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UBC Theses and Dissertations

Identification of Cpn60.2 as a surface ligand of Mycobacterium tuberculosis that facilitates bacterial association with macrophages via CD43. Hickey, Tyler Bruce Malcolm


Mycobacterium tuberculosis bacilli initially contact host cells with elements of their outer cell wall, or capsule. We have shown that capsular proteins from the surface of M. tuberculosis competitively inhibit the non-opsonic binding of whole M. tuberculosis bacilli to macrophages in a dose-dependent manner that is not acting through a global inhibition of macrophage binding. CD43 is a large sialylated glycoprotein that is found on the surface of macrophages and has been shown in previous studies to be necessary for efficient macrophage binding and immunological responsiveness to M. tuberculosis. Using capsular proteins and recombinant CD43, we have employed affinity chromatography to show that Cpn60.2 (Hsp65, GroEL2), and to a lesser extent DnaK (Hsp70), bind to CD43. We subsequently demonstrated that both Cpn60.2 and DnaK can be identified on the surface of M. tuberculosis bacilli. Furthermore, we performed recombinant protein competitive inhibition and polyclonal F(ab')₂ antibody-mediated epitope masking studies to show that Cpn60.2, but not DnaK, acts as a mycobacterial adhesin for macrophage binding. We then compared M. tuberculosis binding of CD43+/+ versus CD43⁻/⁻ macrophages and found that the adhesin function of Cpn60.2 is CD43-dependent. Additionally, the binding between Cpn60.2 and CD43 can be saturated; however the binding affinity is comparatively weak, near one micromolar. 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 mediating bacterial adherence to host cells, distinct from their well-described protein folding activities within the cytosol.

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