Definition of the first mannosylation step in phosphatidylinositol mannoside synthesis -: PimA is essential for growth of mycobacteria
Definition of the first mannosylation step in phosphatidylinositol mannoside synthesis -: PimA is essential for growth of mycobacteria
复制标题
DOI：
10.1074/jbc.m204060200
复制
发表时间：
2002-08-30
影响因子：
4.8
通讯作者：
Jackson, M
中科院分区：
文献类型：
--
作者：
Korduláková, J;Gilleron, M;Jackson, M
我们研究了pimA（Rv 2610 c）基因的功能，该基因位于结核分枝杆菌和耻垢分枝杆菌基因组中磷脂酰肌醇合酶基因附近，该基因编码一种参与磷脂酰肌醇甘露糖苷合成早期步骤的假定甘露糖基转移酶。建立了一种无细胞测定法，其中M.过表达pimA基因的耻垢病将来自GDP-[（14）C]Man的甘露糖掺入二酰化和三酰化磷脂酰肌醇单甘露糖苷中。此外，从大肠杆菌产生的重组PimA蛋白质的粗提取物合成二酰化磷脂酰肌醇单甘露糖苷从GDP-[（14）C]Man和牛磷脂酰肌醇。为了确定PimA是否是分枝杆菌的必需酶，我们构建了分枝杆菌pimA条件突变体。恶臭该突变体合成PimA甘露糖基转移酶的能力依赖于温度敏感性拯救质粒上携带的pimA基因的功能性拷贝的存在。我们在这里证明了pimA突变体不能在较高的温度下生长，在该温度下，拯救质粒丢失。因此，M. smeglycine似乎依赖于PimA，并且对生长至关重要。这项工作提供了磷脂酰肌醇甘露糖苷对分枝杆菌生长的重要性的第一个直接证据。
We examined the function of the pimA (Rv2610c) gene, located in the vicinity of the phosphatidylinositol synthase gene in the genomes of Mycobacterium tuberculosis and Mycobacterium smegmatis, which encodes a putative mannosyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis. A cell-free assay was developed in which membranes from M. smegmatis overexpressing the pimA gene incorporate mannose from GDP-[(14)C]Man into di- and tri-acylated phosphatidylinositol mono-mannosides. Moreover, crude extracts from Escherichia coli producing a recombinant PimA protein synthesized diacylated phosphatidylinositol mono-mannoside from GDP-[(14)C]Man and bovine phosphatidylinositol. To determine whether PimA is an essential enzyme of mycobacteria, we constructed a pimA conditional mutant of M. smegmatis. The ability of this mutant to synthesize the PimA mannosyltransferase was dependent on the presence of a functional copy of the pimA gene carried on a temperature-sensitive rescue plasmid. We demonstrate here that the pimA mutant is unable to grow at the higher temperature at which the rescue plasmid is lost. Thus, the synthesis of phosphatidylinositol mono-mannosides and derived higher phosphatidylinositol mannosides in M. smegmatis appears to be dependent on PimA and essential for growth. This work provides the first direct evidence of the essentiality of phosphatidylinositol mannosides for the growth of mycobacteria.