome sequence survey identifies unique sequences and key virulence genes with unusual rates of amino Acid substitution in bovine Staphylococcus aureus.

Staphylococcus aureus is a major cause of mastitis in bovine and other ruminant species. We here present the results of a comparative genomic analysis between a bovine mastitis-associated clone, RF122, and the recently sequenced human-associated clones, Mu50 and N315, of Staphylococcus aureus. A shotgun sequence survey of approximately 10% of the RF122 genome identified numerous unique sequences and those with elevated rates of nonsynonymous substitution. Taken together, these analyses show that there are notable differences in the genomes of bovine mastitis-associated and human clones of S. aureus and provide a framework for the identification of specific factors associated with host specificity in this major human and animal pathogen.

Amino Acid Substitution↗

PubMed2003

Fibronectin bound to the surface of Staphylococcus aureus induces association of very late antigen 5 and intracellular signaling factors with macrophage cytoskeleton.

Staphylococcus aureus Cowan I and a clinically isolated coagulase-negative Staphylococcus strain, S. saprophyticus 10312, were found to have two fibronectin binding proteins, FnBPA and FnBPB. While both staphylococci bound to serum fibronectin to a similar extent, fibronectin binding significantly increased the phagocytic activity of macrophages against S. aureus (by ca. 150%) but not against S. saprophyticus. This enhancing effect of fibronectin was inhibited by an RGD sequence-containing peptide and also by anti-very late antigen 5 antibody. This suggests that the effect is mediated by very late antigen 5 expressed on macrophages. In macrophages ingesting fibronectin-bound Cowan I, alpha(5) and beta(1) chains were associated with the cytoskeleton. Cytosolic signaling factors such as paxillin, c-Src, and c-Csk were also associated with the cytoskeleton. On the contrary, beta(3) integrin transiently disappeared from the cytoskeleton when macrophages ingested the fibronectin-treated S. aureus Cowan I. Furthermore, the Src kinase family tyrosine kinase Lyn dissociated from the cytoskeleton. These cellular components did not respond in a fibronectin-dependent manner when macrophages phagocytosed S. saprophyticus. This means that only fibronectin-treated S. aureus Cowan I induces the accumulation of very late antigen 5, which in turn induces the association of paxillin and tyrosine kinases. It is thought that the phagocytic activity of macrophages against fibronectin-treated S. aureus was increased by signaling via the activation of very late antigen 5.

Animals↗

PubMed2003

Characterization of the role of the divalent metal ion-dependent transcriptional repressor MntR in the virulence of Staphylococcus aureus.

DtxR-type metal ion-dependent repressors, present in many bacterial pathogens, may regulate expression of virulence genes such as that encoding diphtheria toxin. SirR, a DtxR homologue initially identified in Staphylococcus epidermidis, governs the expression of the adjacent sitABC operon encoding a putative metal ion ABC transporter system. We identified a sirR homologue, mntR, in Staphylococcus aureus and demonstrated by gel shift assay that the corynebacterial repressor DtxR binds to the S. aureus mntABC operator in the presence of Fe(2+) or Mn(2+). Since a mutant DtxR, DtxR(E175K), functions as an iron-independent hyperrepressor in certain settings, we constructed a heterodiploid S. aureus strain expressing dtxR(E175K) from the native mntR promoter. Transcription of the S. aureus mntABC operon was repressed in the presence of Fe(2+) or Mn(2+) in wild-type and heterodiploid S. aureus strains. Under metal ion-limiting conditions, mntABC transcription was reduced but not abolished in S. aureus isolates expressing dtxR(E175K) compared with an isogenic control, suggesting that DtxR(E175K) binds the S. aureus MntR box in vivo. Under all conditions tested, mntABC transcription in the dtxR(E175K)-expressing strain was reduced relative to the isogenic control, indicating that DtxR(E175K) function was constitutively active. In the mouse skin abscess model, dtxR(E175K)-expressing S. aureus recombinants showed significantly reduced CFU levels compared with the isogenic wild-type control. We conclude that the S. aureus MntR box is recognized by corynebacterial DtxR proteins and thus belongs to the DtxR family of metal-dependent operator sites. Moreover, constitutive repression by DtxR(E175K) reduces the virulence of S. aureus in the mouse skin abscess model.

