结核分枝杆菌核糖体蛋白丙氨酸乙酰转移酶过表达菌株的构建及分析

rimI基因编码的核糖体蛋白丙氨酸乙酰转移酶(ribosomal-protein-alanine acetyltransferase，RimI)为结核分枝杆菌GCN5相关N-乙酰转移酶家族成员，其在结核分枝杆菌中的生物学功能尚不十分清楚。为探索RimI的生物学特性及其对结核分枝杆菌致病性的影响，本研究以耻垢分枝杆菌为模式菌，构建过表达结核分枝杆菌rimI基因的重组菌株Msm∷pMV261-rimI。分别培养 Msm∷pMV261-rimI菌株和对照Msm∷pMV261菌株，分析两者生长速率、菌落形态和生物膜形成的差异，以及耐受低氧、低pH值、H₂O₂、二硫苏糖醇(dithiothreitol，DTT)和0.05%～1%十二烷基硫酸钠(sodium dodecyl sulfate，SDS)等逆环境的能力；并将两种菌株分别接种于鼠巨噬细胞RAW264.7，观察两者在巨噬细胞内的存活能力。结果表明，相较于对照菌株，过表达rimI的菌株在生长前中期速率降低，生物膜早期成膜变缓，但不影响生物膜的后期成熟。同时，过表达rimI的菌株抵抗低氧、低pH值、H₂O₂等逆环境的能力增强，在巨噬细胞内的存活能力增强。结果提示，rimI基因对分枝杆菌的生物膜形成、抗逆性及细胞内生存具有重要作用，可能与结核分枝杆菌的毒力密切相关。

Biological characteristics of a recombinant strain overexpressing ribosomal-protein-alanine acetyltransferase from Mycobacterium tuberculosis

Ribosomal-protein-alanine acetyltransferase (RimI) is a member of GCN5-related N-acetyltransferase family of Mycobacterium tuberculosis (M.tb) which is encoded by rimI gene. The biological function of RimI in M．tb is still not clear. In order to explore the biological characteristics of RimI and its role in the virulence of M．tb, Mycobacterium smegmatis (M.sm) was selected as a model strain to construct a recombinant strain Msm∷pMV261-rimI. The difference in growth rate, colony morphology, and biofilm formation between Msm∷pMV261 and Msm∷pMV261-rimI were analyzed in vitro. Their resistance to hypoxia, acidic pH, dithiothreitol (DTT), H₂O_2, sodium dodecyl sulfate (SDS) and intracellular survival ability during the infection of macrophages in Raw264.7 cell line were explored. Compared with the Msm∷pMV261 strain, the growth curve and biofilm forming of the Msm∷pMV261-rimI strain were slowed down in the early and middle growth stages, but the late maturity of biofilm was not affected. Moreover, the Msm∷pMV261-rimI strain not only increased its resistance to acidic or hypoxic stress, its intracellular survival ability was also enhanced during the infection of macrophages in Raw264.7 cell line. Altogether, the results indicated that the rimI gene played an important role in biofilm formation, stress resistance and intracellular survival of M．tb, suggesting that rimI gene may be a virulence factor related to the pathogenicity of M．tb.