Identification and characterization of a surface-associated protein (Ssp) of Staphylococcus saprophyticus.

A 95-kDa protein was isolated from Staphylococcus saprophyticus 7108 grown on dialysis membranes placed on the surface of brain heart infusion agar. Strain CCM883 did not produce this protein. Ultrathin sections revealed the presence of very thin, tuftlike, 50- to 75-nm-long structures on the surface of strain 7108, whereas strain CCM883 was comparably smooth. The surface material could be removed by digestion with proteinase K, suggesting that the surface structures contain protein. High-resolution scanning electron microscopy showed a thick layer of surface material on strain 7108, whereas strain CCM883 appeared smooth. The 95-kDa protein was purified by Sephacryl S-300 chromatography, and an antiserum was raised in rabbits. This antiserum was used in immunogold labeling experiments, which showed that the protein is associated with the surface structures. Our experiments thus demonstrate the presence of a fibrillar protein on the surface of S. saprophyticus (Ssp for S. saprophyticus surface-associated protein).     

Staphylococcus saprophyticus hemagglutinin is a 160-kilodalton surface polypeptide.

Many strains of Staphylococcus saprophyticus cause direct hemagglutination of sheep erythrocytes. For a high proportion of clinical isolates, a surface protein (Ssp) that is apparently not involved in this property has been described. In this study, S. saprophyticus CCM883, a hemagglutinating but Ssp-negative strain, was used for the identification, purification, and characterization of a 160-kDa surface polypeptide that appears to be the major component of the hemagglutinin. Expression of the protein required the addition to the growth medium of EDTA in micromolar quantities, suggesting an inhibitory role for some unidentified metal ion. The protein was purified by means of Sephacryl S-300 chromatography, and antisera were raised in rabbits. Antibody against this protein inhibited the hemagglutination of two other, unrelated strains and was used to demonstrate, by electron microscopy, the presence of the protein on the surface of the cells. In a confirmatory experiment, the purified antigen was incubated with erythrocytes and binding was detected by the Western immunoblot technique with the antibody to the 160-kDa polypeptide. These experiments indicate that this surface protein is the hemagglutinin of S. saprophyticus.     

SdrI, a serine-aspartate repeat protein identified in Staphylococcus saprophyticus strain 7108, is a collagen-binding protein

A gene encoding a serine-aspartate repeat protein of Staphylococcus saprophyticus, an important cause of urinary tract infections in young women, has been cloned and sequenced. In contrast to other SD repeat proteins, SdrI carries 21 additional N-terminal repeats with a consensus sequence of (P/A)ATKE(K/E)A(A/V)(T/I)(A/T/S)EE and has the longest SD(AD)(1-5) repetitive region (854 amino acids) described so far. This highly repetitive sequence contains only the amino acids serine, asparagine, and a distinctly greater amount of alanine (37%) than all other known SD repeat proteins (2.3 to 4.4%). In addition, it is a collagen-binding protein of S. saprophyticus and the second example in this organism of a surface protein carrying the LPXTG motif. We constructed an isogenic sdrI knockout mutant that showed decreased binding to immobilized collagen compared with wild-type S. saprophyticus strain 7108. Binding could be reconstituted by complementation. Collagen binding is specifically caused by SdrI, and the recently described UafA protein, the only LPXTG-containing protein in the genome sequence of the type strain, is not involved in this trait. Our experiments suggest that, as in other staphylococci, the presence of different LPXTG-anchored cell wall proteins is common in S. saprophyticus and support the notion that the presence of matrix-binding surface proteins is common in staphylococci.     

Staphylococcus saprophyticus hemagglutinin binds fibronectin.

Attachment of microorganisms to host tissue is regarded as an important step in the pathogenesis of infections. Staphylococcus saprophyticus adheres to various epithelial cells and hemagglutinates sheep erythrocytes. The hemagglutinin has been identified, but a human target for this surface protein is still not known. In our report, we show that hemagglutinating strains of S. saprophyticus bind to immobilized fibronectin, whereas nonhemagglutinating strains do not. Bacterial binding was inhibited by antibody to the hemagglutinin but not by antibody to Ssp, another surface protein of S. saprophyticus. The purified hemagglutinin but not other surface proteins bound biotin-labeled fibronectin. Binding was saturable and could be inhibited by unbound hemagglutinin, unlabeled fibronectin, and by antibody to the hemagglutinin. We thus conclude that the hemagglutinin of S. saprophyticus may act as a fibronectin receptor in the human host. Heparin, the D3 peptide, or Arg-Gly-Asp-Ser (RGDS) containing peptides did not inhibit binding of fibronectin to the hemagglutinin, indicating that the binding site is different from that of Staphylococcus aureus or Treponema pallidum.     

