Mycobacterium smegmatis laminin‐binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin‐binding haemagglutinin

Abstract

SummaryMycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin±Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.

Mycobacterial heparin-binding hemagglutinin and laminin-binding protein share antigenic methyllysines that confer resistance to proteolysis

“…M. tuberculosis (strain 103), Mycobacterium bovis bacillus Calmette-Guérin (strain 1173P2; World Health Organization), and Mycobacterium smegmatis (strain mc 2 155) were grown in Sauton medium as described (12). E. coli BL21(DE3)(pET-HBHA) (15) and E. coli BL21(DE3) (pET-MSLBP) (17) were grown in LB broth supplemented with 30 g͞ml of kanamycin (19).…”

“…This was evidenced by comparative mass spectrometry analysis of the native and recombinant forms of HBHA and by amino acid sequencing of the C-terminal domain of nHBHA. Previous studies have suggested that nHBHA (13), as well as LBP (17), may be glucosylated, which may be crucial for the recognition of both proteins by mAb 4057D2. The results reported here provide no evidence for glucose covalently bound to nHBHA or nMS-LBP.…”

“…HBHA produced by recombinant M. smegmatis (MS-HBHA) was purified as for nHBHA. Native M. smegmatis LBP (nMS-LBP) and MS-LBP produced by E. coli (rMS-LBP) were purified as described (17).…”

“…Although M. smegmatis does not produce HBHA, a mAb 4057D2-reactive antigen has been detected on its surface and identified as a laminin-binding protein (MS-LBP) (17). Like HBHA, this protein exhibits a lysine-rich C-terminal domain that is also posttranslationally modified.…”

“…Moreover, we show that this modification is performed by enzyme(s) associated with the mycobacterial cell wall and confers increased protease resistance to HBHA. Finally, we show that a similar modification is also borne by the laminin-binding protein (LBP), another mycobacterial adhesin that mediates adherence through cell-surface laminin recognition (17,18).…”