Use of host derived lipoate by Listeria monocytogenes

描述（由申请人提供）：单核增生李斯特菌（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这样的病原体如何适应它们的新陈代谢在宿主体内成功生长的理解仍然很初级。本研究的目的是确定细菌在宿主细胞环境中复制的机制。已知只有两个基因参与Lm细胞内生长，hpt和lplA1。LplA1编码脂酸蛋白连接酶（lpl）；δ - lpla1突变体有250倍的毒力缺陷。Lpls以一种可用于有氧代谢的形式为细胞提供一种关键的辅助因子，硫辛酸（LA）。Lm有两个lpl基因lplA1和lplA2；它们在Lm发病机制中的功能重要性尚不清楚。先前的研究表明，宿主来源的LA （HDLA）对Lm的细胞内生长和毒力至关重要。我们假设LplA1对于HDLA的使用至关重要，而LplA2使用游离LA作为底物。本研究的重点是阐明Lm在感染过程中如何获得和使用LA。1)确定LplA1和LplA2在体外和宿主细胞生长中的作用。将分析缺乏lplA1、lplA2或两者都缺乏的菌株的细胞外和细胞内生长、毒力和抗氧化应激能力。2)确定LplA1和LplA2的底物和靶点。在体外生长的Lm使用不同的宿主衍生的底物将进行检查。此外，我们观察到的细菌蛋白仅在细胞内生长时脂化，将通过质谱法鉴定。3) HDLA的转运体将被识别和表征。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-lplA1表型的Lm突变体。总的来说，这些研究将揭示允许Lm利用宿主细胞生态位的基本适应性。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物，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.