Rv0613c/MSMEG_1285 Interacts with HBHA and Mediates Its Proper Cell-Surface Exposure in Mycobacteria.

Heparin-binding haemagglutinin (HBHA) is a surface-exposed virulence factor of and is involved in the binding of mycobacteria to non-phagocytic cells, allowing for extra-pulmonary dissemination of the bacilli. Despite its surface exposure, HBHA is not produced as a pre-protein containing a typical cleavable N-terminal signal peptide and is thus likely secreted by a Sec-independent, as of yet unknown mechanism. Here, we used the bacterial adenylate cyclase two-hybrid system to identify the proteins encoded by and as being able to interact with HBHA. Our study was focused on Rv0613c, as it showed more consistent interactions with HBHA than MmpL14. Deletion of its orthologous gene in recombinant producing HBHA from resulted in the loss of proper surface exposure of HBHA, as evidenced by atomic force microscopy. Furthermore, the lack of also abolished the clumping phenotype and rough colony morphology of the recombinant and reduced its adherence to A549 epithelial cells. These phenotypes have previously been associated with surface-exposed HBHA. Thus, MSMEG_1285 is directly involved in the proper cell-surface exposure of HBHA. These observations identify MSMEG_1285/Rv0613c as the first accessory protein involved in the cell surface exposure of HBHA.