Cloning and expression of Staphylococcus saprophyticus urease gene sequences in Staphylococcus carnosus and contribution of the enzyme to virulence.

The urease gene of Staphylococcus saprophyticus CCM883 was cloned and expressed in Staphylococcus carnosus TM300. In vitro translation of the cloned DNA sequences revealed six polypeptides (of 70, 47, 29, 27, 20, and 17 kilodaltons) that were associated with enzyme activity. Introduction of the cloned genes into a urease-negative mutant of S. saprophyticus restored the virulence of this strain, confirming our previous suggestion (S. Gatermann, J. John, and R. Marre, Infect. Immun. 57:110-116, 1989) that this enzyme is a major virulence factor of the organism and contributes mainly to cystopathogenicity.     

Expression of Staphylococcus saprophyticus surface properties is modulated by composition of the atmosphere

Expression of two major surface proteins of Staphylococcus saprophyticus, the haemagglutinin and the Staphylococcus saprophyticus surface-associated protein (Ssp), requires carefully defined culture conditions. The Ssp is produced when bacteria are grown on agar, whereas expression of the haemagglutinin requires growth in broth. We sought to identify the environmental signals that are responsible for this modulation. Varying the pH, the osmolarity of the growth medium or the temperature did not influence expression of the proteins. In contrast, growth in an anaerobic atmosphere increased haemagglutination titres and fibronectin binding (both mediated by the haemagglutinin) but suppressed production of the Ssp. As the influence of the CO₂ level could be excluded, we conclude that expression of these surface proteins is probably modulated by the O₂ content of the atmosphere.     

Construction and characterization of an agr deletion mutant of Staphylococcus epidermidis

The physiological significance of the accessory gene regulator (agr) system of Staphylococcus epidermidis was investigated by construction of an agr deletion mutant via allelic replacement with a spectinomycin resistance cassette. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the protein pattern was strongly altered in the mutant; the amounts of most surface proteins were higher, whereas the amounts of most exoproteins were lower. The agr system of S. epidermidis thus appears to have an important impact on growth phase-dependent protein synthesis as has been shown for Staphylococcus aureus. The activity of the exoenzymes lipase and protease, assumed to be involved in staphylococcal pathogenicity, was investigated by agar diffusion assays and SDS-PAGE activity staining. A general reduction of these enzyme activities in the agr mutant was found. The difference in overall lipase activity was small, but zymographic analysis suggested a clear defect in lipase processing in the agr mutant.     

Expression of pls, a gene closely associated with the mecA gene of methicillin-resistant Staphylococcus aureus, prevents bacterial adhesion in vitro

The pls gene, coding for a large surface protein of methicillin-resistant Staphylococcus aureus, was cloned from a strain which adheres poorly to several mammalian proteins. The structure of pls revealed three distinct repeat regions, one of which was a serine-aspartate repeat characteristic of the Clf-Sdr family of surface proteins in staphylococci. The lengths of the repeat regions varied in different clinical strains and could be used as epidemiological markers. pls was found to be closely associated with the mecA gene by pulsed-field gel electrophoresis analysis of SmaI-digested DNA. A pls mutant constructed by allele replacement adhered well to immobilized fibronectin and immunoglobulin G, in contrast to the parental strain, suggesting that Pls could have a role in preventing adhesion at some stages during an infection.     

The hemagglutinin of Staphylococcus saprophyticus is a major adhesin for uroepithelial cells.

The 160-kDa hemagglutinin of Staphylococcus saprophyticus also serves as a fibronectin-binding protein, and the two activities may be present on different parts of the molecule. Bacteria expressing the 160-kDa hemagglutinin bound in large numbers to histological sections of human ureters, whereas nonhemagglutinating bacteria did not bind. Binding was decreased by an antiserum to the 160-kDa protein and by a preparation of sheep erythrocyte membranes. Fibronectin had no effect. We therefore conclude that binding of S. saprophyticus to uroepithelial cells is mediated by the hemagglutinating activity of the 160-kDa surface protein.     

