Fibronectin Bound to the Surface of Staphylococcus aureus Induces Association of Very Late Antigen 5 and Intracellular Signaling Factors with Macrophage Cytoskeleton

Abstract

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, ␣ 5 and ␤ 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, ␤ 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.Microbial pathogens use a number of mechanisms for interacting with their hosts. Adhesins, which are expressed on the surface of bacteria and bind to the surface of host cells, such as epithelial cells, endothelial cells, fibroblasts, and leukocytes, comprise a system that interacts with and colonizes on host tissues in order to invade cells in some cases. It has been demonstrated that bacterial adhesins recognizing integrins are categorized into three groups according their functions: (i) mimicry of a true ligand such as the RGD sequence in fibronectin (FN), (ii) recognition of an ancillary ligand of integrin such as gp63, and (iii) absorption of ligands consisting of extracellular matrix (ECM) (13). Staphylococcus aureus has a number of proteins that bind to extracellular matrix proteins, such as laminin, vitronectin, collagen, FN, elastin, and fibrinogen (9,10,19,20,25,29,32,41). These receptors are thought to play a role in tropism, colonization of host tissues, invasion of host cells, and ingestion by host cells (31). FN-binding protein (FnBP) is a receptor of soluble and assembled FN that is expressed on staphylococci. There are two isoforms, FnBPA and FnBPB, which recognize the N-terminal sequence of FN at region D and also at region Du located in region C (15,16,38). Recently, it has been demonstrated that FnBPA has a third FN-binding site in region B (23). This activity is peculiar to FnBPA, because region B is not found in FnBPB.A previous study showed that S. aureus Cow...

Expression and Distribution of Very Late Antigen‐5 in Mouse Peritoneal Macrophages upon Ingestion of Fibronectin‐Bound Staphylococcus aureus

“…In macrophages ingesting FN-treated S. aureus, VLA-5, paxillin, and other signaling factors such as Src and Csk translocated to the cytoskeleton. Also, anti-VLA-5 immunoprecipitate from macrophages ingesting FNbound bacteria has been shown to contain paxillin (36). These results indicate that VLA-5 is activated during the ingestion of FN-bound bacteria.…”

“…More efficient recognition for ingestion is mediated by binding of opsonins, including complements and antibodies to bacterial surface components. As previously described, FN has an opsonin-like effect, directing inflammatory macrophages to ingest S. aureus (36). Plasma contains 200 to 250 µg/ml of FN dimer, some of which is incorporated into the extracellular matrix.…”

“…Previously, we reported that murine peritoneal inflammatory macrophages display an enhanced ability to ingest FN-bound S. aureus (34), suggesting that FnBP is also utilized for elimination of bacteria by the host defense system. In separate study, we found that this function is mediated by Very Late Antigen-5 (VLA-5) (36). The reason for the development of pneumoniae in a rat model by FnBP inactivated mutant described above is thought to be the impracticality of FN-mediated phagocytosis by alveolar macrophages, because these macrophages are responsible for the first defense system in the lung and constitutively express VLA-5 (19).…”

Mechanisms of TNF induction by heat-killed Staphylococcus aureus differ upon the origin of mononuclear phagocytes

“…S3, online supplement). We also investigated the role of the integrin ␣ 5 ␤ 1 , a transmembrane molecule that interacts with fibrinogen and forms a bridge between the cell and the fibrinogen-binding protein of S. aureus (39). The three cell populations were confirmed to express ␣ 5 ␤ 1 on their surface by flow cytometry (Fig.…”

“…Other receptors on the membrane interact with S. aureus and lead to cellular activation. The scavenger receptor MARCO (macrophage receptor with collagenous structure) (4), the integrin ␣ 5 ␤ 1 (39) and CD36 (40) have been shown to play a role in the response to S. aureus or its uptake. However, the relative role of these receptors has not been clearly established, and the link between S. aureus internalization and the induction of proinflammatory signals still needs to be deciphered.…”