Use of host derived lipoate by Listeria monocytogenes

描述(申请人提供)：单核细胞增生性李斯特菌(Lm)是一种食源性细胞内病原体。每年约有2500例李斯特菌病，死亡率为20-30%。我们对像LM这样的病原体如何调整其新陈代谢以在宿主中成功生长的了解仍处于初级阶段。这项研究的目标是确定细菌在宿主细胞环境中复制的机制。目前已知只有两个基因对LM细胞内生长有贡献，即hpt和lplA1。LplA1编码一种脂酸盐蛋白连接酶(LPL)；Delta-LplA1突变体具有250倍的毒力缺陷。脂多糖为细胞提供了一种关键的辅因子--硫辛酸(LA)，为有氧代谢提供了可用的形式。Lm有两个LPL基因，lplA1和lplA2；对它们在Lm发病机制中的功能重要性知之甚少。以往的工作表明，宿主来源的LA(HDLA)对LM的细胞内生长和毒力是必不可少的。我们假设LplA1是使用HDLA的关键，而LplA2使用游离LA作为底物。这项建议侧重于阐明在感染过程中LM是如何获得和使用LA的。1)LplA1和LplA2在体外和宿主细胞生长中的作用将被确定。缺乏lplA1和/或lplA2的菌株将被分析细胞外和细胞内的生长、毒力和对氧化应激的抵抗力。2)将确定LplA1和LplA2的底物和靶点。将检测使用不同宿主衍生底物的LM的体外生长情况。此外，我们观察到的细菌蛋白质只有在细胞内生长过程中才被脂基化，这一点将通过质谱学进行鉴定。3)将确定和表征HDLA的转运体。HDLA的转运机制尚不清楚。有效的遗传选择将用于鉴定具有类似Delta-lplA1表型的LM突变体。总体而言，这些研究将揭示允许LM利用宿主细胞生态位的基本适应。此外，细胞内生长缺陷的LM突变体的特征将为活疫苗的开发提供新的候选者，并可能确定抗微生物治疗的新靶点。

DESCRIPTION (provided by applicant): Listeria monocytogenes (Lm) is a foodborne intracellular pathogen. There are ~2500 cases of listeriosis/yr and the mortality rate is 20-30%. Our understanding of how pathogens like Lm adapt their metabolism to grow successfully in a host is still rudimentary. The goal of this study is to define mechanisms by which bacteria replicate in the host cell environment. Only two genes are known to contribute to Lm intracellular growth, hpt and lplA1. LplA1 encodes a lipoate protein ligase (lpl); delta-lplA1 mutants have a 250-fold virulence defect. Lpls provide a critical co-factor, lipoic acid (LA), to the cell in a usable form for aerobic metabolism. Lm has two lpl genes, lplA1 and lplA2; little is known about their functional importance in Lm pathogenesis. Previous work showed that host derived LA (HDLA) is essential for intracellular growth and virulence of Lm. We hypothesize that LplA1 is critical for use of HDLA while LplA2 uses free LA as a substrate. This proposal focuses on elucidating how Lm acquires and uses LA during infection. 1) The roles of LplA1 and LplA2 in growth in vitro and in host cells will be determined. Strains deficient in lplA1, lplA2 or both will be analyzed for extracellular and intracellular growth, virulence, and resistance to oxidative stress. 2) The substrates and targets for LplA1 and LplA2 will be identified. In vitro growth of Lm using different host derived substrates will be examined. In addition, bacterial proteins that we have observed are lipoylated only during intracellular growth will be identified by mass spectrometry. 3) The transporter for HDLA will be identified and characterized. The mechanism of transport of HDLA is unknown. A validated genetic selection will be used to identify Lm mutants that have a phenotype like delta-lplA1. Overall, these studies will reveal essential adaptations that permit Lm to exploit the host cell niche. In addition, the characterization of Lm mutants that are defective in intracellular growth will provide new candidates for live vaccine development and may identify new targets for anti-microbial therapeutics.