Ultrastructure of Mutants of Streptococcus mutans with Reference to Agglutination, Adhesion, and Extracellular Polysaccharide

An electron microscopic study of S. mutans 6715-13 wild type and representatives of three distinct classes of glucan synthesis-defective mutants (which fail to form adherent microbial plaques but agglutinate normally in the presence of exogenous glucans) disclosed the presence of two sucrose-dependent, glucanase-sensitive, extracellular components. In the wild type, these extracellular glucans had predominantly fibrillar (with some globular) morphologies. However, in the mutant strains, there was a consistent reduction in or loss of the fibrillar components and dramatic increases in globular forms. A cell surface-associated fuzzy coat was consistently seen, and it was neither sucrose-dependent nor glucanase-sensitive. The data indicated that in vitro and in vivo adhesion and virulence at smooth tooth surfaces (all these properties dramatically reduced in the mutants) were causally and functionally related to the extracellular, fibrillar, glucan component, whereas in vitro glucan-mediated agglutination may be related to the cell-associated surface fuzzy coat.     

Hyperproduction of alpha-toxin by Staphylococcus aureus results in paradoxically reduced virulence in experimental endocarditis: a host defense role for platelet microbicidal proteins.

Staphylococcal alpha-toxin targets several cell types which are important components of cardiac vegetations in endocarditis, including platelets, erythrocytes, and endothelial cells. We evaluated the in vivo role of Staphylococcus aureus alpha-toxin in experimental endocarditis by using isogenic strains differing in the capacity to produce functional alpha-toxin, including 8325-4 (wild-type strain), DU-1090 (a mutant strain with allelic replacement of the alpha-toxin gene [hla]), DU1090(pH35L) (a mutant strain producing a target cell-binding but nonlytic toxin), DU1090(pDU1212) (a variant of DU1090 carrying the cloned hla gene on a multicopy plasmid), and DU1090(pCL84::hla) (a variant of DU1090 with a single copy of the hla gene cloned into the chromosomal lipase locus). In vitro, wild-type alpha-toxin (from parental strain 8325-4) extensively lysed both erythrocytes and platelets. In contrast, mutant alpha-toxin [from strain DU1090(pH35L)] lysed neither cell type. Following exposure to the wild-type alpha-toxin, platelet lysates were found to contain microbicidal activity against Bacillus subtilis (but not against Micrococcus luteus), as well as against the parental and alpha-toxin variant S. aureus strains noted above. Furthermore, lysate microbicidal activity was heat stable, neutralized by polyanionic filters or compounds, and recoverable from anionic filter membranes by hypertonic saline elution. These characteristics are consistent with those of cationic platelet microbicidal proteins (PMPs). Reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis confirmed the presence of three distinct PMPs (1, 2, and 3) in platelet lysates. In experimental endocarditis, the two variant staphylococcal strains producing either minimal alpha-toxin or nonlytic alpha-toxin in vitro [strains DU1090 and DU1090(pH35L), respectively] exhibited significantly lower virulence in vivo than the parental strain (decreased intravegetation staphylococcal densities). Paradoxically, the two variant staphylococcal strains producing alpha-toxin at supraparental levels in vitro [strains DU1090(p1212) and DU1090(pCL84::hla)] also exhibited significantly decreased induction rates and intravegetation staphylococcal densities in experimental endocarditis versus the parental strain. The reduced in vivo virulence of the latter variant staphylococcal strains could not be explained by differences in bacteremic clearance or initial adherence to sterile vegetations (compared to the parental strain). These findings suggest that the reduced virulence exhibited by the variant staphylococcal strains in this model was related to pathogenetic events subsequent to bacterial adherence to the damaged endocardium. Excess intravegetation secretion of alpha-toxin, leading to increased PMP release (secondary to either increased platelet secretion or lysis), may well explain the reduced virulence observed in experimental endocarditis.     