Abscess↗

PubMed2004

Role of siderophore biosynthesis in virulence of Staphylococcus aureus: identification and characterization of genes involved in production of a siderophore.

Molecular determinants underlying the production of siderophores in the human and animal pathogen Staphylococcus aureus and the contribution of siderophore production to the virulence of this bacterium have, until now, remained undefined. Here, we show that S. aureus strains RN6390 and Newman produce siderophore when the cells are starved for iron. We further identified and characterized a nine-gene, iron-regulated operon, designated sbn and situated between sirABC and galE on the S. aureus chromosome, that is involved in the production of a siderophore. Mutation of the sbnE gene, in both RN6390 and Newman, eliminates the ability of these strains to produce a siderophore under iron-limited growth conditions, while introduction of multicopy sbnE into sbnE mutants complemented the inability of the mutants to produce the siderophore. sbnE mutants, in both the RN6390 and Newman backgrounds, displayed a drastic growth deficiency, compared to the wild type, in iron-restricted growth medium, whereas no such deficiency was observed during growth in iron-replete medium. Complemented mutants showed a restored ability to grow under iron restriction. We further showed that an sbnE mutant was compromised in a murine kidney abscess model of S. aureus infection, illustrating the importance of siderophore production to the pathogenicity of S. aureus. sbn genes were present in all S. aureus strains tested (and all S. aureus genome sequences) but were undetectable in any of the 13 coagulase-negative staphylococci tested, including Staphylococcus epidermidis.

Abscess↗

PubMed2004

Truncation of fibronectin-binding proteins in Staphylococcus aureus strain Newman leads to deficient adherence and host cell invasion due to loss of the cell wall anchor function.

Staphylococcus aureus fibronectin-binding proteins (FnBPs) play a critical role in S. aureus pathogenesis. FnBPs mediate adhesion to fibronectin and invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, by fibronectin bridging to the host cell fibronectin receptor integrin (alpha(5))beta(1). Strain Newman is a laboratory strain frequently used for genetic, functional, and in vivo studies. However, despite pronounced production of FnBPs, strain Newman is only weakly adherent to immobilized Fn and weakly invasive. We examined whether these effects are due to a structural difference of FnBPs. Here, we show that both fnbA(Newman) and fnbB(Newman) contain a centrally located point mutation resulting in a stop codon. This leads to a truncation of both FnBPs at the end of the C domain at identical positions. Most likely, the stop codon occurred first in fnbB(Newman) and was subsequently transferred to fnbA(Newman) by replacement of the entire region encompassing the C, D, and W domains with the respective sequence of fnbB(Newman). Using heterologous expression in Staphylococcus carnosus, we found that truncated FnBPs were completely secreted into the culture medium and not anchored to the cell wall, since they lack the sortase motif (LPETG). Consequently, this led to a loss of FnBP-dependent functions, such as strong adhesion to immobilized fibronectin, binding of fibrinogen, and host cell invasion. This mutation may explain some of the earlier reported conflicting data with strain Newman. Thus, care should be taken when drawing negative conclusions about the role of FnBPs as a virulence factor in a given model.

Adhesins, Bacterial↗

PubMed2004

RsbU-dependent regulation of Staphylococcus epidermidis biofilm formation is mediated via the alternative sigma factor sigmaB by repression of the negative regulator gene icaR.

