IDENTIFICATION OF PHOSPHATIDYLINOSITOL MANNOSIDE AS A MYCOBACTERIAL ADHESIN MEDIATING BOTH DIRECT AND OPSONIC BINDING TO NONPHAGOCYTIC MAMMALIAN-CELLS

The molecular basis for the binding of Mycobacterium tuberculosis to n onphagocytic cells, which are readily infected in vitro, and the in vi vo significance of this interaction are incompletely understood, Of si x cell types tested, we found that only two, Chinese hamster ovary (CH O) fibroblasts and primary porcine aortic endothelial cells, were able to bind M. tuberculosis H37Rv efficiently in vitro, Binding to both C HO and endothelial cells was markedly (three-to fivefold) enhanced by 10 to 20% human or bovine serum, suggesting that the bacteria were coa ted by a serum opsonin, Preincubation with individual candidate opsoni ns revealed that recombinant human mannose-binding protein (rMBP), fib ronectin, and transferrin were each able to enhance binding threefold, Preincubation of bacteria in serum depleted of mannan-binding lectins or in genetic MBP-deficient serum resulted in enhancements that were only similar to 60 and 58%, respectively, of that produced by preincub ation in control serum, In contrast, serum depleted of fibronectin or transferrin retained its opsonizing capacity, suggesting that the latt er two are not significant opsonins in whole serum, Binding of M. tube rculosis and Mycobacterium smegmatis to both CHO and endothelial cells in the presence or absence of serum was blocked (60 to 70%) by a mono clonal antibody, MAb 1D1, selected for recognition of intact bacilli, The 1D1 antigen was purified from mycobacterial cell walls and chemica lly identified as a polar phosphatidylinositol mannoside (PIM), Latex beads coated with purified 1D1 antigen bound to CHO cells, which was e nhanced threefold by serum and abolished by periodate treatment, sugge sting a requirement for the PIM mannoses in opsonic adhesion, This was likely mediated, at least in part, by serum MBP, as rMBP bound strong ly to 1D1 antigen in both thin-layer chromatography overlay and plate binding assays, the latter in a mannan-inhibitable manner, This is the first demonstration that mycobacterial PIMs can function as adhesins for binding to nonphagocytic cells, both directly and after opsonizati on with serum proteins, including MBP.