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Single-Molecule Force Spectroscopy of Mycobacterial Adhesin-Adhesin Interactions

Unité de Chimie des Interfaces, Université Catholique de Louvain, Croix du Sud 2/18, B-1348 Louvain-la-Neuve, Belgium,¹ INSERM, U629, Mécanismes Moléculaires de la Pathogénie Microbienne, Lille, France,² Institut Pasteur de Lille, 1 rue du Professeur Calmette, F-59019 Lille Cedex, France,³ IFR 142, Molecular and Cellular Medicine, Lille, France,⁴ CNRS UMR 8161, IBL, Lille, France⁵

Received 10 August 2007/ Accepted 28 September 2007

The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule. Here, we used single-molecule atomic-force microscopy to measure individual homophilic HBHA-HBHA interaction forces. Force curves recorded between tips and supports derivatized with HBHA proteins exposing their N-terminal domains showed a bimodal distribution of binding forces reflecting the formation of dimers or multimers. Moreover, the binding peaks showed elongation forces that were consistent with the unfolding of -helical coiled-coil structures. By contrast, force curves obtained for proteins exposing their lysine-rich C-terminal domains showed a broader distribution of binding events, suggesting that they originate primarily from intermolecular electrostatic bridges between cationic and anionic residues rather than from specific coiled-coil interactions. Notably, similar homophilic HBHA-HBHA interactions were demonstrated on live mycobacteria producing HBHA, while they were not observed on an HBHA-deficient mutant. Together with the fact that HBHA mediates bacterial aggregation, these observations suggest that the single homophilic HBHA interactions measured here reflect the formation of multimers that may promote mycobacterial aggregation.

Published ahead of print on 12 October 2007.