Reniere Michelle L, Whiteley Aaron T, Portnoy Daniel A
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, United States of America.
Graduate Group in Infectious Diseases and Immunity, School of Public Health, University of California, Berkeley, Berkeley, California, United States of America.
PLoS Pathog. 2016 Jul 14;12(7):e1005741. doi: 10.1371/journal.ppat.1005741. eCollection 2016 Jul.
Listeria monocytogenes is an environmental saprophyte and facultative intracellular bacterial pathogen with a well-defined life-cycle that involves escape from a phagosome, rapid cytosolic growth, and ActA-dependent cell-to-cell spread, all of which are dependent on the master transcriptional regulator PrfA. The environmental cues that lead to temporal and spatial control of L. monocytogenes virulence gene expression are poorly understood. In this study, we took advantage of the robust up-regulation of ActA that occurs intracellularly and expressed Cre recombinase from the actA promoter and 5' untranslated region in a strain in which loxP sites flanked essential genes, so that activation of actA led to bacterial death. Upon screening for transposon mutants that survived intracellularly, six genes were identified as necessary for ActA expression. Strikingly, most of the genes, including gshF, spxA1, yjbH, and ohrA, are predicted to play important roles in bacterial redox regulation. The mutants identified in the genetic selection fell into three broad categories: (1) those that failed to reach the cytosolic compartment; (2) mutants that entered the cytosol, but failed to activate the master virulence regulator PrfA; and (3) mutants that entered the cytosol and activated transcription of actA, but failed to synthesize it. The identification of mutants defective in vacuolar escape suggests that up-regulation of ActA occurs in the host cytosol and not the vacuole. Moreover, these results provide evidence for two non-redundant cytosolic cues; the first results in allosteric activation of PrfA via increased glutathione levels and transcriptional activation of actA while the second results in translational activation of actA and requires yjbH. Although the precise host cues have not yet been identified, we suggest that intracellular redox stress occurs as a consequence of both host and pathogen remodeling their metabolism upon infection.
单核细胞增生李斯特菌是一种环境腐生菌和兼性胞内细菌病原体，具有明确的生命周期，包括从吞噬体逃逸、在胞质中快速生长以及依赖肌动蛋白聚合蛋白A（ActA）的细胞间传播，所有这些都依赖于主要转录调节因子PrfA。导致单核细胞增生李斯特菌毒力基因表达的时空控制的环境信号尚不清楚。在本研究中，我们利用细胞内发生的ActA的强烈上调，在一个loxP位点位于必需基因两侧的菌株中，从actA启动子和5'非翻译区表达Cre重组酶，这样actA的激活会导致细菌死亡。在筛选细胞内存活的转座子突变体时，鉴定出六个基因是ActA表达所必需的。引人注目的是，大多数基因，包括gshF、spxA1、yjbH和ohrA，预计在细菌氧化还原调节中起重要作用。在遗传筛选中鉴定出的突变体分为三大类：（1）那些未能到达胞质区室的；（2）进入胞质但未能激活主要毒力调节因子PrfA的突变体；（3）进入胞质并激活actA转录但未能合成它的突变体。液泡逃逸缺陷突变体的鉴定表明，ActA的上调发生在宿主胞质中而不是液泡中。此外，这些结果为两种非冗余的胞质信号提供了证据；第一种通过增加谷胱甘肽水平导致PrfA的变构激活和actA的转录激活，而第二种导致actA的翻译激活并需要yjbH。尽管尚未确定确切的宿主信号，但我们认为细胞内氧化还原应激是宿主和病原体在感染时重塑其代谢的结果。
