Involvement of alpha5beta1-integrin and TNF-alpha in Staphylococcus aureus alpha-toxin-induced death of epithelial cells

Staphylococcus aureus causes suppurative infections which are often associated with tissue destruction and cell death. In the present study, we investigated the molecular and cellular basis of S. aureus-induced apoptosis and death in a human lung epithelial cell line (A549). We found that staphylococcal alpha-toxin is an important mediator of cytotoxicity in these epithelial cells. Specifically, we found that downregulating alpha-toxin production eliminated the cytotoxicity of S. aureus, whereas the addition of alpha-toxin to the cell culture medium significantly increased cell death in a dose-dependent manner. Importantly, we found that alpha-toxin-mediated cell death may partially function through alpha5beta1-integrin, because both the beta1-integrin antibody and the ligand fibronectin inhibited the cytotoxicity of alpha-toxin. Furthermore, we found that the overexpression of the inflammatory cytokine interferon (TNF)-alpha is associated with alpha-toxin-induced cell death, because both the TNF-alpha release inhibitor and antibody effectively inhibited the cytotoxicity of alpha-toxin. In contrast, the cytotoxicity of alpha-toxin was enhanced by the inhibition of the MAPK p38 and NF-kappaB pathways. Taken together, our results suggest that the activation of the MAPK p38 and NF-kappaB pathways are stress responses for survival, rather than direct contributes to alpha-toxin-induced cell death, and that the interaction of alpha-toxin with alpha5beta1-integrin and overproduction of TNF-alpha may contribute to destruction of epithelial cells during S. aureus infection.     

Integrin-mediated invasion of Staphylococcus aureus into human cells requires Src family protein-tyrosine kinases

Staphylococcus aureus, a common cause of nosocomial infections, is able to invade eukaryotic cells by indirectly engaging beta1 integrin-containing host receptors, whereas non-pathogenic Staphylococcus carnosus is not invasive. Here, we identify intracellular signals involved in integrin-initiated internalization of S. aureus. In particular, the host cell actin cytoskeleton and Src family protein-tyrosine kinases (PTKs) are essential to mediate S. aureus invasion. Src PTKs are activated in response to pathogenic S. aureus, but not S. carnosus. In addition, pharmacological and genetic interference with Src PTK function reduces bacterial internalization. Importantly, Src PTK-deficient cells are resistant to S. aureus invasion, demonstrating the essentiality of host Src PTKs in integrin-mediated uptake of this pathogen.     

Staphylococcus aureus alpha-toxin induces apoptosis in endothelial cells

The internalization of Staphylococcus aureus by cultured human umbilical vein endothelial cells was recently shown to induce apoptosis. We examined the role of alpha-toxin, a major pore-forming toxin secreted by S. aureus, in causing apoptosis in vitro. Purified alpha-toxin, at sublytic concentrations, induced apoptosis in endothelial cell monolayers. Comparisons of two alpha-toxin (hla)-positive S. aureus strains and their isogenic hla-deficient mutants in the invasion assay of endothelial cells demonstrated that the capacity to produce alpha-toxin was associated with a greater propensity for apoptosis in endothelial cells. These results demonstrate for the first time that expression of alpha-toxin during endothelial cell invasion by S. aureus enhances apoptosis.     

Staphylococcus aureus extracellular adherence protein serves as anti-inflammatory factor by inhibiting the recruitment of host leukocytes

Staphylococcus aureus is a human pathogen that secretes proteins that contribute to bacterial colonization. Here we describe the extracellular adherence protein (Eap) as a novel anti-inflammatory factor that inhibits host leukocyte recruitment. Due to its direct interactions with the host adhesive proteins intercellular adhesion molecule 1 (ICAM-1), fibrinogen or vitronectin, Eap disrupted beta(2)-integrin and urokinase receptor mediated leukocyte adhesion in vitro. Whereas Eap-expressing S. aureus induced a 2 3-fold lower neutrophil recruitment in bacterial peritonitis in mice as compared with an Eap-negative strain, isolated Eap prevented beta(2)-integrin-dependent neutrophil recruitment in a mouse model of acute thioglycollate-induced peritonitis. Thus, the specific interactions with ICAM-1 and extracellular matrix proteins render Eap a potent anti-inflammatory factor, which may serve as a new therapeutic substance to block leukocyte extravasation in patients with hyperinflammatory pathologies.     

