Major autolysin (Atl) of Staphylococcus aureusis a cell surface associated peptidoglycan hydrolase with amidase and glucosaminidase domains. Atl enzymes (amidase and glucosaminidase) are known to participate in biofilm formation and also can bind with host matrices. Earlier studies demonstrated the binding of Atlwithfibronectin, thrombospondin 1, vitronectin and heat shock cognate protein Hsc70. Here, we have shown, Atl mediates attachment of S.aureus to heparin and gelatine as well. The atl mutant strain demonstrated around 2.5 fold decreased adherence with fibronectin, gelatin and heparin coated microtiter plates. The microscopic studies confirmed the reduced binding of atl mutant with them compared to its parental wild type and complemented mutant strains. Amidase and glucosaminidase were expressed as N-terminal histidine tagged proteins from Escherichia coli, purified and refolded. We found refolded amidase bind with fibronectin, gelatin and heparin; whereas refolded glucosaminidase binds with only fibronectin and heparin but not gelatin. These results reemphasize Atl as one of the crucial proteins from Staphylococcus that facilitate their binding with multiple host cellular components during colonization and infection.

Background: Advanced glycation end products (AGEs) have been linked to pathogenic mechanisms of diabetes mellitus. However, little is known about the contribution of protein glycation to periodontal disease in patients with diabetes. Therefore, this study investigated whether glycation of type I collagen (COLI) and fibronectin (FN) modified the behavior of human gingival fibroblasts (hGFs) and periodontal ligament fibroblasts (hPDLs).Methods: Procedures for rapid in vitro glycation of COLI and FN used methylglyoxal (MG). Formation of AGEs was analyzed by changes in protein migration using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and Western blotting with antibodies specific for MG‐glycated proteins. Experiments then characterized the effects of glycated FN and COLI on the behavior of hGFs and hPDLs.Results: MG glycated COLI and FN in <6 hours. Confirming the specificity of the reactions, antibodies specific for MG‐induced AGEs reacted with glycated FN and COLI but not with control proteins. In cell culture experiments, glycated FN was significantly less efficient in supporting the attachment of hGFs and hPDLs (P <0.05). Moreover, the morphologic parameters, including length, area, perimeter, and shape factor, were altered (P <0.001) for cells on both glycated proteins. Finally, cell migration was reduced on glycated FN and COLI (P <0.001).Conclusions: MG treatment efficiently glycated COLI and FN, providing a new tool to study the effects of diabetes on periodontal disease. The substantial effects of glycated COLI and FN on hGF and hPDL behavior indicated that protein glycation contributed to the pathogenesis and altered periodontal wound healing observed in patients with diabetes.

Background: Fibronectin (FN) undergoes fragmentation in periodontal disease sites and in poorly healing diabetic wounds. The biologic effects of FN fragments on wound healing remain unresolved. This study characterized the pattern of FN fragmentation and its effects on cellular behavior compared to intact FN.Methods: Polyclonal antibodies were raised against FN and three defined recombinant segments of FN and used to analyze gingival crevicular fluid from periodontal disease sites in systemically healthy subjects and in subjects with diabetes, as well as chronic leg and foot wound exudates from subjects with diabetes. Subsequently, the behavior of human gingival fibroblasts (hGFs) and HT1080 reference cells were analyzed by measuring cell attachment, migration, and chemotaxis in the presence of intact FN or recombinant FN fragments.Results: FN fragmentation was evident in fluids from periodontal disease sites and diabetic leg and foot wounds. However, no fragmentation pattern distinguished systemically healthy subjects from subjects with diabetes. hGFs and HT1080 cells required significantly higher concentrations of FN fragments to achieve attachment comparable to intact FN. Cells cultured on FN fragments also were morphologically different from cells cultured on full‐length FN. Migration was reduced for hGFs cultured on FN fragments relative to full‐length FN. In contrast, FN fragments increased HT1080 fibrosarcoma cell migration over intact FN.Conclusions: FN fragmentation is a prominent feature of periodontal and chronic leg and foot wounds in diabetes. Furthermore, cell culture assays confirmed the hypothesis that exposure to defined FN fragments significantly alters cell behavior.

Recent literature has suggested a novel symbiotic relationship between bacteriophage and metazoan host that provides antimicrobial defense protecting mucosal surface by binding to host matrix mucin glycoproteins. Here, we isolated and studied different bacteriophages that specifically interact with human extracellular matrix molecules such as fibronectin, gelatin, heparin and demonstrated their potency for protection to host against microbial infections. We showed that subpopulations of bacteriophages that work against clinical isolates of Escherichia coli can bind to pure gelatin, fibronectin and heparin and reduced bacterial load in human colon cell line HT29. The bacteriophages were characterized with respect to their genome sizes, melting curve patterns and host tropism (cross-reactivity with different hosts). Since, the bacteriophages are non-toxic to the host and can effectively reduce bacterial load in HT29 cell line their therapeutic potency against bacterial infection could be explored.

Platelet-derived growth factor BB and its receptor (PDGFRβ) play a pivotal role in the development of renal glomerular mesangial cells. Their roles in increased mesangial cell proliferation during mesangioproliferative glomerulonephritis have long been noted, but the operating logic of signaling mechanisms regulating these changes remains poorly understood. We examined the role of a recently identified MAPK, Erk5, in this process. PDGF increased the activating phosphorylation of Erk5 and tyrosine phosphorylation of proteins in a time-dependent manner. A pharmacologic inhibitor of Erk5, XMD8-92, abrogated PDGF-induced DNA synthesis and mesangial cell proliferation. Similarly, expression of dominant negative Erk5 or siRNAs against Erk5 blocked PDGF-stimulated DNA synthesis and proliferation. Inhibition of Erk5 attenuated expression of cyclin D1 mRNA and protein, resulting in suppression of CDK4-mediated phosphorylation of the tumor suppressor protein pRb. Expression of cyclin D1 or CDK4 prevented the dominant negative Erk5- or siErk5-mediated inhibition of DNA synthesis and mesangial cell proliferation induced by PDGF. We have previously shown that phosphatidylinositol 3-kinase (PI3-kinase) contributes to PDGF-induced proliferation of mesangial cells. Inhibition of PI3-kinase blocked PDGF-induced phosphorylation of Erk5. Since PI3-kinase acts through Akt, we determined the role of Erk5 on Akt phosphorylation. XMD8-92, dominant negative Erk5, and siErk5 inhibited phosphorylation of Akt by PDGF. Interestingly, we found inhibition of PDGF-induced Erk5 phosphorylation by a pharmacological inhibitor of Akt kinase and kinase dead Akt in mesangial cells. Thus our data unfold the presence of a positive feedback microcircuit between Erk5 and Akt downstream of PI3-kinase nodal point for PDGF-induced mesangial cell proliferation.