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目的将肝素结合血凝素(HBHA)重组表达于耻垢分枝杆菌(MS),鉴定其对A549细胞自噬作用的影响。方法将HBHA克隆于穿梭表达质粒pMV261,构建HBHA重组载体,电转化至MS;感染A549细胞,Western blot法鉴定A549细胞微管相关蛋白轻链3(LC3)的表达情况,并检测LC3Ⅱ/LC3Ⅰ比值的变化可估计自噬的水平;利用单丹磺酰尸胺(MDC)将自噬体染色后,激光共聚焦观察细胞内自噬体变化的情况;通过MS、rMS-HBHA感染A549细胞18 h,计算胞内活菌数,鉴定感染状况。结果 rMS-HBHA经42℃热诱导成功表达HBHA蛋白;相对野生型MS,重组表达HBHA蛋白的MS(rMS-HBHA)可以显著抑制A549细胞中的LC3Ⅰ和LC3Ⅱ的表达,rMS-HBHA组的LC3Ⅱ/LC3Ⅰ比值为0.625显著低于MS组2.025,表明rMS-HBHA组自噬体形成受到抑制;并且rMS-HBHA感染A549细胞18 h后胞内活菌为6.3×106,明显高于MS组1×105。结论外源性HBHA可以通过抑制A549细胞的自噬作用增强MS的感染能力。
Objective To recombinantly express Heparin-binding hemagglutinin (HBHA) in Mycobacterium smegmatis (MS) and identify its effect on the autophagy of A549 cells. Methods HBHA was cloned into the shuttle plasmid pMV261 to construct HBHA recombinant vector and electrotransformed into MS. A549 cells were infected with HBHA and the expression of LC3 Ⅱ / LC3 Ⅰ was detected by Western blot. The changes of autophagy were assessed by autophagy staining. Autophagosomes were stained with dansyl cadaverine (MDC) and the changes of autophagosomes were observed by laser confocal laser scanning. A549 cells were infected by MS or rMS-HBHA for 18 h , Calculate the intracellular viable count and identify the infection status. Results HBMS protein was successfully induced by heat-induced at 42 ℃ in rMS-HBHA. Compared with wild-type MS, rMS-HBHA recombinantly expressing HBHA protein could significantly inhibit the expression of LC3Ⅰ and LC3Ⅱ in A549 cells. The ratio of LC3Ⅰwas 0.625, which was significantly lower than that of MS group (2.025), indicating that autophagosome formation was inhibited in rMS-HBHA group. The viable cell count of rMS-HBHA-infected A549 cells after 18 h was 6.3 × 106, significantly higher than that of MS group . Conclusion Exogenous HBHA can enhance the infection ability of MS by inhibiting the autophagy of A549 cells.