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

单核细胞增生李斯特菌是一种兼性胞内革兰氏阳性菌，可引起单核细胞增生。这种细菌，像其他细胞内病原体一样，通过产生不同的毒力因子来操纵宿主细胞功能。L.单核细胞增多症表达一种分泌的磷酸酶，命名为利帕，特别是在感染期间。我们以前的研究表明，利帕脱磷酸化的磷酸酪氨酸以及磷脂酰肌醇磷酸（PIP）在体外无细胞系统中，并且利帕（Δ利帕）缺陷的细菌的毒力在体内被严重削弱，而Δ利帕L的生命周期被破坏。巨噬细胞或上皮细胞中的单核细胞增多症突变体保持完整。本申请旨在（1）确定感染期间受利帕影响的细胞类型和（2）鉴定感染的宿主细胞中利帕的底物。为此，我们将检验（1a）利帕通过抑制NADPH氧化酶的PIP依赖性组装来损害中性粒细胞氧化爆发的假设;（1b）利帕对其他先天性免疫细胞功能的影响;（2a）感染宿主细胞中利帕靶向的PIP的身份和定位;（2b）利帕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L.这种方法不仅可以抑制单核细胞增多症以逃避免疫应答，而且还具有定义一类新的可适合作为药物靶标的细菌毒力因子的潜力。

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.