结核分枝杆菌蛋白Rv3425对细菌表型及毒力影响的研究

The effect of Mycobacterium tuberculosis protein Rv3425 in Mycobacterium smegmatis

为探索蛋白Rv3425在结核分枝杆菌(Mycobacterium tuberculosis,M.tuberculosis)中的功能,本研究以耻垢分枝杆菌(Mycobacterium smegmatis,M.smegmatis)为模式菌株,构建重组了耻垢分枝杆菌Ms-Rv3425。分别将构建菌株(Ms-Rv3425)、野生株(Ms)及空载对照(Ms-Pact)接种于7H9-OADC培养基中37℃培养,观察Ms-Rv3425与Ms及Ms-Pact之间在生长速率、菌落形态、生物膜以及聚集度方面的差异。分别用低pH值以及含有十二烷基磺酸钠(sodium dodecyl sulfate,SDS)、氨苄西林、异烟肼及利福平的培养基进行培养,计算存活率以分析抗逆和抗药能力;用上述压力条件培养结核分枝杆菌标准株H37Ra,分析Rv3425内源表达量的变化;进行THP-1细胞感染和BALB/c小鼠攻毒实验分析菌株的毒性变化。结果显示,与Ms及Ms-Pact相比,Ms-Rv3425的菌落形态更为粗糙且隆起,成膜及聚集能力增强;在压力条件下,Ms-Rv3425表现出更高的抗逆和抗药能力,H37Ra中Rv3425的表达量也显著上调;胞内存活率及小鼠致死率更高,各脏器病理损伤更为严重。综上所述,过表达Rv3425能够改变耻垢分枝杆菌的表型,提高抗逆性、抗药性和毒力。深入探讨PPE家族蛋白Rv3425的功能,将为结核病的防治带来新的视角。

Rv3425 encodes the PE/PPE family protein PPE57,which is a candidate antigen for vaccine construction that was tested in mice and rhesus monkeys in early studies of this laboratory.To study the function of Rv3425 of Mycobacterium tuberculosis(M.tuberculosis),Mycobacterium smegmatis(M.smegmatis)was selected as a model strain,and the recombinant strain Ms-Rv3425 was constructed.Compared with wild type,the colony morphology of Ms-Rv3425 was rougher and more bulging,and the ability of pellicle and aggregate formation was stronger.Ms-Rv3425 showed higher resistance to stress when cultured in acidic,sodium dodecyl sulfate(SDS),ampicillin,isoniazid,and rifampin conditions.The expression level of Rv3425 in the attenuated strain of M.tuberculosis H37 Ra cultured under these adverse conditions was also significantly up-regulated.Infection of macrophages showed that Ms-Rv3425 enhanced the intracellular survival of M.smegmatis in THP-1 cell line.It led to higher mortality in BALB/c mice,and the bacterial retention and pathological damage of each organ were also higher than that of the control group.In summary,over expression of Rv3425 in M.smegmatis enhanced stress resistance,drug resistance and virulence.