Yang Shufeng, Zhang Fei, Kang Jian, Zhang Wenli, Deng Guoying, Xin Yi, Ma Yufang
Department of Biochemistry and Molecular Biology, Dalian Medical University, 9 W Lushun South Road, Dalian 116044, China.
BMC Microbiol. 2014 Jun 30;14:174. doi: 10.1186/1471-2180-14-174.
Many bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG) deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been identified.
In this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096 protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG deacetylase towards M. smegmatis PG as a substrate were as follows: Km, 0.910 ± 0.007 mM; Vmax, 0.514 ± 0.038 μMmin-1; and Kcat = 0.099 ± 0.007 (S-1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096 protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein, M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis.
We have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG may be involved in evasion of host immune defenses by M. tuberculosis.
许多细菌通过肽聚糖（PG）去乙酰化来调节和逃避宿主的免疫防御。肺炎链球菌、单核细胞增生李斯特菌和乳酸乳球菌的PG去乙酰化酶已得到表征。然而，迄今为止，结核分枝杆菌的PG去乙酰化酶尚未被鉴定。
在本研究中，我们从结核分枝杆菌H37Rv菌株中克隆了Rv1096基因，并在大肠杆菌和耻垢分枝杆菌中表达了Rv1096蛋白。结果表明，纯化的Rv1096蛋白具有金属依赖性PG去乙酰化酶活性，在Co2+存在下活性增加。以耻垢分枝杆菌PG为底物时，PG去乙酰化酶的动力学参数如下：Km，0.910±0.007 mM；Vmax，0.514±0.038 μMmin-1；Kcat = 0.099±0.007（S-1）。此外，在溶菌酶处理后，过表达Rv1096蛋白的情况下，耻垢分枝杆菌的活力比野生型耻垢分枝杆菌高109倍。另外，光学显微镜和扫描电子显微镜显示，在过表达Rv1096蛋白的情况下，耻垢分枝杆菌保持其规则形状，细胞壁未受损且表面光滑。这些结果表明，Rv1096导致细胞壁PG去乙酰化，从而使耻垢分枝杆菌产生溶菌酶抗性。
我们确定结核分枝杆菌Rv1096是一种PG去乙酰化酶。Rv1096的PG去乙酰化酶活性有助于耻垢分枝杆菌产生溶菌酶抗性。我们的研究结果表明，细胞壁PG去乙酰化可能参与结核分枝杆菌逃避宿主免疫防御的过程。
