Wu Shiping, Barnes Peter F, Samten Buka, Pang Xiuhua, Rodrigue Sébastien, Ghanny Saleena, Soteropoulos Patricia, Gaudreau Luc, Howard Susan T
Department of Microbiology and Immunology, Center for Pulmonary and Infectious Disease Control, University of Texas Health Science Center at Tyler, 11937 US Hwy 271, Tyler, TX 75708-3154, USA.
Départment of Medicine, University of Texas Health Science Center at Tyler, 11937 US Hwy 271, Tyler, TX 75708-3154, USA.
Microbiology (Reading). 2009 Apr;155(Pt 4):1272-1281. doi: 10.1099/mic.0.024638-0.
There is growing evidence that strains of Mycobacterium tuberculosis differ in pathogenicity and transmissibility, but little is understood about the contributory factors. We have previously shown that increased expression of the principal sigma factor, SigA, mediates the capacity of M. tuberculosis strain 210 to grow more rapidly in human monocytes, compared with other strains. Strain 210 is part of the widespread W-Beijing family of M. tuberculosis strains and includes clinical isolate TB294. To identify genes that respond to changes in SigA levels and that might enhance intracellular growth, we examined RNA and protein expression patterns in TB294-pSigA, a recombinant strain of TB294 that overexpresses sigA from a multicopy plasmid. Lysates from broth-grown cultures of TB294-pSigA contained high levels of Eis, a protein known to modulate host-pathogen interactions. DNA microarray analysis indicated that the eis gene, Rv2416c, was expressed at levels in TB294-pSigA 40-fold higher than in the vector control strain TB294-pCV, during growth in the human monocyte cell line MonoMac6. Other genes with elevated expression in TB294-pSigA showed much smaller changes from TB294-pCV, and the majority of genes with expression differences between the two strains had reduced expression in TB294-pSigA, including an unexpected number of genes associated with the DNA-damage response. Real-time PCR analyses confirmed that eis was expressed at very high levels in TB294-pSigA in monocytes as well as in broth culture, and further revealed that, like sigA, eis was also more highly expressed in wild-type TB294 than in the laboratory strain H37Rv, during growth in monocytes. These findings suggested an association between increased SigA levels and eis activation, and results of chromatin immunoprecipitation confirmed that SigA binds the eis promoter in live TB294 cells. Deletion of eis reduced growth of TB294 in monocytes, and complementation of eis reversed this effect. We conclude that SigA regulates eis, that there is a direct correlation between upregulation of SigA and high expression levels of eis, and that eis contributes to the enhanced capacity of a clinical isolate of M. tuberculosis strain 210 to grow in monocytes.
越来越多的证据表明，结核分枝杆菌菌株在致病性和传播性方面存在差异，但对其影响因素却知之甚少。我们之前已经表明，主要的σ因子SigA表达增加介导了结核分枝杆菌菌株210与其他菌株相比在人类单核细胞中更快生长的能力。菌株210是广泛存在的结核分枝杆菌W-北京家族的一部分，包括临床分离株TB294。为了鉴定对SigA水平变化有反应且可能增强细胞内生长的基因，我们检测了TB294-pSigA中的RNA和蛋白质表达模式，TB294-pSigA是TB294的重组菌株，它从多拷贝质粒上过表达sigA。来自TB294-pSigA肉汤培养物的裂解物含有高水平的Eis，一种已知可调节宿主-病原体相互作用的蛋白质。DNA微阵列分析表明，在人类单核细胞系MonoMac6中生长期间，eis基因（Rv2416c）在TB294-pSigA中的表达水平比载体对照菌株TB294-pCV高40倍。在TB294-pSigA中表达升高的其他基因与TB294-pCV相比变化要小得多，并且两株菌之间存在表达差异的大多数基因在TB294-pSigA中的表达降低，包括数量意外的与DNA损伤反应相关的基因。实时PCR分析证实，eis在单核细胞以及肉汤培养的TB294-pSigA中表达水平非常高，并且进一步揭示，与sigA一样，在单核细胞生长期间，野生型TB294中的eis表达也比实验室菌株H37Rv中的更高。这些发现表明SigA水平升高与eis激活之间存在关联，染色质免疫沉淀结果证实SigA在活的TB294细胞中结合eis启动子。缺失eis会降低TB294在单核细胞中的生长，而补充eis可逆转这种效应。我们得出结论，SigA调节eis，SigA上调与eis高表达水平之间存在直接相关性，并且eis有助于结核分枝杆菌菌株210的临床分离株在单核细胞中增强生长的能力。
