RGD Motif of Lipoprotein T,Involved in Adhesion of Mycoplasma conjunctivae to Lamb Synovial Tissue Cells

Lipoprotein T (LppT), a membrane-located 105-kDa lipoprotein of Mycoplasma conjunctivae, the etiological agent of infectious keratoconjunctivitis (IKC) of domestic sheep and wild Caprinae, was characterized. LppT was shown to promote cell attachment to LSM 192 primary lamb joint synovial cells. Adhesion of M. conjunctivae to LSM 192 cells is inhibited by antibodies directed against LppT. The RGD (Arg-Gly-Asp) motif of LppT was found to be a specific site for binding of M. conjunctivae to these eukaryotic host cells. Recombinant LppT fixed to polymethylmethacrylate slides binds LSM 192 cells, whereas LppT lacking the RGD site is deprived of binding capacity to LSM 192, and LppT containing RGE rather than RGD shows reduced binding. Synthetic nonapeptides derived from LppT containing RGD competitively inhibit binding of LSM 192 cells to LppT-coated slides, whereas nonapeptides containing RAD rather than RGD do not inhibit. RGD-containing, LppT-derived nonapeptides are able to directly inhibit binding of M. conjunctivae to LSM 192 cells by competitive inhibition, whereas the analogous nonapeptide containing RAD rather than RGD or the fibronectin-derived RGD hexapeptide has no inhibitory effect. These results reveal LppT as the first candidate of a RGD lectin in Mycoplasma species that is assumed to bind to β integrins.Mycoplasma conjunctivae, the etiological agent of infectious keratoconjunctivitis (IKC), causes severe ocular infections that lead to blindness and perforation of the cornea, particularly in Alpine ibex (Capra ibex ibex) and chamois (Rupicapra rupicapra rupicapra) (4). In view of the harsh physiochemical conditions that protect the eye from being colonized and infected by pathogenic microorganisms, M. conjunctivae is expected to exhibit efficient adhesion functions in order to avoid being flooded off by lachrymal fluid. Adhesion is thought to play a central role in the pathogenicity of bacteria in general and of Mycoplasma species in particular, both directly as a basic condition of colonization (10, 23, 42, 43) and indirectly by adherence coupled to cytopathic functions. In the latter, adhering mycoplasmas may induce oxidative damage to the host cell by targeted release of peroxide and oxygen radical species (7, 27) or disrupt K⁺ channels of ciliated bronchial epithelial cells, which leads to ciliostasis (13). Extracellular matrix proteins and glycosaminoglycans play important roles as receptors for adhesion of bacterial pathogens, including those of Mycoplasma species. In Mycoplasma hyopneumoniae, protein P159 has recently been identified as a heparin binding protein that promotes adherence to eukaryotic cells (10). Furthermore, the R1 region near the carboxy terminus of protein P97 of M. hyopneumoniae has been shown to mediate adherence to swine cilia (23, 41). Mycoplasmal adhesion structures have extensively been studied in virulent Mycoplasma pneumoniae, where two surface proteins, P1 of 169 kDa and P30 of 30 kDa, are densely clustered to form the tip organelle that provides strong polarity to the cytoadherence process (12, 20). Moreover, a putative cytoskeleton-forming protein with a proline-rich, acidic domain was speculated to be involved in the formation of the adhesion tip (28). In contrast to the well-structured adherence organelle of M. pneumoniae, adhesins of most other Mycoplasma species appear to be distributed on the mycoplasmal surface, and no particular receptor-ligand mechanisms have to date been identified (29).In M. conjunctivae, a serine-rich membrane-located lipoprotein, LppS, was found to be involved in the adhesion to LSM 192 lamb joint synovial cells. LppS was shown to have sequence similarity to the fibrinogen binding protein, clumping factor A (ClfA) of Staphylococcus aureus, which has a repeated serine-aspartate domain at the analogous polyserine location (6). In the lamb joint synovial cell model, adherence of M. conjunctivae was inhibited using Fab fragments from immunoglobulin G (IgG) directed against recombinant purified LppS (6). Lipoprotein T (LppT) of M. conjunctivae, which is encoded by the same bicistronic operon downstream of lppS, shows significant similarity to the heparin binding protein P159, protein P102, and Mhp494 of M. hyopneumoniae, which are involved in adhesion to swine cilia (10, 17, 19, 38). We report here the characterization of LppT and its role in adhesion. LppT contains an RGD cell attachment motif that consists of the amino acids Arg-Gly-Asp, which is shown to be directly involved in binding to primary lamb joint synovial cells. RGD adhesins belong to a large class of integrin binding proteins that bind the extracellular matrix and which are known to induce important biological events such as cell differentiation, malignant transformation, immune recognition, and blood coagulation (25, 31).