Staphylococcus aureus fibronectin binding protein-A induces motile attachment sites and complex actin remodeling in living endothelial cells

Staphylococcus aureus fibronectin binding protein-A (FnBPA) stimulates alpha5beta1-integrin signaling and actin rearrangements in host cells. This eventually leads to invasion of the staphylococci and their targeting to lysosomes. Using live cell imaging, we found that FnBPA-expressing staphylococci induce formation of fibrillar adhesion-like attachment sites and translocate together with them on the surface of human endothelial cells (velocity approximately 50 microm/h). The translocating bacteria recruited cellular actin and Rab5 in a cyclic and alternating manner, suggesting unsuccessful attempts of phagocytosis by the endothelial cells. Translocation, actin recruitment, and eventual invasion of the staphylococci was regulated by the fibrillar adhesion protein tensin. The staphylococci also regularly produced Neural Wiskott-Aldrich syndrome protein-controlled actin comet tails that further propelled them on the cell surface (velocity up to 1000 microm/h). Thus, S. aureus FnBPA produces attachment sites that promote bacterial movements but subvert actin- and Rab5 reorganization during invasion. This may constitute a novel strategy of S. aureus to postpone invasion until its toxins become effective.     

Dynamic interaction between airway epithelial cells and Staphylococcus aureus

Staphylococcus aureus is a major cause of pulmonary infection, particularly in cystic fibrosis (CF) patients. However, few aspects of the interplay between S. aureus and host airway epithelial cells have been investigated thus far. We investigated by videomicroscopy the time- and bacterial concentration-dependent (10(4), 10(6), and 10(8) CFU/ml) effect of S. aureus on adherence, internalization, and the associated damage of the airway epithelial cells. The balance between the secretion by S. aureus of the alpha-toxin virulence factor and by the airway cells of the antibacterial secretory leukoproteinase inhibitor (SLPI) was also analyzed. After 1 h of interaction, whatever the initial bacterial concentration, a low percentage of S. aureus (<8%) adhered to airway cells, and no airway epithelial cell damage was observed. In contrast, after 24 h of incubation, more bacteria adhered to airway epithelial cells, internalized bacteria were observed, and a bacterial concentration-dependent effect on airway cell damage was observed. At 24 h, most airway cells incubated with bacteria at 10(8) CFU/ml exhibited a necrotic phenotype. The necrosis was preceded by a transient apoptotic process. In parallel, we observed a time- and bacterial concentration-dependent decrease in SLPI and increase in alpha-toxin expression. These results suggest that airway cells can defend against S. aureus in the early stages of infection. However, in later phases, there is a marked imbalance between the bactericidal capacity of host cells and bacterial virulence. These findings reinforce the potential importance of S. aureus in the pathogenicity of airway infections, including those observed early in CF patients.     

Lipid rafts mediate internalization of beta1-integrin in migrating intestinal epithelial cells

Intestinal mucosal inflammation is associated with epithelial wounds that rapidly reseal by migration of intestinal epithelial cells (IECs). Cell migration involves cycles of cell-matrix adhesion/deadhesion that is mediated by dynamic turnover (assembly and disassembly) of integrin-based focal adhesions. Integrin endocytosis appears to be critical for deadhesion of motile cells. However, mechanisms of integrin internalization during remodeling of focal adhesions of migrating IECs are not understood. This study was designed to define the endocytic pathway that mediates internalization of beta(1)-integrin in migrating model IECs. We observed that, in SK-CO15 and T84 colonic epithelial cells, beta(1)-integrin is internalized in a dynamin-dependent manner. Pharmacological inhibition of clathrin-mediated endocytosis or macropinocytosis and small-interfering RNA (siRNA)-mediated knock down of clathrin did not prevent beta(1)-integrin internalization. However, beta(1)-integrin internalization was inhibited following cholesterol extraction and after overexpression of lipid raft protein, caveolin-1. Furthermore, internalized beta(1)-integrin colocalized with the lipid rafts marker cholera toxin, and siRNA-mediated knockdown of caveolin-1 and flotillin-1/2 increased beta(1)-integrin endocytosis. Our data suggest that, in migrating IEC, beta(1)-integrin is internalized via a dynamin-dependent lipid raft-mediated pathway. Such endocytosis is likely to be important for disassembly of integrin-based cell-matrix adhesions and therefore in regulating IEC migration and wound closure.     

Bacterial internalization mediated by beta 1 chain integrins is determined by ligand affinity and receptor density.

