Agarwal Nisheeth, Woolwine Samuel C, Tyagi Sandeep, Bishai William R
Department of Medicine, Johns Hopkins School of Medicine, CRB2 Room 1.08, 1550 Orleans Street, Baltimore, Maryland 21231-1044, USA.
Infect Immun. 2007 Jan;75(1):452-61. doi: 10.1128/IAI.01395-06. Epub 2006 Nov 6.
Alternate sigma factors have been implicated in the survival of mycobacteria in response to specific stresses. To characterize the role of SigM in Mycobacterium tuberculosis, a sigM deletion mutant was generated by allelic exchange in the virulent CDC1551 strain. Comparing the wild-type and Delta sigM strains by complete genomic microarray, we observed a low level of baseline expression of sigM in wild-type M. tuberculosis and no significant differences in the gene expression patterns between these two strains. Alternatively, a SigM-overexpressing M. tuberculosis strain was constructed and microarray profiling revealed SigM-dependent expression of a relatively small group of genes, which included four esat-6 homologues: esxE, esxF, esxT, and esxU. An assessment of SigM-dependent promoters from the microarray analysis revealed a putative consensus sequence for M. tuberculosis SigM of -35 GGAAC and -10 CGTCR. In vitro expression studies showed that M. tuberculosis sigM transcripts accumulate slightly in stationary phase and following heat shock. To understand the role of SigM in pathogenesis, the M. tuberculosis sigM deletion strain was compared with the isogenic wild-type strain and the complemented mutant strain for survival in murine macrophages and in the mouse model. The mutant was found to have similar abilities to survive in both the resting and activated J774A.1 macrophages. Mouse organ bacterial burdens indicated that the mutant proliferated and persisted at the same level as that of the wild-type and complemented strains in lung and spleen tissues. In time-to-death experiments in the mouse model, the Delta sigM mutant exhibited lethality times comparable to those observed for the wild-type and complemented strains. These data indicate that M. tuberculosis SigM governs the expression of a small set of genes, including four esat-6 homologues, and that the loss of sigM does not confer a detectable virulence defect in the macrophages and mouse models of infection.
交替的σ因子与分枝杆菌在应对特定应激时的存活有关。为了表征SigM在结核分枝杆菌中的作用，通过在强毒株CDC1551中进行等位基因交换构建了sigM缺失突变体。通过全基因组微阵列比较野生型和ΔsigM菌株，我们观察到野生型结核分枝杆菌中sigM的基线表达水平较低，并且这两种菌株之间的基因表达模式没有显著差异。另外，构建了过表达SigM的结核分枝杆菌菌株，微阵列分析显示一小部分基因的表达依赖于SigM，其中包括四个esat-6同源物：esxE、esxF、esxT和esxU。对微阵列分析中SigM依赖性启动子的评估揭示了结核分枝杆菌SigM的假定共有序列为-35 GGAAC和-10 CGTCR。体外表达研究表明，结核分枝杆菌sigM转录本在稳定期和热休克后略有积累。为了了解SigM在发病机制中的作用，将结核分枝杆菌sigM缺失菌株与同基因野生型菌株和互补突变菌株在小鼠巨噬细胞和小鼠模型中的存活情况进行了比较。发现该突变体在静止和活化的J774A.1巨噬细胞中具有相似的存活能力。小鼠器官细菌载量表明，该突变体在肺和脾组织中的增殖和持续水平与野生型和互补菌株相同。在小鼠模型的致死时间实验中，ΔsigM突变体的致死时间与野生型和互补菌株相当。这些数据表明，结核分枝杆菌SigM调控一小部分基因的表达，包括四个esat-6同源物，并且sigM的缺失在巨噬细胞和感染小鼠模型中未导致可检测到的毒力缺陷。
