Mycobacterium tuberculosis, the causative agent of tuberculosis, initially contacts host cells with elements of its outer cell wall, or capsule. We have shown that capsular material from the surface of M. tuberculosis competitively inhibits the nonopsonic binding of whole M. tuberculosis bacilli to macrophages in a dosedependent manner that is not acting through a global inhibition of macrophage binding. We have further demonstrated that isolated M. tuberculosis capsular proteins mediate a major part of this inhibition. Twodimensional polyacrylamide gel electrophoresis analysis of the capsular proteins showed the presence of a wide variety of protein species, including proportionately high levels of the Cpn60.2 (Hsp65, GroEL2) and DnaK (Hsp70) molecular chaperones. Both of these proteins were subsequently detected on the bacterial surface. To determine whether these molecular chaperones play a role in bacterial binding, recombinant Cpn60.2 and DnaK were tested for their ability to inhibit the association of M. tuberculosis bacilli with macrophages. We found that recombinant Cpn60.2 can inhibit ϳ57% of bacterial association with macrophages, while DnaK was not inhibitory at comparable concentrations. Additionally, when polyclonal F(ab) 2 fragments of anti-Cpn60.2 and anti-DnaK were used to mask the surface presentation of these molecular chaperones, a binding reduction of ϳ34% was seen for anti-Cpn60.2 F(ab) 2 , while anti-DnaK F(ab) 2 did not significantly reduce bacterial association with macrophages. Thus, our findings suggest that while M. tuberculosis displays both surfaceassociated Cpn60.2 and DnaK, only Cpn60.2 demonstrates adhesin functionality with regard to macrophage interaction.The initiation of a tuberculous infection involves the adherence and phagocytosis of Mycobacterium tuberculosis bacilli by host cells. It is generally thought that the primary host niche of M. tuberculosis is the alveolar macrophage (M). To access this cell, ligands on the outer surface of the M. tuberculosis bacillus must come in contact with surface receptors of the M. Although a significant amount of information concerning the M receptors involved in this interaction is available (15, 71), the identities of the mycobacterial cell surface components that mediate this binding are less well understood. However, evidence for the involvement of mycobacterial lipoarabinomannans (57), capsular polysaccharides (8), glycopeptidolipids (72), 19-kDa antigen (9), mycotin (21), and Apa glycoprotein (47) has been reported previously.For any of the aforementioned moieties to be involved in the binding of mycobacteria to host cells, they would have to be located on the surface of the bacterium. Early reports suggested that the outer surface of mycobacteria was composed of mycosides (10,11). Later studies indicated the presence of an outer polysaccharide-rich layer (45, 50), which could explain the presence of the so-called electron-transparent zone often seen in electron micrographs of mycobacteria inside M (13, 18) and more recently in axe...

SummaryCD43 is a large sialylated glycoprotein found on the surface of haematopoietic cells and has been previously shown to be necessary for efficient macrophage binding and immunological responsiveness to Mycobacterium tuberculosis. Using capsular material from M. tuberculosis and recombinant CD43-Fc, we have employed affinity chromatography to show that Cpn60.2 (Hsp65, GroEL), and to a lesser extent DnaK (Hsp70), bind to CD43. Competitive inhibition using recombinant protein and polyclonal F(abЈ) 2 antibody-mediated epitope masking studies were used to evaluate M. tuberculosis binding to CD43 +/+ versus CD43 -/-macrophages. Results showed that Cpn60.2, but not DnaK, acts as a CD43-dependent mycobacterial adhesin for macrophage binding. Assessment of the specific binding between Cpn60.2 and CD43 showed it to be saturable, with a comparatively weak affinity in the low micromolar range. We have also shown that the ability of Cpn60.2 to competitively inhibit M. tuberculosis binding to macrophages is shared by the Escherichia coli homologue, GroEL, but not by the mouse and human Hsp60 homologues. These findings add to a growing field of research that implicates molecular chaperones as having extracellular functions, including bacterial adherence to host cells. Thus, CD43 may act as a Pattern Recognition Receptor (PRR) for bacterial homologues of the 60 kDa molecular chaperone.

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