Binding of bacteria to beta 1 chain integrin receptors results in either bacterial adherence or uptake by cultured cells (Isberg, 1991). In this report we show that Staphylococcus aureus coated with high affinity ligands for the beta 1 chain integrin family can be internalized efficiently, whereas bacteria coated with low affinity ligands are poorly internalized. Overproduction of the alpha 5 beta 1 integrin increased the efficiency of bacterial internalization, indicating that the uptake efficiency is directly related to the level of expression of the receptor. By using latex beads or S. aureus coated with mAbs directed against the alpha 5 beta 1 integrin, a roughly semi-logarithmic correlation was observed between the affinity of the receptor-ligand interaction and the rate of bacterial internalization. Evidence is presented that high affinity binding of the bacterium allows the microorganism to compete efficiently with receptor-ligand interactions at the basolateral surface of the cell.     

Adherence and internalization of Streptococcus gordonii by epithelial cells involves beta1 integrin recognition by SspA and SspB (antigen I/II family) polypeptides

Streptococcus gordonii is a commensal bacterium that colonizes the hard and soft tissues present in the human mouth and nasopharynx. The cell wall-anchored polypeptides SspA and SspB expressed by S. gordonii mediate a wide range of interactions with host proteins and other bacteria. In this article we have determined the role of SspA and SspB proteins, which are members of the streptococcal antigen I/II (AgI/II) adhesin family, in S. gordonii adherence and internalization by epithelial cells. Wild-type S. gordonii DL1 expressing AgI/II polypeptides attached to and was internalized by HEp-2 cells, whereas an isogenic AgI/II- mutant was reduced in adherence and was not internalized. Association of S. gordonii DL1 with HEp-2 cells triggered protein tyrosine phosphorylation but no significant actin rearrangement. By contrast, Streptococcus pyogenes A40 showed 50-fold higher levels of internalization and this was associated with actin polymerization and interleukin-8 upregulation. Adherence and internalization of S. gordonii by HEp-2 cells involved beta1 integrin recognition but was not fibronectin-dependent. Recombinant SspA and SspB polypeptides bound to purified human alpha5beta1 integrin through sequences present within the NAV (N-terminal) region of AgI/II polypeptide. AgI/II polypeptides blocked interactions of S. gordonii and S. pyogenes with HEp-2 cells, and S. gordonii DL1 cells expressing AgI/II proteins inhibited adherence and internalization of S. pyogenes by HEp-2 cells. Conversely, S. gordonii AgI/II- mutant cells did not inhibit internalization of S. pyogenes. The results suggest that AgI/II proteins not only promote integrin-mediated internalization of oral commensal streptococci by host cells, but also potentially influence susceptibility of host tissues to more pathogenic bacteria.     

Src kinase has a central role in in vitro cellular internalization of Staphylococcus aureus

Traditionally recognized as an extracellular pathogen, the Gram-positive bacterium Staphylococcus aureus can also be internalized by a variety of cell types in vitro. Internalization is known to involve binding of the host extracellular protein fibronectin to the bacterium, recognition of the fibronectin-coated bacterium by the fibronectin-binding integrin alpha5beta1 on the host cell surface, and integrin-mediated internalization. Here we examine elements of mammalian cell signalling pathways involved in S. aureus internalization. The mouse fibroblast cell line GD25, in which the gene encoding the beta1 integrin subunit is inactivated, has been complemented with a beta1 integrin cDNA encoding a tyrosine (Y) to phenylalanine (F) mutation in each of the two beta1 integrin intracellular NPXY motifs. This cell line, GD25beta1 A Y783/795F, is defective in migration on fibronectin coated surfaces and intracellular signalling activities involving the tyrosine kinase Src. GD25beta1 A Y783/795F cells have a decreased ability to internalize S. aureus compared to GD25beta1 A cells expressing wild-type beta1 integrins. Furthermore, using mouse embryo fibroblasts in which different members of the Src family kinases are genetically inactivated, we demonstrate that optimal internalization is dependent on expression of Src kinase. Interferon, which has been implicated in repression of the effects of the viral homologue of Src inhibits internalization of S. aureus indicating that internalization may be blocked by inhibitors of Src kinase function. We then demonstrate that Src family kinase specific inhibitors effectively block S. aureus internalization into HeLa cells leading to the conclusion that a function unique to Src is required for optimal internalization of S. aureus in vitro.     

Quantification of bacterial invasion into adherent cells by flow cytometry

Quantification of invasive, intracellular bacteria is critical in many areas of cellular microbiology and immunology. We describe a novel and fast approach to determine invasion of bacterial pathogens in adherent cell types such as epithelial cells or fibroblasts based on flow cytometry. Using the CEACAM-mediated uptake of Opa-expressing Neisseria gonorrhoeae as a well-characterized model of bacterial invasion, we demonstrate that the flow cytometry-based method yields results comparable to a standard antibiotic protection assay. Furthermore, the quantification of intracellular bacteria by the novel approach is not biased by intracellular killing of the microbes and correctly discriminates between cell-associated extracellular and bona fide intracellular bacteria. As flow cytometry-based quantification is also applicable to other pathogen-host interactions such as the integrin-mediated internalization of Staphylococcus aureus, this approach provides a fast and convenient alternative for the quantification of bacterial uptake and should be particularly useful in elucidating the molecular mechanisms of pathogen-triggered host cell invasion.     

