Insertional Inactivation of eap in Staphylococcus aureus Strain Newman Confers Reduced Staphylococcal Binding to Fibroblasts

Abstract

In contrast, the mutant adhered to a significantly lesser extent to cultured fibroblasts (P < 0.001) than did the wild type, while adherence was restorable upon complementation. Furthermore, adherence to both epithelial cells (P < 0.05) and fibroblasts (not significant) could be blocked with antibodies against Eap, whereas preimmune serum was not active. In conclusion, Eap may contribute to pathogenicity by promoting adhesion of whole staphylococcal cells to complex eukaryotic substrates.Staphylococcus aureus continues to be a major cause of human disease, accounting for superficial skin infections as well as for serious invasive infections, such as endocarditis, osteomyelitis, and septic shock (22). Adherence of S. aureus to components of host tissues is an important first step in colonization and subsequent infection (10, 35) and is mediated by specific interactions between adhesins on the bacterial cell surface and host cell receptors (6). In addition to the wellcharacterized bacterial surface-located proteins (28) conferring adhesion to various extracellular matrix proteins (6) and invasion of eukaryotic cells (1, 31, 32), other adhesive S. aureus proteins are secreted. Three of these molecules, i.e., coagulase (29), the extracellular fibrinogen (Fg)-binding protein Efb (26), and the extracellular adherence protein Eap (24), have been shown to bind Fg. Eap of S. aureus Newman has been cloned and sequenced (14) and has previously been shown to bind to additional plasma proteins, including fibronectin (Fn) and prothrombin (Pt) (24). Eap can form oligomers, and by rebinding to the staphylococcal cell surface, it mediates bacterial agglutination. It also enhances binding to epithelial cells and fibroblasts by its dual affinity for eukaryotic components and the S. aureus surface (24). While these observations have been made with purified Eap, further precision in describing the role of Eap, as well as of the related molecule Map (for major histocompatibility complex class II analogous protein [19]), in intact S. aureus cells has been hampered by the lack of availability of defined Eap-negative mutants. Thus, we have constructed an eap-deficient mutant by allelic replacement, and here we report the genotypic and phenotypic characteristics of the mutant compared to the parent strain. MATERIALS AND METHODSBacterial strains and media. S. aureus Newman (kindly provided by T. Foster, Dublin, Ireland) was used to generate the ⌬eap mutant. Recombinant plasmids cloned in Escherichia coli were passaged in a restriction-negative S. aureus strain, SA113 (16), before electroporation to S. aureus Newman. Staphylococcus carnosus TM300 (11) was used as an intermediate host in the construction of a complemented strain. The following strains of E. coli were used as cloning hosts: E. coli DH5␣, E. coli TG1, and E. coli SCS 110 (Stratagene, La Jolla, Calif.).For cultivation of S. aureus, tryptic soy broth and agar (Difco, Detroit, Mich.), brain heart broth and agar (Merck, Darmstadt, Germany), Mueller-Hinton broth and agar (Mast, Me...

eapGene as Novel Target for Specific Identification ofStaphylococcus aureus

“…For S. aureus, secreted proteins such as Eap have pleiotropic effects, mediating adherence to host extracellular matrix components (e.g., fibronectin, fibrinogen, vitronectin, collagens, and elastin) and thereby potentially contributing to bacterial colonization of host tissues (10) and, in part, to invasion of nonprofessional phagocytes (19)(20)(21). In addition, Eap has antiinflammatory and immunomodulatory functions in the host, indicating that Eap constitutes a potent virulence factor in the course of staphylococcal infections (1,8,15,18,25).…”

“…The anchorless extracellular adherence protein (Eap) of S. aureus, also designated major histocompatibility complex class II analogous protein (Map), selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules (16,21,23). This broad-spectrum adhesin shows structural homology to the C-terminal domain of bacterial superantigens but lacks superantigen activity (11,28).…”

Staphylococcus aureus colonization and non-influenza respiratory viruses: Interactions and synergism mechanisms

“…EAP belongs to Secreted Expanded Repertoire Adhesive Molecules (SERAMs) and is involved in bacterial aggregation [65], adhesion and invasion of epithelial and fibroblastic cells [66–68], and in preventing neutrophil recruitment [64,69–72]. In addition to ICAM-1, EAP can also bind a large variety of extracellular matrix and plasma proteins including collagen, fibrinogen, fibronectin, vitronectin, laminin, thrombospondin and prothrombin [64,65,73–75].…”

The Staphylococcus aureus Map protein is an immunomodulator that interferes with T cell–mediated responses

“…The resulting plasmid was called pMap9. Plasmid pMap9 was introduced into SA strain RN4220 by electroporation and then plated on TSA containing 10 µg/ml tetracycline and incubated at 30°C (34,35). The temperature sensitivity of plasmid pMap9 in RN4220 was confirmed.…”