Use of host derived lipoate by Listeria monocytogenes

描述（申请方提供）：单核细胞增生李斯特菌（Lm）是一种食源性胞内病原体。每年约有2500例淋病，死亡率为20- 30%。我们对Lm这样的病原体如何适应它们的新陈代谢以在宿主中成功生长的理解仍然是初步的。本研究的目的是确定细菌在宿主细胞环境中复制的机制。已知只有两个基因有助于Lm细胞内生长，hpt和lplA 1。LplA 1编码一种硫辛酸蛋白连接酶（lpl）; delta-lplA 1突变体具有250倍的毒力缺陷。LPL以可用于有氧代谢的形式向细胞提供关键辅因子，硫辛酸（LA）。Lm有两个lpl基因，lplA 1和lplA 2;关于它们在Lm发病机制中的功能重要性知之甚少。以往的研究表明，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LplA 1对于HDLA的使用是关键的，而LplA 2使用游离LA作为底物。该提案的重点是阐明Lm在感染期间如何获得和使用LA。1)将确定LplA 1和LplA 2在体外生长和宿主细胞中的作用。将分析lplA 1、lplA 2或两者缺陷的菌株的细胞外和细胞内生长、毒力和对氧化应激的抗性。2)将鉴定LplAl和LplA 2的底物和靶标。将检查使用不同宿主来源的底物的Lm的体外生长。此外，我们观察到的细菌蛋白质仅在细胞内生长期间被脂酰化，将通过质谱法鉴定。3)将对HDLA转运蛋白进行鉴定和表征。HDLA的转运机制尚不清楚。经过验证的遗传选择将用于鉴定具有类似delta-lplA 1表型的Lm突变体。总的来说，这些研究将揭示允许Lm利用宿主细胞小生境的必要适应。此外，Lm突变体的表征是有缺陷的细胞内生长将提供新的候选人的活疫苗的发展，并可能确定新的目标，抗微生物治疗。

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.