Transposon mutagenesis of rsbU leads to a biofilm-negative phenotype in Staphylococcus epidermidis. However, the pathway of this regulatory mechanism was unknown. To investigate the role of RsbU in the regulation of the alternative sigma factor sigma(B) and biofilm formation, we generated different mutants of the sigma(B) operon in S. epidermidis strains 1457 and 8400. The genes rsbU, rsbV, rsbW, and sigB, as well as the regulatory cascade rsbUVW and the entire sigma(B) operon, were deleted. Transcriptional analysis of sarA and the sigma(B)-dependent gene asp23 revealed the functions of RsbU and RsbV as positive regulators and of RsbW as a negative regulator of sigma(B) activity, indicating regulation of sigma(B) activity similar to that characterized for Bacillus subtilis and Staphylococcus aureus. Phenotypic characterization of the mutants revealed that the dramatic decrease of biofilm formation in rsbU mutants is mediated via sigma(B), indicating a crucial role for sigma(B) in S. epidermidis pathogenesis. However, biofilm formation in mutants defective in sigma(B) or its function could be restored in the presence of subinhibitory ethanol concentrations. Transcriptional analysis revealed that icaR is up-regulated in mutants lacking sigma(B) function but that icaA transcription is down-regulated in these mutants, indicating a sigma(B)-dependent regulatory intermediate negatively regulating IcaR. Supplementation of growth media with ethanol decreased icaR transcription, leading to increased icaA transcription and a biofilm-positive phenotype, indicating that the ethanol-dependent induction of biofilm formation is mediated by IcaR. This icaR-dependent regulation under ethanol induction is mediated in a sigma(B)-independent manner, suggesting at least one additional regulatory intermediate in the biofilm formation of S. epidermidis.

Bacterial Proteins↗

PubMed2005

The surface-associated protein of Staphylococcus saprophyticus is a lipase.

Staphylococcus saprophyticus surface-associated protein (Ssp) was the first surface protein described for this organism. Ssp-positive strains display a fuzzy layer of surface-associated material in electron micrographs, whereas Ssp-negative strains appear to be smooth. The physiologic function of Ssp, however, has remained elusive. To clone the associated gene, we determined the N-terminal sequence, as well as an internal amino acid sequence, of the purified protein. We derived two degenerate primers from these peptide sequences, which we used to identify the ssp gene from genomic DNA of S. saprophyticus 7108. The gene was cloned by PCR techniques and was found to be homologous to genes encoding staphylococcal lipases. In keeping with this finding, strains 7108 and 9325, which are Ssp positive, showed lipase activity on tributyrylglycerol agar plates, whereas the Ssp-negative strain CCM883 did not. Association of enzyme activity with the cloned DNA was proven by introducing the gene into Staphylococcus carnosus TM300. When wild-type strain 7108 and an isogenic mutant were analyzed by transmission electron microscopy, strain 7108 exhibited the fuzzy surface layer, whereas the mutant appeared to be smooth. Lipase activity and the surface appendages could be restored by reintroduction of the cloned gene into the mutant. Experiments using immobilized collagen type I did not provide evidence for the involvement of Ssp in adherence to this matrix protein. Our experiments thus provided evidence that Ssp is a surface-associated lipase of S. saprophyticus.

Amino Acid Sequence↗

PubMed2005

Comparative opsonic and protective activities of Staphylococcus aureus conjugate vaccines containing native or deacetylated Staphylococcal Poly-N-acetyl-beta-(1-6)-glucosamine.

