Functional analysis of the LipA phosphatase, a new virulence factor of Listeria monocytogene

单核增生李斯特菌是一种兼性细胞内革兰氏阳性细菌，负责李斯特菌病。这种细菌，像其他细胞内病原体一样，通过产生多种毒力因子来操纵宿主细胞功能。单核增生乳杆菌在感染过程中表达一种名为LipA的分泌磷酸酶。我们之前的研究表明，在体外无细胞系统中，LipA使磷酸酪氨酸和磷脂酰肌醇磷酸（PIP）去磷酸化，并且体内缺乏LipA的细菌（ΔlipA）的毒力受到严重损害，而巨噬细胞或上皮细胞中ΔlipA单核增生乳杆菌突变体的生命周期保持完整。本应用旨在(1)确定感染过程中受LipA影响的细胞类型；(2)鉴定受感染宿主细胞中LipA的底物。为此，我们将检验（1a） LipA通过抑制pip依赖性NADPH氧化酶组装而损害中性粒细胞氧化爆发的假设；（1b） LipA对其他先天免疫细胞功能的影响；（2a） LipA在感染宿主细胞中靶向PIPs的识别和定位；（2b） LipA对宿主细胞蛋白磷酸化水平的影响。该项目不仅将提高我们对单核增生乳杆菌逃避免疫反应机制的理解，而且有可能定义一类新的李斯特菌毒力因子，这些毒力因子可能适合作为药物靶点。

Listeria monocytogenes is a facultative intracellular Gram-positive bacterium responsible for listeriosis. This bacterium, like other intracellular pathogens, manipulates host cell functions by producing diverse virulence factors. L. monocytogenes expresses a secreted phosphatase, named LipA, specifically during infection. We previously showed that LipA dephosphorylated phosphotyrosines as well as phosphatidylinositol phosphates (PIP) in cell-free in vitro systems and that the virulence of bacteria deficient for LipA (ΔlipA) was strongly impaired in vivo, whereas the life cycle of ΔlipA L. monocytogenes mutants in macrophages or epithelial cells remained intact. This application aims to (1) define the cell types affected by LipA during infection and (2) to identify the substrates of LipA in infected host cells. To this end, we will test (1a) the hypothesis that LipA impairs the oxidative burst of neutrophils by inhibiting the PIP-dependent assembly of NADPH oxidase; (1b) the impact of LipA on the function of other innate immune cells; (2a) the identity and localization of PIPs targeted by LipA in infected host cells; (2b) the effect of LipA on the level of host cell protein phosphorylation. The project will not only improve our understanding of the mechanisms developed by L. monocytogenes to escape the immune response, but also has the potential to define a novel class of listerial virulence factors which could be suitable as drug targets.