Dubnau Eugenie, Fontán Patricia, Manganelli Riccardo, Soares-Appel Sonia, Smith Issar
TB Center, The Public Health Research Institute, Newark, New Jersey 07103, USA.
Infect Immun. 2002 Jun;70(6):2787-95. doi: 10.1128/IAI.70.6.2787-2795.2002.
We identified Mycobacterium tuberculosis genes preferentially expressed during infection of human macrophages using a promoter trap adapted for this pathogen. inhA encodes an enoyl-acyl carrier protein reductase that is required for mycolic acid biosynthesis (A. Quemard et al., Biochemistry 34:8235-8241, 1995) and is a major target for isoniazid (INH) in mycobacterial species (A. Banerjee et al., Science 263:227-230, 1994). Since overexpression of inhA confers INH resistance in Mycobacterium smegmatis (Banerjee et al., Science 263:227-230, 1994), we designed a promoter trap based on this gene. A library of clones, containing small fragments of M. tuberculosis DNA cloned upstream of inhA in a plasmid vector, was electroporated into M. tuberculosis, and the resulting culture was used to infect the human monocytic THP-1 cell line. Selection was made for clones surviving INH treatment during infection but retaining INH sensitivity on plates. The DNA upstream of inhA was sequenced in each clone to identify the promoter driving inhA expression. Thirteen genes identified by this method were analyzed by quantitative reverse transcription-PCR (R. Manganelli et al., Mol. Microbiol. 31:715-724, 1999), and eight of them were found to be differentially expressed from cultures grown in macrophages compared with broth-grown cultures. Several of these genes are presumed to be involved in fatty acid metabolism; one potentially codes for a unique DNA binding protein, one codes for a possible potassium channel protein, and the others code for proteins of unknown function. Genes which are induced during infection are likely to be significant for survival and growth of the pathogen; our results lend support to the view that fatty acid metabolism is essential for the virulence of M. tuberculosis.
我们利用适用于该病原体的启动子捕获技术，鉴定了在人类巨噬细胞感染期间优先表达的结核分枝杆菌基因。inhA编码一种烯酰-酰基载体蛋白还原酶，该酶是分枝菌酸生物合成所必需的（A. Quemard等人，《生物化学》34:8235 - 8241，1995年），并且是分枝杆菌属中异烟肼（INH）的主要作用靶点（A. Banerjee等人，《科学》263:227 - 230，1994年）。由于inhA的过表达赋予耻垢分枝杆菌对INH的抗性（Banerjee等人，《科学》263:227 - 230，1994年），我们基于该基因设计了一种启动子捕获技术。将一个克隆文库（包含在质粒载体中克隆于inhA上游的结核分枝杆菌DNA小片段）电穿孔导入结核分枝杆菌，然后用所得培养物感染人类单核细胞THP - 1细胞系。在感染期间选择在INH处理下存活但在平板上仍保持对INH敏感性的克隆。对每个克隆中inhA上游的DNA进行测序，以鉴定驱动inhA表达的启动子。通过定量逆转录PCR分析了用这种方法鉴定出的13个基因（R. Manganelli等人，《分子微生物学》31:715 - 724，1999年），发现其中8个基因在巨噬细胞中生长的培养物与在肉汤中生长的培养物相比存在差异表达。这些基因中的几个被推测参与脂肪酸代谢；一个可能编码一种独特的DNA结合蛋白，一个编码一种可能的钾通道蛋白，其他的编码功能未知的蛋白。在感染期间被诱导的基因可能对病原体的存活和生长至关重要；我们的结果支持了脂肪酸代谢对结核分枝杆菌毒力至关重要的观点。