Staphylococcus aureus and Staphylococcus epidermidis both synthesize the surface polysaccharide poly-N-acetyl-beta-(1-6)-glucosamine (PNAG), which is produced in vitro with a high level (>90%) of the amino groups substituted by acetate. Here, we examined the role of the acetate substituents of PNAG in generating opsonic and protective antibodies. PNAG and a deacetylated form of the antigen (dPNAG; 15% acetylation) were conjugated to the carrier protein diphtheria toxoid (DT) and used to immunize animals. Mice responded in a dose-dependent fashion to both conjugate vaccines, with maximum antibody titers observed at the highest dose and 4 weeks after the last of three weekly immunizations. PNAG-DT and dPNAG-DT vaccines were also very immunogenic in rabbits. Antibodies raised to the conjugate vaccines in rabbits mediated the opsonic killing of various staphylococcal strains, but the specificity of the opsonic killing was primarily to dPNAG, as this antigen inhibited the killing of S. aureus strains by both PNAG- and dPNAG-specific antibodies. Passive immunization of mice with anti-dPNAG-DT rabbit sera showed significant levels of clearance of S. aureus from the blood (54 to 91%) compared to control mice immunized with normal rabbit sera, whereas PNAG-specific antibodies were ineffective at clearing S. aureus. Passive immunization of mice with a goat antiserum raised to the dPNAG-DT vaccine protected against a lethal dose of three different S. aureus strains. Overall, these data show that immunization of animals with a conjugate vaccine of dPNAG elicit antibodies that mediated opsonic killing and protected against S. aureus infection, including capsular polysaccharide types 5 and 8 and an untypable strain.

Acetylation↗

PubMed2005

The multifunctional Staphylococcus aureus autolysin aaa mediates adherence to immobilized fibrinogen and fibronectin.

Staphylococci can cause a wide spectrum of infections, including endocarditis, osteomyelitis, and sepsis, which is reflected by the numerous virulence factors they produce, among them a recently identified new class of adhesins, namely, the multifunctional autolysins/adhesins. Here we report the identification and molecular characterization of Aaa, a novel autolysin/adhesin from Staphylococcus aureus. The gene encoding Aaa was cloned from the clinical isolate Staphylococcus aureus 4074. DNA sequence analysis revealed that aaa encodes a deduced protein of 334 amino acids with a predicted molecular mass of 35.8 kDa. Aaa contains three N-terminal repetitive sequences that comprise features of a peptidoglycan-binding domain, the LysM domain. The expression of aaa by Escherichia coli and its subsequent characterization revealed that Aaa possesses bacteriolytic activity as well as adhesive properties, such as binding to extracellular matrix proteins. Real-time biomolecular interaction analysis demonstrated that the interaction of Aaa with fibrinogen, fibronectin, and vitronectin is dose dependent and saturable and occurs with a high affinity. Furthermore, we demonstrate that Aaa binds to the Aalpha and Bbeta chains of fragment D of fibrinogen. Immunofluorescence microscopy revealed that Aaa is located at the cell surface. Finally, an aaa knockout mutant showed reduced adherence to surface-adsorbed fibrinogen and fibronectin, strongly suggesting a role for Aaa in the colonization of host factor-coated polymer surfaces and/or host tissue.

Adhesins, Bacterial↗

PubMed2006

Staphylococcus aureus mutants with increased lysostaphin resistance.

Staphylococcus simulans secretes lysostaphin, a bacteriolytic enzyme that specifically binds to the cell wall envelope of Staphylococcus aureus and cleaves the pentaglycine cross bridges of peptidoglycan, thereby killing staphylococci. The study of S. aureus mutants with resistance to lysostaphin-mediated killing has revealed biosynthetic pathways for cell wall assembly. To identify additional genes involved in cell wall envelope biosynthesis, we have screened a collection of S. aureus strain Newman transposon mutants for lysostaphin resistance. Bursa aurealis insertion in SAV2335, encoding a polytopic membrane protein with predicted protease domain, caused a high degree of lysostaphin resistance, similar to the case for a previously described femAB promoter mutant. In contrast to the case for this femAB mutant, transposon insertion in SAV2335, herein named lyrA (lysostaphin resistance A), did not cause gross alterations of cell wall cross bridges such as truncations of pentaglycine to tri- or monoglycine. Also, inactivation of LyrA in a methicillin-resistant S. aureus strain did not precipitate a decrease in beta-lactam resistance as observed for fem (factor essential for methicillin resistance) mutants. Lysostaphin bound to the cell wall envelopes of lyrA mutants in a manner similar to that for wild-type staphylococci. Lysostaphin resistance of lyrA mutants is attributable to altered cell wall envelope properties and may in part be due to increased abundance of altered cross bridges. Other lyr mutants with intermediate lysostaphin resistance carried bursa aurealis insertions in genes specifying GTP pyrophosphokinase or enzymes of the purine biosynthetic pathway.

