Phenotypic Profiling of Mycobacterium tuberculosis EspA Point Mutants Reveals that Blockage of ESAT-6 and CFP-10 Secretion In Vitro Does Not Always Correlate with Attenuation of Virulence
Phenotypic Profiling of Mycobacterium tuberculosis EspA Point Mutants Reveals that Blockage of ESAT-6 and CFP-10 Secretion In Vitro Does Not Always Correlate with Attenuation of Virulence
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DOI：
10.1128/jb.00967-13
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发表时间：
2013-12-01
影响因子：
3.2
通讯作者：
Cole, Stewart T.
中科院分区：
文献类型：
--
作者：
Chen, Jeffrey M.;Zhang, Ming;Cole, Stewart T.
结核分枝杆菌的EspA蛋白对于递送主要毒力因子ESAT-6和CFP-10的VII型ESX-1蛋白分泌装置是必需的。在这项研究中，EspA的定点突变进行，以阐明其对ESX-1系统的影响。在EspA的W-X-G基序中，用甘氨酸取代Trp（55）（W55）或Gly（57）（G57）残基，在体外削弱ESAT-6和CFP-10的分泌，并减弱M.结核分别用精氨酸和丙氨酸取代W-X-G基序侧翼的Phe（50）（F50）和Lys（62）（K62）残基，使EspA不稳定，在体外消除ESAT-6和CFP-10分泌，并减弱M.结核同样地，分别用精氨酸和丙氨酸替换Phe（5）（F5）和Lys（41）（K41）残基也使EspA不稳定并在体外阻断ESAT-6和CFP-10分泌。然而，这两个特殊的突变并没有减弱M。在细胞感染模型中或在小鼠的急性感染期间，因此，我们已经鉴定了EspA中对于促进ESAT-6和CFP-10分泌和毒力重要的氨基酸残基。然而，我们的数据也首次表明，M。结核杆菌ESAT-6和CFP-10在体外的分泌和减毒是相互排斥的。
The EspA protein of Mycobacterium tuberculosis is essential for the type VII ESX-1 protein secretion apparatus, which delivers the principal virulence factors ESAT-6 and CFP-10. In this study, site-directed mutagenesis of EspA was performed to elucidate its influence on the ESX-1 system. Replacing Trp(55) (W55) or Gly(57) (G57) residues in the putative W-X-G motif of EspA with arginines impaired ESAT-6 and CFP-10 secretion in vitro and attenuated M. tuberculosis. Replacing the Phe(50) (F50) and Lys(62) (K62) residues, which flank the W-X-G motif, with arginine and alanine, respectively, destabilized EspA, abolished ESAT-6 and CFP-10 secretion in vitro, and attenuated M. tuberculosis. Likewise, replacing the Phe(5) (F5) and Lys(41) (K41) residues with arginine and alanine, respectively, also destabilized EspA and blocked ESAT-6 and CFP-10 secretion in vitro. However, these two particular mutations did not attenuate M. tuberculosis in cellular models of infection or during acute infection in mice. We have thus identified amino acid residues in EspA that are important for facilitating ESAT-6 and CFP-10 secretion and virulence. However, our data also indicate for the first time that blockage of M. tuberculosis ESAT-6 and CFP-10 secretion in vitro and attenuation are mutually exclusive.