Abstract
Heparin-binding haemagglutinin (HBHA) is a surface-exposed virulence factor of Mycobacterium tuberculosis 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 rv0613c and mmpL14 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 MSMEG_1285 in recombinant Mycobacterium smegmatis producing HBHA from M. tuberculosis resulted in the loss of proper surface exposure of HBHA, as evidenced by atomic force microscopy. Furthermore, the lack of MSMEG_1285 also abolished the clumping phenotype and rough colony morphology of the recombinant M. smegmatis 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.
Highlights
Tuberculosis (TB) remains the leading global cause of infection-related mortality and morbidity, with 1.7 million deaths and an estimated incidence of 10.4 million cases in 2016 [1]
As an approach to unravel Heparin-binding haemagglutinin (HBHA) cell surface exposure, we identified the proteins encoded by rv0613c and mmpL14, as the first proteins able to interact with HBHA
To identify protein partners able to interact with HBHA, we used the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system with pKT25_hbhA as a bait to screen a Mycobacterium tuberculosis (Mtb) Erdman library contained in pUT18C [11]
Summary
Tuberculosis (TB) remains the leading global cause of infection-related mortality and morbidity, with 1.7 million deaths and an estimated incidence of 10.4 million cases in 2016 [1]. The Heparin-binding haemagglutinin (HBHA) is a surface-exposed mycobacterial adhesin [2,3] that is able to bind heparan sulfate [4]. HBHA is dispensable for initial pulmonary colonization, the protein is required for extrapulmonary dissemination of the bacilli in mice [5]. It is a highly methylated protein in its C-terminal part [7] and its complex methylation profile plays a direct role in the immunogenicity and recognition of HBHA by T cells from Mtb-infected subjects [8]. Despite its cell surface exposure in Mtb [2], HBHA lacks a canonical signal peptide, usually required for protein transport through bacterial plasma membranes. HBHA crosses the mycobacterial membrane by an unknown mechanism that is independent of the classical signal sequences peptides
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