Amino Acid Sequence↗

PubMed1969

Pseudocompact-type growth and conversion of growth types of strains of Staphylococcus aureus in vitro and in vivo.

Some strains of Staphylococcus aureus grew as compact colonies in Brain Heart Infusion-serum-soft agar but as diffuse colonies in a modified Staphylococcus 110-serum-soft agar. These strains were designated "pseudocompact." Strains showing compact-type colonial morphology in both media were designated "compact," whereas strains showing diffuse-type growth in both media were designated "diffuse." It was observed that the most recently isolated strains of S. aureus were of the pseudocompact type, whereas most stock culture strains tested were of the compact type. Using cultures recently isolated from clinical material, it was shown that pseudocompact strains convert to compact-type growth after prolonged incubation. Interconversion of compact, diffuse, and pseudocompact growth forms could be induced in vitro by appropriate cultural conditions, and conversion of growth type was also observed in vivo. Femoral abscesses produced in mice by four different compact-type strains showed conversion to diffuse or pseudocompact-type growth during the course of the infection.

Abscess↗

PubMed1970

Lysostaphin-induced, osmotically fragile Staphylococcus aureus cells.

Staphylococcus aureus FDA 209P cells when suspended in 24% (w/v) NaCl were rendered osmotically fragile by exposure to lysostaphin for time intervals ranging from 2 to 60 min. Such cells were analyzed chemically and serologically for evidence of residual cell wall material, were plated in hypertonic sucrose medium to determine revertibility to normal, and were subjected to manometric studies to determine metabolic capabilities. Most of the cells (95%) which were exposed to lysostaphin (0.5 or 1.0 unit/ml) for 2 min, although osmotically fragile, retained their cell wall hexosamine and were capable of reverting to osmotically normal cells when plated in hypertonic medium. Cells exposed to lysostaphin for 5 and 10 min also retained much of their cell wall hexosamine, but lost their ability to revert to normal staphylococci. Cells exposed to lysostaphin for 2 to 10 min continued to react with staphylococcus anti-k antiserum. Complete removal of cell wall hexosamine was attained only after exposure to lysostaphin for 20 min or more; these cells failed to react with k antiserum. Lysostaphin-induced L-type colonies were extremely rare in our experiments, even if incubation times and media were optimal for their detection. Lysostaphin-induced staphylococcal protoplasts were as active metabolically in manometric studies as were untreated staphylococci.

Agar↗

PubMed1971

Electron microscopy during release and purification of mesosomal vesicles and protoplast membranes from Staphylococcus aureus.

The mesosomes of log-phase Staphylococcus aureus ATCC 6538P and Staphylococcus aureus phage-type 80/81, as seen in situ in ultrathin sections, were of the vesicular type. The constituent vesicles ranged from 35 to 50 nm in diameter when the glutaraldehyde-osmium-uranium-lead sequence of fixation and staining was used. During protoplasting in hypertonic buffer containing a muralytic enzyme, vesicles of the same size were extruded and required magnesium ion to maintain structural integrity. The vesicles, purified from the protoplasting supernatant medium by density gradient centrifugation, maintained size and configuration in a homogeneous preparation. Cytoplasmic membranes, produced by osmotic shock and nuclease treatment of protoplasts, were similarly concentrated in gradients. However, they were not free of membrane-associated ribosomes nor of mesosomal vesicles except when prepared in the absence of magnesium.

Acetates↗

PubMed1973

Protein and fatty acid composition of mesosomal vesicles and plasma membranes of Staphylococcus aureus.

