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

Abstract

Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Gué rin produce a heparin-binding hemagglutinin adhesin (HBHA) required for extrapulmonary dissemination and a laminin-binding protein (LBP) involved in cytoadherence through laminin recognition. These adhesins bear posttranslational modifications that are not present when the proteins are produced in a recombinant (r) form in Escherichia coli. Mass spectrometry analysis of HBHA revealed that the posttranslational modifications are borne by the C-terminal moiety, which comprises the heparinbinding domain made of repeated lysine-rich motifs. Amino acid sequencing showed that these modifications consist of mono-and dimethyllysines within these motifs. The methyllysine-containing repeats were recognized by mAb 4057D2 and were also detected in LBP, which is equally recognized by mAb 4057D2. This Ab does not recognize the recombinant forms of these proteins. However, when rHBHA and rLBP were subjected to NaBH4 and formalin treatment to induce lysine methylation, reactivity with mAb 4057D2 was recovered. Methylated rHBHA displayed enhanced resistance to proteolysis compared with rHBHA, as previously observed for native HBHA. S-adenosylmethionine-dependent HBHA methyltransferase activity was detected in the cell-wall fractions of M. bovis bacillus Calmette-Gué rin and of Mycobacterium smegmatis, a species that produces LBP but naturally lacks hbhA, suggesting that the same enzyme(s) methylate(s) both LBP and HBHA. This hypothesis was confirmed by the fact that HBHA produced by recombinant M. smegmatis was also methylated. These results show that mycobacteria use enzymatic methylation of lysines to ensure greater stability of their adhesins.A lthough tuberculosis remains a major cause of morbidity and mortality worldwide with 3 million deaths and 10 million new cases per year (1), relatively little is known about the virulence factors expressed by its etiologic agent, Mycobacterium tuberculosis. M. tuberculosis infection begins by inhalation of droplet nuclei smaller than 0.5 m and containing one to three bacilli that reach the lung alveoli (2, 3). Dissemination of viable bacilli from the alveolus lumen into the lymph or circulatory systems is an important step in the pathogenesis of tuberculosis (4, 5). Because M. tuberculosis exhibits a tropism for macrophages and has evolved mechanisms to survive and multiply within the macrophage phagosome, it is generally accepted that these professional phagocytes are the main target cells of the tubercle bacillus (6-8).However, besides its interaction with macrophages, M. tuberculosis is also able to invade and replicate within other cell types, including epithelial cells (9-11). Adherence of the tubercle bacillus to epithelial cells is predominantly mediated by the heparin-binding hemagglutinin adhesin (HBHA), a 199-residue surface-exposed protein that is also involved in mycobacterial agglutination (12). The adherence mediated by HBHA relies on the interaction of its C-terminal domain composed of lysine-rich m...

Difference in TB10.4 T‐cell epitope recognition following immunization with recombinant TB10.4, BCG or infection with Mycobacterium tuberculosis

“…It also indicates that vaccines may not have to induce responses against dominant infection-driven T-cell epitopes in order to be protective. This may be important for future vaccine design as discussed below in the concluding remarks of the Discussion section.It has been demonstrated that post-translational modifications and native folding of Ag can alter immunogenicity of a protein and even mask or unmask certain epitopes in an Ag compared with the recombinant version of the same antigen [29,30]. However, we found that immunization with native TB10.4 did not alter the epitope pattern compared to immunization with recombinant TB10.4 produced in E. coli (Fig.…”

Towards a New Challenge in TB Control: Development of Antibody-Based Protection

“…Monoclonal antibodies 3921E4 (IgG2a) and 4057D2 (IgG3) directed against HBHA were used to coat mycobacteria before administration to mice. In this study, spleen CFUs were reduced while lung CFUs did not [58]. These results suggest that binding of these antibodies to HBHA impede mycobacterial dissemination.…”

Haemophilus influenzae Protein F Mediates Binding to Laminin and Human Pulmonary Epithelial Cells

“…Mycobacterium spp, have also been reported to interact with Ln and thereby augment their virulence [9][10][11][12][13][14][15][16]. Binding to ECM-components such as Ln is thus most likely an important mechanism for bacterial pathogenesis.…”

“…It is involved in an array of physiological functions such as cellular proliferation, migration and structural scaffolding in tissues [6][7][8]. During bacterial pathogenesis, Ln is frequently targeted for adherence by respiratory tract pathogens [9][10][11][12][13][14][15][16]. NTHi has previously been reported to bind Ln via the Haemophilus adhesion and penetration protein (Hap) and Protein E (PE) [17,18].…”