M Protein-Associated Adherence of Streptococcus pyogenes to Epithelial Surfaces: Prerequisite for Virulence

Virulent strains of Streptococcus pyogenes containing M protein were found to adhere well to human cheek epithelial cells in vitro, whereas an avirulent M - mutant strain adhered feebly. Pretreatment of M + strains with trypsin to remove their M protein surface coating or reacting them with type-specific antiserum markedly impaired their abilities to attach to epithelial cells. Electron microscopy revealed that the attachment of an M + strain to germfree rat epithelial cells was mediated by a fuzzy surface structure previously shown to contain M protein. When mixtures of streptomycin-resistant M + and M - strains were introduced into the mouths of mice, the proportions of the M + strain increased on tongue and cheek surfaces relative to its M - mutant. These data indicate that the surface fuzz of S. pyogenes which contains M protein functions in the attachment of the organism to epithelial surfaces, thereby permitting its colonization.     

Granulocyte activation by a cell surface complex of Staphylococcus saprophyticus: a receptor-mediated phenomenon

High molecular weight cell surface complex (CSC) from Staphylococcus saprophyticus strain S 1 could be shown to be a potent stimulator of human polymorphonuclear leukocyte (PMN) chemiluminescence whereas human monocytes were not activated. Heating of the CSC (100 degrees C for 5 min) as well as protease treatment significantly (p less than 0.001) inhibited the PMN activating process suggesting that the protein part of the molecule mediates its biological activity. Data on the biochemical character of the CSC are given. Preincubation of PMNs with CSC inhibited another chemiluminescence response to this substance and to homologous opsonized S. saprophyticus, respectively. However, restimulation with formylmethionyl peptides (fMLP) or non-opsonized staphylococci suggested that the PMN function is a receptor-mediated phenomenon. These data were substantiated since fMLP activated PMNs could be evidently re-stimulated with CSC but not with analogue peptides. Evaluation of the bactericidal capacity of human PMNs yielded comparable results.     

Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction.

A simple and reliable method using a polymerase chain reaction (PCR) was devised to identify methicillin-resistant staphylococci. By using lysates of the strain to be tested as templates and 22-mer oligonucleotides as primers, a 533-bp region of mecA, the structural gene of a low-affinity penicillin-binding protein (PBP 2'), was amplified by PCR and detected by agarose gel electrophoresis. Results obtained by this method were compared with those obtained by broth microdilution MIC determination for 210 and 100 clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, respectively. Of 99 mecA-negative S. aureus isolates, 100% of the strains were methicillin susceptible and 98% of the strains were oxacillin susceptible. Three strains (3%) of 111 mecA-positive S. aureus isolates exhibited almost the same susceptibility to beta-lactams as the mecA-negative ones and did not produce detectable amounts of PBP 2' despite the presence of the mecA gene. One of them yielded typically methicillin-resistant variants at a low frequency with concomitant recovery of PBP 2' production. The mecA gene was also found in coagulase-negative Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, and Staphylococcus caprae and conferred resistance on most of the bacteria.     

The production of fatty acid modifying enzyme (FAME) and lipase by various staphylococcal species.

Eighty-six strains encompassing 11 species of coagulase-negative staphylococci were examined for the production of fatty acid modifying enzyme (FAME) and lipase. Staphylococcus schleiferi and S. saprophyticus most closely resembled S. aureus in that 80% of the strains produced both enzymes. In contrast, no strains of S. lugdunensis and S. haemolyticus tested produced these enzymes. S. simulans was unusual in that eight of 10 strains produced FAME, but only one produced lipase. Among the other species the proportion of strains producing both enzymes ranged from 10 to 60%. Generally there was a strong correlation between FAME and lipase production.     

The hemagglutinin of Staphylococcus saprophyticus binds to a protein receptor on sheep erythrocytes

Staphylococcus saprophyticus, an important cause of urinary tract infections, produces two major surface proteins, the S. saprophyticus surface-associated protein (Ssp) and the hemagglutinin, which mediates fibronectin binding and also functions as the major adhesin of the organism. The hemagglutinating and fibronectin binding functions probably reside on different parts of the molecule. To identify a receptor on eukaryotic cells, binding and inhibition studies with acidic and neutral glycosphingolipids, carbohydrates, and proteins of sheep erythrocyte membranes were conducted. S. saprophyticus did not bind to any glycosphingolipid and no inhibition was observed when hemagglutination assays were done in the presence of carbohydrates or fibronectin. Neither treatment of erythrocytes with galactose oxidase or neuraminidase and galactose oxidase nor mild periodate oxidation of erythrocytes reduced hemagglutination. However, proteinase-treated erythrocytes were no longer agglutinated. Similarly, untreated erythrocyte membranes inhibited hemagglutination, whereas proteinase-treated membranes did not. In addition, only hemagglutinating strains bound to 60- and 21-kDa sheep erythrocyte membrane proteins on ligand blots, and these proteins inhibited hemagglutination. Our data indicate that, in contrast to many other hemagglutinins, the receptor on sheep erythrocytes for S. saprophyticus is a protein. Received: 27 March 1997     