Type 1 pilus-mediated bacterial invasion of bladder epithelial cells

Most strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili. We have determined that the type 1 pilus adhesin, FimH, mediates not only bacterial adherence, but also invasion of human bladder epithelial cells. In contrast, adherence mediated by another pilus adhesin, PapG, did not initiate bacterial internalization. FimH-mediated invasion required localized host actin reorganization, phosphoinositide 3-kinase (PI 3-kinase) activation and host protein tyrosine phosphorylation, but not activation of Src-family tyrosine kinases. Phosphorylation of focal adhesin kinase (FAK) at Tyr397 and the formation of complexes between FAK and PI 3-kinase and between alpha-actinin and vinculin were found to correlate with type 1 pilus-mediated bacterial invasion. Inhibitors that prevented bacterial invasion also blocked the formation of these complexes. Our results demonstrate that UPEC strains are not strictly extracellular pathogens and that the type 1 pilus adhesin FimH can directly trigger host cell signaling cascades that lead to bacterial internalization.     

Involvement of integrins in fimbriae-mediated binding and invasion by Porphyromonas gingivalis

Interaction between the major fimbriae of Porphyromonas gingivalis and gingival epithelial cells is important for bacterial adhesion and invasion. In this study, we identified integrins as an epithelial cell cognate receptor for P. gingivalis fimbriae. Immunoprecipitation and direct binding assays revealed a physical association between recombinant fimbrillin and beta1 integrins. In vitro adhesion and invasion assays demonstrated inhibition of binding and invasion of P. gingivalis by beta1 integrin antibodies. In contrast, invasion of a fimbriae-deficient mutant of P. gingivalis was not affected by integrin antibodies. Infection of gingival epithelial cells with wild-type P. gingivalis induced tyrosine phosphorylation of the 68 kDa focal adhesion protein paxillin, whereas the fimbriae-deficient mutant failed to evoke similar changes. Interestingly, activation of paxillin was not accompanied by an increase in the phosphorylation of focal adhesion kinase (FAK). These results provide evidence that P. gingivalis fimbriae promote adhesion to gingival epithelial cells through interaction with beta1 integrins, and this association represents a key step in the induction of the invasive process and subsequent cell responses to P. gingivalis infection.     

The fibronectin binding proteins of Staphylococcus aureus are required for adhesion to and invasion of bovine mammary gland cells

We recently described adhesion to and invasion of bovine mammary gland cells by Staphylococcus aureus in vitro. Here, we show that the levels of adhesion and invasion are dependent on the bacterial growth phase and are controlled by the agr locus. Incubation of exponential growth phase cells of S. aureus with mammary gland cells resulted in bacterial cell clumping. Strains of S. aureus deficient in expression of the fibronectin binding proteins (FnBPA and FnBPB) failed to clump and their ability to adhere to and to invade the bovine mammary gland cells is strongly reduced. This indicates that the fibronectin binding proteins are essential for S. aureus adhesion to and invasion of bovine mammary gland cells.     

Inhibition of β1 integrin induces its association with MT1-MMP and decreases MT1-MMP internalization and cellular invasiveness

Integrin signaling plays a fundamental role in the establishment of focal adhesions and the subsequent formation of invadopodia in malignant cancer cells. Invadopodia facilitate localized adhesion and degradation of the extracellular matrix (ECM), which promote tumour cell invasion and metastasis. Degradation of ECM components is often driven by membrane type-1 matrix metalloproteinase (MT1-MMP), and we have recently shown that regulation of enzyme internalization is dependent on signaling downstream of β1 integrin. Phosphorylation of the cytoplasmic tail of MT1-MMP is required for its internalization and delivery to Rab5-marked early endosomes, where it is then able to be recycled to new sites of invadopodia formation and promote invasion. Here we found that inhibition of β1 integrin, using the antibody AIIB2, inhibited the internalization and recycling of MT1-MMP that is necessary to support long-term cellular invasion. MT1-MMP and β1 integrin were sequestered at the cell surface when β1-integrin was inhibited, and their association under these conditions was detected using immunoprecipitation and mass spectrometry analyses. Sequestration of β1 integrin and MT1-MMP at the cell surface resulted in the formation of large invadopodia and local ECM degradation; however, the impaired internalization and recycling of MT1-MMP and β1 integrin ultimately led to a loss of invasive behaviour.     