Qualitatively, the protein and fatty acid composition of purified mesosomal vesicles and the plasma membrane isolated from Staphylococcus aureus ATCC 6538P are identical, the major difference between these two cellular components being only quantitative in nature. Mesosomal vesicles and plasma membranes, when subjected to acidic or neutral disk gel electrophoresis, exhibited more than 22 bands of protein. With urea-acetic acid gels, the plasma membrane had a higher concentration of "slower-migrating proteins" whereas "faster-migrating proteins" predominated in the mesosomal vesicles. With neutral disk gel electrophoresis, mesosomal vesicles exhibited one prominent protein band with an approximate molecular weight of 35,000 and which was four times greater than that found in the corresponding region on gels of the plasma membrane. Finally, fatty acid analyses by capillary column gas chromatography showed that although the fatty acid composition is the same, the fatty acid content in mesosomal vesicles is 48% greater than that of the plasma membrane. The dominant fatty acids in both of these cellular components are the iso and anteiso branched methyl C15, C17, and C19 fatty acids and comprise at least 85% of the total fatty acids extracted. These results show that distinct chemical differences exist between the mesosomal vesicles and the plasma membrane of Staphylococcus aureus.

Bacterial Proteins↗

PubMed1974

Surface-bound nuclease of Staphylococcus aureus: localization of the enzyme.

The cellular localization of staphylococcus nuclease, previously known as an exoenzyme, was investigated, and the following results were obtained. (i) When Staphylococcus aureus cells were converted to protoplasts by cell wall lytic enzyme L-11 (a bacteriolytic enzyme purified from Flavobacterium sp. which specifically hydrolyzes amide and peptide linkages of murein layers), over 80% of the cell-bound nuclease was released into the surrounding sucrose medium. (ii) The cell-bound nuclease was associated with the cell-wall membrane fraction of mechanically disrupted cells. (iii) The nuclease activity of cell-wall membrane fractions from cells during early and late stages of protoplast formation were compared. Less activity was found in the late stage. These results suggest that nuclease may be located at or near the surface of the cells. The distribution of cell-bound nuclease in the cell-wall membrane fraction varied with the growth conditions of S. aureus. The activity of alkaline phosphatase, another surface enzyme, was also investigated. Less of this enzyme than nuclease was released when the cells were converted to protoplasts.

Alkaline Phosphatase↗

PubMed1974

Role of menaquinone in nitrate respiration in Staphylococcus aureus.

Two menaquinone-deficient and one aromatic-deficient mutants of Staphylococcus aureus were unable to reduce nitrate to nitrite. Reinitiation of menaquinone synthesis in the aromatic-deficient mutant by growing it with shikimic acid restored its nitrate respiratory activity. The results clearly demonstrate a role for menaquinone in nitrate respiration in Staphylococcus aureus.

Anaerobiosis↗

PubMed1985

Nucleotide sequence of ermA, a macrolide-lincosamide-streptogramin B determinant in Staphylococcus aureus.

The complete nucleotide sequence of ermA, the prototype macrolide-lincosamide-streptogramin B resistance gene from Staphylococcus aureus, has been determined. The sequence predicts a 243-amino-acid protein that is homologous to those specified by ermC, ermAM, and ermD, resistance determinants from Staphylococcus aureus, Streptococcus sanguis, and Bacillus licheniformis, respectively. The ermA transcript, identified by Northern analysis and S1 mapping, contains a 5' leader sequence of 211 bases which has the potential to encode two short peptides of 15 and 19 amino acids; the second, longer peptide has 13 amino acids in common with the putative regulatory leader peptide of ermC. The coding sequence for this peptide is deleted in several mutants in which macrolide-lincosamide-streptogramin B resistance is constitutively expressed. Potential secondary structures available to the leader sequence of the wild-type (inducible) transcript and to constitutive deletion, insertion, and point mutations provide additional support for the translational attenuation model for induction of macrolide-lincosamide-streptogramin B resistance.

Amino Acid Sequence↗