Surface-Expressed Mig Protein Protects Streptococcus dysgalactiae against Phagocytosis by Bovine Neutrophils

The mig gene of Streptococcus dysgalactiae, a major bovine mastitis pathogen, encodes two plasma protein-binding receptors, alpha2-macroglobulin (alpha2-M) and immunoglobulin G (IgG). In this study, the mig gene from one S. dysgalactiae isolate was cloned and expressed in Escherichia coli. The IgG receptor region encoded by mig was conserved in 16 S. dysgalactiae strains. An isogenic mig mutant was constructed by allele replacement mutagenesis of the wild-type gene in S. dysgalactiae. The IgG-binding activity was lost in the mig mutant strain, whereas the alpha2-M receptor activity was still expressed but was detected only in the culture supernatant. In flow cytometry phagocytosis and bacterial-colony-counting bactericidal assays, the wild-type strain was found to be significantly more resistant to phagocytosis and killing by bovine neutrophils (PMNs) than the mig mutant strain when bacteria were preincubated with bovine serum. We therefore speculate that the Mig protein of S. dysgalactiae plays a role in virulence of the bacteria by binding to the plasma protein alpha2-M or IgG and thus preventing phagocytosis by bovine PMNs.     

Adhesiveness and invasiveness of staphylococcal species in a cell culture model

A model was established for the study of adhesiveness and invasiveness of staphylococcal species. Five collection strains from each of the species Staphylococcus aureus, S. epidermidis, and S. saprophyticus and 26 fresh isolates from patients with urinary tract infections were tested for adhesiveness and invasiveness in HEp-2 cell cultures. All the strains of S. saprophyticus were able to invade the cells and localize intracellularly in the cultures, whereas the invasive potential among the strains of S. aureus and S. epidermidis was lower. The number of adhesive bacteria was also highest among the S. saprophyticus strains, whereas S. epidermidis was the least adhesive. The model may be suitable for further study of urinary tract infection strains.     

Staphylococcus saprophyticus urease: characterization and contribution to uropathogenicity in unobstructed urinary tract infection of rats.

We studied the biochemical properties of the urease of Staphylococcus saprophyticus and the possible role of the urease in experimental urinary tract infections. For this purpose, the nonhemagglutinating and nonadherent strain 9325, which was isolated from a case of symptomatic urinary tract infection, was used. The urease was shown to have a Km of 6.64 mM urea and a Vmax of 4.59 mumol NH3.min-1.mg-1. The enzyme was inhibited by acetohydroxamic acid in a noncompetitive manner. By means of Sephacryl S-300 column chromatography, we determined a mean molecular weight (+/- standard error of the mean) of 420,000 +/- 16,000. To assess the contribution of S. saprophyticus urease to uropathogenicity, a urease-negative mutant was constructed by nitrosoguanidine mutagenesis. In the rat model of ascending unobstructed urinary tract infection, higher numbers of CFU.gram of tissue-1 and more-severe lesions were detected with the parent strain. Moreover, bladder stones were found in animals infected with the urease-positive strain only. Interestingly, the difference in mean bacterial counts of the bladders was found to be significant by the Wilcoxon two-sample test (P less than 0.05), whereas that between the kidney bacterial counts was not. Immunoblot studies revealed a faint antibody response in rats infected with the mutant strain, although bacteria could still be detected in the kidneys after 7 days. Sera of animals challenged with the parent strain reacted strongly with many antigens of S. saprophyticus. Our data indicate that urease is a major factor for invasiveness of S. saprophyticus, especially in the tissue of the bladder, whereas persistence in the urinary tract and nephropathogenicity of this organism are governed by factors other than urease.