Mycobacterium tuberculosis Cpn60.2 and DnaK Are Located on the Bacterial Surface,Where Cpn60.2 Facilitates Efficient Bacterial Association with Macrophages

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 dose-dependent 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. Two-dimensional 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 ∼57% 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 ∼34% 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 surface-associated 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 (Mφ). To access this cell, ligands on the outer surface of the M. tuberculosis bacillus must come in contact with surface receptors of the Mφ. Although a significant amount of information concerning the Mφ 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 Mφ (13, 18) and more recently in axenically grown bacteria (17, 42, 43, 51). Support for this contention has come from studies describing the presence of an outer surface capsule on M. tuberculosis (12). Carbohydrates make up 85% of the capsule, and the predominant sugar is glucan (approximately 70% of all sugars present). Arabinomannan and mannan are also present in significant quantities, as are a number of proteins, some of which are glycosylated. While about 10% of the capsule is composed of proteins, there is very little lipid present (31, 37). No evidence for the presence of lipoarabinomannan in the capsule was found, though phosphatidylinositol mannoside (PIM) was identified (38). The presence of a glycan-rich capsule surrounding intracellular mycobacteria has been confirmed using specific monoclonal antibodies (MAbs) against arabinomannan and glucan (58, 59).We have shown previously that mechanical removal of capsular material from M. tuberculosis results in a 10-fold increase in bacterial binding to Mφ, suggesting that the capsule can act as an antiphagocytic barrier that limits the interaction of M. tuberculosis with Mφ (65). However, even though the capsule reduces binding of M. tuberculosis to Mφ, it does not eliminate it, and it is clear that at least some bacteria maintain the capacity to bind to Mφ. These observations, along with our earlier studies showing that only certain populations of Mφ efficiently bind M. tuberculosis (64, 67), suggest that the M. tuberculosis capsule modulates the interaction of bacteria with host cells, preventing uptake by some populations of Mφ and directing the bacteria to specific Mφ types or particular receptor-ligand interactions. In this study, we have evaluated the diversity of proteins present in the M. tuberculosis capsule and assessed the role of two bacterial molecular chaperones, Cpn60.2 and DnaK, in host cell binding. Using both competitive-inhibition and epitope-masking strategies, we have shown that while both Cpn60.2 and DnaK are present on the bacterial surface, only Cpn60.2 appears to be necessary to facilitate efficient bacterial association with Mφ.