Abstract：
Rapid phagosomal escape mediated by listeriolysin O (LLO) is a prerequisite for Listeria monocytogenes intracellular replication and pathogenesis. Escape takes place within minutes after internalization from vacuoles that are negative to the early endosomal Rab5 GTPase and positive to the late endosomal Rab7. Using mutant analysis, we found that the listerial invasin InlB was required for optimal intracellular proliferation of L. monocytogenes. Starting from this observation, we determined in HeLa cells that InlB promotes early phagosomal escape and efficient Rab7 acquisition by the Listeria-containing vacuole (LCV). Recruitment of the class III phosphoinositide 3-kinase (PI3K) Vps34 to the LCV and accumulation of its lipid product, phosphatidylinositol 3-phosphate (PI3P), two key endosomal maturation mediators, were also dependent on InlB. Small interfering RNA (siRNA) knockdown experiments showed that Vps34 was required for Rab7 recruitment and early (LLO-mediated) escape and supported InlB-dependent intracellular proliferation. Together, our data indicate that InlB accelerates LCV conversion into an escape-favorable Rab7 late phagosome via subversion of class III PI3K/Vps34 signaling. Our findings uncover a new function for the InlB invasin in Listeria pathogenesis as an intracellular proliferation-promoting virulence factor. IMPORTANCE Avoidance of lysosomal killing by manipulation of the endosomal compartment is a virulence mechanism assumed to be largely restricted to intravacuolar intracellular pathogens. Our findings are important because they show that cytosolic pathogens like L. monocytogenes, which rapidly escape the phagosome after internalization, can also extensively subvert endocytic trafficking as part of their survival strategy. They also clarify that, instead of delaying phagosome maturation (to allow time for LLO-dependent disruption, as currently thought), via InlB L. monocytogenes appears to facilitate the rapid conversion of the phagocytic vacuole into an escape-conducive late phagosome. Our data highlight the multifunctionality of bacterial virulence factors. At the cell surface, the InlB invasin induces receptor-mediated phagocytosis via class I PI3K activation, whereas after internalization it exploits class III PI3K (Vsp34) to promote intracellular survival. Systematically elucidating the mechanisms by which Listeria interferes with PI3K signaling all along the endocytic pathway may lead to novel anti-infective therapies.
摘要：
李斯特菌溶血素O（LLO）介导的快速吞噬体逃逸是单核细胞增生李斯特菌细胞内复制和发病的先决条件。在从对早期内体Rab5GTP酶呈阴性且对晚期内体Rab7呈阳性的空泡内化后几分钟内发生逃逸。使用突变分析，我们发现单核细胞增生李斯特菌的最佳细胞内增殖需要李斯特菌侵入素InlB。从这个观察开始,我们在HeLa细胞中确定InlB促进含李斯特菌液泡（LCV）的早期吞噬体逃逸和有效的Rab7获取。向LCV募集III类磷酸肌醇3激酶（PI3K）Vps34并积累其脂质产物，磷脂酰肌醇3-磷酸酯(PI3P),两个关键的内体成熟介质，也依赖于InlB。小干扰RNA(siRNA)敲低实验表明,Rab7募集和早期(LLO介导的)逃逸需要Vps34,并支持InlB依赖性细胞内增殖。一起,我们的数据表明，InlB通过破坏III类PI3K/Vps34信号传导加速LCV转化为逃逸有利的Rab7晚期吞噬体。我们的发现揭示了InlBinvasin在李斯特菌发病机制中作为细胞内促进增殖的毒力因子的新功能。重要性通过操纵内体区室避免溶酶体杀死是一种被认为主要限于针内细胞内病原体的毒力机制。我们的发现很重要，因为它们表明细胞溶质病原体，如单核细胞增生李斯特菌，内化后迅速逃离吞噬体，作为生存策略的一部分，也可以广泛颠覆内吞贩运。他们还澄清说，而不是延迟吞噬体成熟(为LLO依赖性破坏留出时间，正如目前所认为的)，通过InlBL.单核细胞增多性细胞似乎有助于吞噬液泡快速转化为有利于逃逸的晚期吞噬体。我们的数据强调了细菌毒力因子的多功能性。在细胞表面，InlBinvasin通过I类PI3K激活诱导受体介导的吞噬作用，而在内化后，它利用III类PI3K（Vsp34）来促进细胞内存活。系统地阐明李斯特菌在整个胞吞途径中干扰PI3K信号传导的机制可能会导致新的抗感染疗法。
