The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host
The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host
复制标题
土拉弗朗西斯菌 LVS 的蛋白酶位点是哺乳动物宿主的应激耐受和感染所必需的
DOI：
10.1128/iai.00076-16
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发表时间：
2016-05-01
影响因子：
3.1
通讯作者：
Zhang, Jing-Ren
中科院分区：
文献类型：
--
作者：
He, Lihong;Nair, Manoj Kumar Mohan;Zhang, Jing-Ren
摘要 土拉弗朗西斯菌是土拉热病的病原体，也是 A 类潜在生物恐怖因子，但土拉弗朗西斯菌的致病机制尚不清楚。我们之前的转座子诱变筛选鉴定了 95 个与肺部感染相关的土拉弗朗西斯基因，包括编码 Lon 和 ClpP 蛋白酶的基因。本研究通过使用土拉弗氏菌活疫苗株 (LVS) 的未标记缺失突变体，验证了 Lon 和 ClpP 在巨噬细胞内生长和哺乳动物宿主感染中的重要性。进一步的实验表明，lon 和 clpP 也是 F. tularensis 对应激条件的耐受性所必需的。热应激 LVS 和同基因 Lon 缺陷突变体之间的定量蛋白质组比较鉴定出 29 种假定的 Lon 底物蛋白。后续蛋白质降解实验鉴定了土拉弗朗西斯 Lon 蛋白酶的五种底物（FTL578、FTL663、FTL1217、FTL1228 和 FTL1957）。 FTL578（鸟氨酸环脱氨酶）、FTL663（热休克蛋白）和 FTL1228（铁硫激活剂复合物亚基 SufD）先前已被描述为土拉弗拉菌的毒力相关因子。因此，这些 Lon 底物的鉴定为进一步了解土拉弗拉菌应激反应和发病机制提供了重要线索。本研究开发的高通量方法可用于系统鉴定其他原核和真核生物中的 Lon 底物。
ABSTRACT Francisella tularensis is the causative agent of tularemia and a category A potential agent of bioterrorism, but the pathogenic mechanisms of F. tularensis are largely unknown. Our previous transposon mutagenesis screen identified 95 lung infectivity-associated F. tularensis genes, including those encoding the Lon and ClpP proteases. The present study validates the importance of Lon and ClpP in intramacrophage growth and infection of the mammalian host by using unmarked deletion mutants of the F. tularensis live vaccine strain (LVS). Further experiments revealed that lon and clpP are also required for F. tularensis tolerance to stressful conditions. A quantitative proteomic comparison between heat-stressed LVS and the isogenic Lon-deficient mutant identified 29 putative Lon substrate proteins. The follow-up protein degradation experiments identified five substrates of the F. tularensis Lon protease (FTL578, FTL663, FTL1217, FTL1228, and FTL1957). FTL578 (ornithine cyclodeaminase), FTL663 (heat shock protein), and FTL1228 (iron-sulfur activator complex subunit SufD) have been previously described as virulence-associated factors in F. tularensis. Identification of these Lon substrates has thus provided important clues for further understanding of the F. tularensis stress response and pathogenesis. The high-throughput approach developed in this study can be used for systematic identification of the Lon substrates in other prokaryotic and eukaryotic organisms.