Li Ming-Shi, Monahan Irene M, Waddell Simon J, Mangan Joseph A, Martin Steve L, Everett Martin J, Butcher Philip D
Department of Medical Microbiology, St George's Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, UK1.
Glaxo Wellcome Research and Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK2.
Microbiology (Reading). 2001 Aug;147(Pt 8):2293-2305. doi: 10.1099/00221287-147-8-2293.
Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell-intracellular pathogen interactions. To identify such genes a cDNA-total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds in total bacterial RNA. The subtraction products were used to screen a high-density gridded Mycobacterium tuberculosis genomic library. Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase (mas). Mas is an enzyme involved in the synthesis of multi-methylated long-chain fatty acids that are part of phthiocerol dimycocerosate, a major component of the complex mycobacterial cell wall. Northern blotting and primer extension data confirmed up-regulation of mas in intracellular mycobacteria and also revealed a putative extended -10 promoter structure and a long untranslated upstream region 5' of the mas transcripts, containing predicted double-stranded structures. Furthermore, clones containing overlapping sequences for furB, groEL-2, rplE and fadD28 were identified and the up-regulation of these genes was confirmed by Northern blot analysis. The cDNA-RNA subtractive hybridization enrichment and high density gridded library screening, combined with selective extraction of bacterial mRNA represents a valuable approach to the identification of genes expressed during intra-macrophage residence for bacteria such as M. bovis BCG and the pathogenic mycobacterium, M. tuberculosis.
鉴定经巨噬细胞吞噬后牛分枝杆菌卡介苗差异表达的基因，将有助于理解宿主细胞与细胞内病原体相互作用的分子机制。为了鉴定此类基因，已采用一种cDNA-总RNA消减杂交策略，该策略规避了感染模型中细菌RNA可用性有限以及细菌总RNA中高rRNA背景这两个问题。消减产物用于筛选高密度网格化的结核分枝杆菌基因组文库。从19个差异克隆中获得了序列数据，其中5个包含编码霉菌酸合酶（mas）基因的重叠序列。Mas是一种参与多甲基化长链脂肪酸合成的酶，多甲基化长链脂肪酸是结核菌酸二霉菌酸酯的一部分，结核菌酸二霉菌酸酯是分枝杆菌复杂细胞壁的主要成分。Northern印迹和引物延伸数据证实了mas在细胞内分枝杆菌中的上调，还揭示了一个推定的延伸-10启动子结构以及mas转录本5'端的一个长的非翻译上游区域，该区域包含预测的双链结构。此外，鉴定出了包含furB、groEL-2、rplE和fadD28重叠序列的克隆，并通过Northern印迹分析证实了这些基因的上调。cDNA-RNA消减杂交富集和高密度网格化文库筛选，结合细菌mRNA的选择性提取，是鉴定牛分枝杆菌卡介苗和致病性分枝杆菌结核分枝杆菌等细菌在巨噬细胞内驻留期间表达的基因的一种有价值的方法。
