Use of host derived lipoate by Listeria monocytogenes

单核细胞增生李斯特菌使用宿主来源的硫辛酸

基本信息

批准号：
7590330
负责人：
Mary O'Riordan
金额：
$ 25.44万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-04-15 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7590330
关键词：
Animals Attenuated Vaccines Bacillus subtilis Bacteria Bacterial Proteins Biological Assay Biological Models Candidate Disease Gene Cell Respiration Cells Complement Cytosol Defect Disease Environment Enzymes Epitopes Eukaryota Exhibits Genes Goals Growth Immunocompromised Host In Vitro Individual Infection Laboratories Laboratory culture Life Ligase Ligase Gene Listeria monocytogenes Listeriosis Mass Spectrum Analysis Metabolic Metabolism Modeling Molecular Mutation Oxidative Stress Pathogenesis Phenotype Predisposition Pregnant Women Prokaryotic Cells Proteins Research Personnel Resistance Role Soil Testing Therapeutic Thioctic Acid Time Virulence Work antimicrobial base extracellular foodborne genetic selection interest lipoate lipoate-protein ligase mortality mouse model mutant pathogen pathogenic bacteria prevent programs research study vaccine development

项目摘要

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.

描述（由申请人提供）：单核细胞增生李斯特氏菌（Lm）是一种食源性细胞内病原体。每年约有 2500 例李斯特菌病病例，死亡率为 20-30%。我们对 Lm 等病原体如何调整其新陈代谢以在宿主体内成功生长的理解仍然很初级。这项研究的目的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有助于 Lm 细胞内生长：hpt 和 lplA1。 LplA1 编码硫辛酸蛋白连接酶 (lpl)； delta-lplA1 突变体的毒力缺陷是原来的 250 倍。 Lpls 以可用于有氧代谢的形式为细胞提供关键的辅助因子硫辛酸 (LA)。 Lm有两个lpl基因，lplA1和lplA2；人们对它们在 Lm 发病机制中的功能重要性知之甚少。先前的工作表明，宿主衍生的 LA (HDLA) 对于 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。该提案的重点是阐明 Lm 在感染过程中如何获取和使用 LA。 1) 将确定LplA1和LplA2在体外和宿主细胞生长中的作用。将分析缺乏 lplA1、lplA2 或两者的菌株的细胞外和细胞内生长、毒力和对氧化应激的抵抗力。 2) 将鉴定LplA1和LplA2的底物和靶标。将检查使用不同宿主衍生底物的 Lm 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硫辛酰化，将通过质谱法进行鉴定。 3) HDLA 的转运蛋白将被鉴定和表征。 HDLA 的运输机制尚不清楚。经过验证的遗传选择将用于鉴定具有 delta-lplA1 等表型的 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性。此外，细胞内生长缺陷的 Lm 突变体的表征将为活疫苗开发提供新的候选者，并可能确定抗微生物治疗的新靶点。