[Heparin-binding hemagglutinin enhances Mycobacterium smegmatis infection by inhibiting autophagy in A549 cells].

OBJECTIVE

To construct recombinant Mycobacterium smegmatis (MS) expressing heparin-binding hemagglutinin (HBHA) (rMS-HBHA) and identify its impact on autophagy in the A549 cells.

METHODS

The HBHA was cloned into the shuttle expression plasmid pMV261. The HBHA recombinant vector was constructed and electrotransformed into MS. Western blotting was performed to detect the expression level of the microtubule-associated protein light chain 3 (LC3) in A549 cells infected with rMS-HBHA, and the LC3-II/LC3I ratio was calculated to evaluate the level of autophagy. Then, autophagosomes were detected using monodansylcadaverine (MDC) staining and identified through laser confocal microscopy. Finally, the A549 cells that were infected with MS and rMS-HBHA for 18 hours were measured for the intracellular survival MS and rMS.

RESULTS

HBHA protein was successfully expressed in rMS-HBHA under 42 °C heat-induction. Compared with wild-type MS, rMS-HBHA significantly inhibited the expressions of LC3I and LC3II in A549 cells. And LC3II/LC3I ratio of the rMS-HBHA group (0.625) was significantly lower than that of the MS group (2.025), indicating that autophagosome formation of the rMS-HBHA group was inhibited. In addition, the intracellular survival rMS-HBHA (6.3 × 10⁶) was significantly higher than that of the MS group (1 × 10⁵) in the infected A549 cells after 18 hours.

CONCLUSION

The exogenous HBHA could enhance the infection ability of MS in A549 cells through inhibiting autophagy.