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 nonphagocytic cells, which are readily infected in vitro, and the in vivo significance of this interaction are incompletely understood. Of six cell types tested, we found that only two, Chinese hamster ovary (CHO) fibroblasts and primary porcine aortic endothelial cells, were able to bind M. tuberculosis H37Rv efficiently in vitro. Binding to both CHO and endothelial cells was markedly (three- to fivefold) enhanced by 10 to 20% human or bovine serum, suggesting that the bacteria were coated by a serum opsonin. Preincubation with individual candidate opsonins revealed that recombinant human mannose-binding protein (rMBP), fibronectin, 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 approximately 60 and 58%, respectively, of that produced by preincubation in control serum. In contrast, serum depleted of fibronectin or transferrin retained its opsonizing capacity, suggesting that the latter two are not significant opsonins in whole serum. Binding of M. tuberculosis and Mycobacterium smegmatis to both CHO and endothelial cells in the presence or absence of serum was blocked (60 to 70%) by a monoclonal antibody, MAb 1D1, selected for recognition of intact bacilli. The 1D1 antigen was purified from mycobacterial cell walls and chemically identified as a polar phosphatidylinositol mannoside (PIM). Latex beads coated with purified 1D1 antigen bound to CHO cells, which was enhanced threefold by serum and abolished by periodate treatment, suggesting a requirement for the PIM mannoses in opsonic adhesion. This was likely mediated, at least in part, by serum MBP, as rMBP bound strongly 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 opsonization with serum proteins, including MBP.