Study On Function And Mechanism Of HBHA In MTB Interacte With AECⅡ

Mycobacterium tuberculosis, one of the most devastating microbial pathogens for humans, infects roughly one-third of the population worldwide and results in nearly two million deaths and eight million new cases every year. So far very few M. tuberculosis virulence factors have been identified. The heparin-binding hemagglutinin adhesin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a the 28-kD surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesin to nonphagocytic cells and in extrapulmonary dissemination. But the functions of HBHA during MTB infection need to further investigate. Here, we expressed rHBHA and extracted nHBHA, and then studied on the mechanism of interaction of HBHA with epithelial cells.1 Expression and purification of Mycobacterium tuberculosis HBHA protein.MTB HBHA nucleotide sequences were searched in GenBank. The specific primers was designed by using the software of Primer Premier 5.0 . The genome DNA of MTB were extracted and then amplified the 600bp fragment by PCR. The sequence was cloned into BamHⅠand HindIII of expression pQE-80L vector. After confirmed by DNA sequencing, the recombinant plasmid was transformed into E.coli BL-21(DE3). The recombinant bacteria was induced at 37℃using 0.3mmol?L-1 IPTG. The expression of recombinant protein HBHA was analysed by SDS-PAGE and its moleculer weight was 28-kD. The recombinant protein was purified by His Gravitrap chromatography column. And the concentration of purified recombinant protein was 10.98mg/mL.Then the polyclonal antibody against recombinant HBHA was prepared by immunizing rabbit.2 Extraction of nature HBHA (nHBHA) and preliminary studying on the biological function of protein HBHA.After growing BCG to the stationary status in the 7H9 liquid medium, we collected BCG bacteria by centrifugation, and then resuspended with PBS, intermittently sonicating the BCG bacteria. The supernatant was passed through a Heparin Sepharose CL-6B column which was equilibrated with PBS. The column was then washed with 100mL PBS and the bound material was eluted by a 0--959 mmol?L-1 NaCl gradient in PBS. Extracted nature HBHA was analysed by SDS-PAGE and Western blot. Obtained nHBHA had a maximum concentration of 1.016mg/mL. We incubated different concentrations of purified nature or recombinant HBHA with dispersed BCG cells in 7H9 liquid culture medium. After 36 hours, the aggregation effect of BCG. was observed. Meanwhile, the HBHA protein and anti-HBHA antibodies were incubated for one hour at 37℃. Then the incubated protein was added to the BCG medium. And after 36 hours, we observed the aggregation effect. The results showed that both the nature and recombinant protein had induced BCG aggregation. And the ability of nature HBHA was better than recombinant HBHA. Such aggregation can be inhibited by anti-rHBHA polyclonal antibody.3 Study on function and mechanism of HBHA in MTB interacte with TypeⅡalveolar epithelial cells (AECⅡ).We incubated rHBHA or nHBHA with A-549 cell. And then LC3 autophagy-related molecules was detected by RT-PCR and Western blot. The results showed that when the concentration of HBHA increased, the expression of A-549 cells LC3 gradually decreased. Both rHBHA and nHBHA groups had this trend. This result indicated that HBHA inhibited the autophagy of epithelial cells. Both rHBHA and nHBHA had the same ability. It is one of the important mechanism in the course of M. tuberculosis infection.