The expression of Staphylococcus aureus adhesins in Lactococcus lactis identified clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) as critical for valve colonization in rats with experimental endocarditis. This study further analyzed their role in disease evolution. Infected animals were followed for 3 d. ClfA-positive lactococci successfully colonized damaged valves, but were spontaneously eradicated over 48 h. In contrast, FnBPA-positive lactococci progressively increased bacterial titers in vegetations and spleens. At imaging, ClfA-positive lactococci were restricted to the vegetations, whereas FnBPA-positive lactococci also invaded the adjacent endothelium. This reflected the capacity of FnBPA to trigger cell internalization in vitro. Because FnBPA carries both fibrinogen- and fibronectin-binding domains, we tested the role of these functionalities by deleting the fibrinogen-binding domain of FnBPA and supplementing it with the fibrinogen-binding domain of ClfA in cis or in trans. Deletion of the fibrinogen-binding domain of FnBPA did not alter fibronectin binding and cell internalization in vitro. However, it totally abrogated valve infectivity in vivo. This ability was restored in cis by inserting the fibrinogen-binding domain of ClfA into truncated FnBPA, and in trans by coexpressing full-length ClfA and truncated FnBPA on two separate plasmids. Thus, fibrinogen and fibronectin binding could cooperate for S. aureus valve colonization and endothelial invasion in vivo.
Staphylococcus aureus is a Gram-positive human pathogen that is readily internalized by professional phagocytes such as macrophages and neutrophils but also by non-professional phagocytes such as epithelial or endothelial cells. Intracellular bacteria have been proposed to play a role in evasion of the innate immune system and may also lead to dissemination within migrating phagocytes. Further, S. aureus efficiently lyses host cells with a battery of cytolytic toxins. Recently, phenol-soluble modulins (PSM) have been identified to comprise a genus-specific family of cytolytic peptides. Of these the PSMα peptides have been implicated in killing polymorphonuclear leukocytes after phagocytosis. We questioned if the peptides were active in destroying endosomal membranes to avoid lysosomal killing of the pathogen and monitored integrity of infected host cell endosomes by measuring the acidity of the intracellular bacterial microenvironment via flow cytometry and by a reporter recruitment technique. Isogenic mutants of the methicillin-resistant S. aureus (MRSA) strains USA300 LAC, USA400 MW2 as well as the strongly cytolytic methicillin-sensitive strain 6850 were compared to their respective wild type strains. In all three genetic backgrounds, PSMα mutants were unable to escape from phagosomes in non-professional (293, HeLa, EAhy.926) and professional phagocytes (THP-1), whereas mutants in PSMβ and δ-toxin as well as β-toxin, phosphatidyl inositol-dependent phospholipase C and Panton Valentine leukotoxin escaped with efficiencies of the parental strains. S. aureus replicated intracellularly only in presence of a functional PSMα operon thereby illustrating that bacteria grow in the host cell cytoplasm upon phagosomal escape.
Staphylococcus aureus is generally considered an extracellular pathogen (12, 44). However, it was shown in vitro over several years that S. aureus can invade a variety of nonprofessional phagocytes of different species. Many studies have used cells of nonhuman origin, mainly bovine endothelial cells. S. aureus invasion of human cells has been described so far for endothelial cells (8,38), epithelial cells (7,47,50), osteoblasts (24), and fibroblasts (50).S. aureus is among the most important bacterial pathogens for humans. It commonly causes superficial skin infections. Upon bloodstream dissemination or by continuous spread, it can readily survive in various deep tissues and cause, among others, abscess formation, osteomyelitis, endocarditis, and sepsis. Since these S. aureus infections are frequently long lasting, are difficult to eradicate, and often relapse after antibiotic treatment, prolonged treatment, including the use of cell-permeating antimicrobial drugs, is often required (30, 31). Invasion of nonprofessional phagocytes may provide a protected niche for S. aureus, but the precise role of invasion of host cells in S. aureus pathology in vivo is still not known.We have previously analyzed the process of cellular S. aureus invasion and elucidated the molecular mechanism of the internalization step (50). Bacterial entry proceeds by a "zippertype" mechanism, which resembles professional phagocytosis, and requires bacterial surface proteins, namely, adhesins (also termed microbial surface components recognizing adhesive matrix molecules [MSCRAMMs] [13]). The internalization process if F actin dependent, as demonstrated by complete inhibition by cytochalasin D. By use of isogenic deletion mutants, we identified fibronectin-binding protein (FnBP) as the necessary S. aureus invasin. Using competition experiments, we mapped the region of interaction with the host cell to the major fibronectin-binding domain of the FnBP, the D1 to D4 repeats. Fibronectin acts as a bridging molecule between the FnBP and the fibronectin receptor integrin ␣ 5  1 , an adhesion molecule on the host cell. Cellular invasion is highly conserved in clinical S. aureus isolates (15 out of 15 tested were invasive), and the mechanism elucidated for the epithelial cell model of 293 cells is also valid for ECV304 cells, skin fibroblasts (50), and human umbilical vein endothelial cells (B. Sinha, unpublished data). These data are compatible with the results obtained by four different groups, who found that FnBPs are essential either for adhesion to or invasion of human endothelial cells (42), bovine mammary epithelial cells (9, 27), and mouse fibroblastic cells (14).Our previous data did not support a role for S. aureus invasins other than FnBPs but could not exclude necessary (S. aureus-specific) coreceptors. However, they largely excluded an efficient second invasin in S. aureus (50), unlike the situation found for streptococcal M proteins. This study was performed to address the questions of whether S. aureus FnBPs are sufficient to confer...
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