Maciag Anna, Piazza Aurora, Riccardi Giovanna, Milano Anna
Department of Genetics and Microbiology, University of Pavia, Pavia, Italy.
BMC Microbiol. 2009 Mar 4;9:48. doi: 10.1186/1471-2180-9-48.
The ESAT-6 (early secreted antigenic target, 6 kDa) family collects small mycobacterial proteins secreted by Mycobacterium tuberculosis, particularly in the early phase of growth. There are 23 ESAT-6 family members in M. tuberculosis H37Rv. In a previous work, we identified the Zur- dependent regulation of five proteins of the ESAT-6/CFP-10 family (esxG, esxH, esxQ, esxR, and esxS). esxG and esxH are part of ESAT-6 cluster 3, whose expression was already known to be induced by iron starvation.
In this research, we performed EMSA experiments and transcriptional analysis of ESAT-6 cluster 3 in Mycobacterium smegmatis (msmeg0615-msmeg0625) and M. tuberculosis. In contrast to what we had observed in M. tuberculosis, we found that in M. smegmatis ESAT-6 cluster 3 responds only to iron and not to zinc. In both organisms we identified an internal promoter, a finding which suggests the presence of two transcriptional units and, by consequence, a differential expression of cluster 3 genes. We compared the expression of msmeg0615 and msmeg0620 in different growth and stress conditions by means of relative quantitative PCR. The expression of msmeg0615 and msmeg0620 genes was essentially similar; they appeared to be repressed in most of the tested conditions, with the exception of acid stress (pH 4.2) where msmeg0615 was about 4-fold induced, while msmeg0620 was repressed. Analysis revealed that in acid stress conditions M. tuberculosis rv0282 gene was 3-fold induced too, while rv0287 induction was almost insignificant.
In contrast with what has been reported for M. tuberculosis, our results suggest that in M. smegmatis only IdeR-dependent regulation is retained, while zinc has no effect on gene expression. The role of cluster 3 in M. tuberculosis virulence is still to be defined; however, iron- and zinc-dependent expression strongly suggests that cluster 3 is highly expressed in the infective process, and that the cluster contributes to the antigenic profile during the course of infection. Moreover, cluster 3 induction in acid stress conditions strengthens the hypothesis that cluster 3 is expressed in the course of infection.In M. smegmatis, the expression of msmeg0615 and msmeg0620 genes is broadly similar in differing growth phases and in stress conditions, with the exception of acid stress (pH 4.2). Differences in expression between cluster 3 genes can be explained by the presence of internal promoters, both in M. smegmatis and M. tuberculosis.
ESAT-6（早期分泌抗原靶标，6 kDa）家族包含结核分枝杆菌分泌的小分子蛋白质，尤其是在生长早期阶段。结核分枝杆菌H37Rv中有23个ESAT-6家族成员。在之前的研究中，我们确定了Zur对ESAT-6/CFP-10家族的五种蛋白质（esxG、esxH、esxQ、esxR和esxS）的依赖性调控。esxG和esxH是ESAT-6簇3的一部分，已知其表达受铁饥饿诱导。
在本研究中，我们对耻垢分枝杆菌（msmeg0615-msmeg0625）和结核分枝杆菌中的ESAT-6簇3进行了电泳迁移率变动分析（EMSA）实验和转录分析。与我们在结核分枝杆菌中观察到的情况相反，我们发现耻垢分枝杆菌中的ESAT-6簇3仅对铁有反应，而对锌无反应。在这两种生物体中，我们都鉴定出了一个内部启动子，这一发现表明存在两个转录单元，因此簇3基因存在差异表达。我们通过相对定量PCR比较了msmeg0615和msmeg0620在不同生长和应激条件下的表达。msmeg0615和msmeg0620基因的表达基本相似；它们在大多数测试条件下似乎受到抑制，酸应激（pH 4.2）除外，在酸应激条件下msmeg0615约被诱导4倍，而msmeg0620受到抑制。分析表明，在酸应激条件下，结核分枝杆菌rv0282基因也被诱导3倍，而rv0287的诱导几乎不明显。
与关于结核分枝杆菌的报道相反，我们的结果表明，在耻垢分枝杆菌中仅保留了IdeR依赖性调控，而锌对基因表达没有影响。簇3在结核分枝杆菌毒力中的作用仍有待确定；然而，铁和锌依赖性表达强烈表明簇3在感染过程中高度表达，并且该簇在感染过程中有助于抗原谱。此外，酸应激条件下簇3的诱导强化了簇3在感染过程中表达的假设。在耻垢分枝杆菌中，msmeg0615和msmeg0620基因的表达在不同生长阶段和应激条件下大致相似，酸应激（pH 4.2）除外。簇3基因之间的表达差异可以通过耻垢分枝杆菌和结核分枝杆菌中都存在内部启动子来解释。
Zotero 插件