Role of alphavbeta5 integrins and vitronectin in Pseudomonas aeruginosa PAK interaction with A549 respiratory cells

Bacterial adherence to mammalian cells and their internalization are thought to participate in Pseudomonas aeruginosa pathogenicity. In this study, we explored the role of alpha5beta1 and alphavbeta5 integrins and their natural ligands, fibronectin (Fn) and vitronectin (Vn), in P. aeruginosa interaction with epithelial cells by using the PAK reference bacterial strain, A549 respiratory, and SKOV-3 human ovarian cell lines. The host cell cytoskeleton and cellular tyrosine kinases seem to be solicited during the PAK-respiratory cell interaction: cytochalasin D and genistein decreased the bacterial adherence and internalization. Blocking antibodies to alphavbeta5 integrins were the only antibodies tested to have inhibitory activity against PAK adherence to A549 cells. PAK internalization by A549 and SKOV-3 cells was markedly decreased in the presence of blocking antibodies to Vn and alphavbeta5 integrins. Addition of Vn in excess restored PAK invasion of both A549 and SKOV-3 cells in the presence of anti-Vn antibodies. Immunofluorescence experiments revealed that, in the presence of bacteria, the Vn fibrillar network disappeared, and alphavbeta5 staining was concentrated in sites where adherent bacteria were present. Taken together, these findings suggest that alphavbeta5 integrins, and their natural ligand Vn, are involved in PAK entry into human epithelial cells.     

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8–11 and SCR19–20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19–20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.     

Methylselenol generated from selenomethionine by methioninase downregulates integrin expression and induces caspase-mediated apoptosis of B16F10 melanoma cells

Melanoma is a highly metastatic cancer resistant to current chemotherapeutic and radiotherapeutic approaches. Several studies have shown that interactions between cancer cells and the extracellular matrix (ECM) are critical for the survival and invasion of metastatic cancer cells. In this study, we examine the effects of methylselenol generated from selenomethionine (SeMet) by methioninase (METase) on cell proliferation, adhesion, and expression of integrins in murine melanoma B16F10 cells, which are metastatic in the lungs of syngeneic C57BL/6J mice. Combined treatment with SeMet-METase decreased the expression of integrins alpha(4), beta(1), alpha(nu), and beta(3), and inhibited melanoma-ECM adhesion. Caspase-mediated apoptosis was induced following loss of cell adherence. Phosphorylation of focal adhesion kinase (FAK) and Akt, related to integrin-mediated survival, were decreased upon treatment with SeMet-METase while phosphorylation of p38, PKC-delta, and IkappaBalpha increased. In the presence of specific inhibitors of p38, PKC-delta, and NF-kappaB, expression of integrins and cell adhesion to ECM were maintained and cell apoptosis was prevented in SeMet-METase-treated melanoma cells. Treatment with caspase inhibitors restored cell viability and blocked poly (ADP-ribose) polymerase (PARP) cleavage, but did not restore integrin expression and cell adhesion to ECMs reduced by SeMet-METase. Based on these results, we propose that combined treatment with SeMet-METase induces caspase-mediated apoptosis in melanoma cells by altering integrin expression and adhesion. Furthermore, activation of p38, PKC-delta, and NF-kappaB is a prerequisite for the down-regulation of integrin expression, followed by detachment-mediated apoptosis.     

Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components

Episialin (MUC1) is a transmembrane molecule with a large mucin-like extracellular domain protruding high above the cell surface. The molecule is located at the apical side of most glandular epithelial cells, whereas in carcinoma cells it is often present at the entire surface and it is frequently expressed in abnormally large quantities. We have previously shown that overexpression of episialin reduces cell- cell interactions. Here we show that the integrin-mediated adhesion to extracellular matrix of transfectants of a melanoma cell line (A375), a transformed epithelial cell line (MDCK-ras-e) and a human breast epithelial cell line (HBL-100) is reduced by high levels of episialin. This reduction can be reversed by inducing high avidity of the beta 1 integrins by mAb TS2/16 (at least for beta 1-mediated adhesion). The adhesion can also be restored by redistribution of episialin on the cell surface by monoclonal antibodies into patches or caps. Similarly, capping of episialin on ZR-75-1 breast carcinoma cells, growing in suspension, caused adherence and spreading of these cells. We propose that there is a delicate balance between adhesion and anti-adhesion forces in episialin expressing cells, which can be shifted towards adhesion by strengthening the integrin-mediated adhesion, or towards anti-adhesion by increasing the level of expression of episialin.