Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages.
Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages.
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DOI：
10.1371/journal.ppat.1005915
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发表时间：
2016-10
期刊：
影响因子：
6.7
通讯作者：
Voth DE
中科院分区：
文献类型：
--
作者：
Colonne PM;Winchell CG;Graham JG;Onyilagha FI;MacDonald LJ;Doeppler HR;Storz P;Kurten RC;Beare PA;Heinzen RA;Voth DE
伯氏柯克斯体是一种细胞内细菌病原体，可引起人类Q热，这是一种急性流感样疾病，可发展为慢性心内膜炎以及肝脏和骨骼感染。人类通常通过气溶胶介导的传播感染，伯氏梭菌最初以肺泡巨噬细胞为目标，病原体在被称为寄生虫空泡(PV)的吞噬溶酶体样小生境中复制。伯氏梭菌通过操纵宿主cAMP依赖的蛋白激酶(PKA)信号来促进PV的形成、细胞存活和细菌复制。在这项研究中，我们发现肌动蛋白调节蛋白血管扩张剂刺激的磷酸蛋白(Vasp)是一种PKA底物，在伯氏梭菌感染过程中，它在S157和S239处被越来越多地磷酸化。无毒力和强毒力的伯氏卡氏杆菌引起巨噬细胞样THP-1细胞和原代人肺泡巨噬细胞中Vasp磷酸化水平升高，这一事件需要PKA的Cα亚单位。Vasp的磷酸化还需要细菌通过IV型分泌系统合成和分泌效应蛋白，这表明病原菌主动触发了长时间的Vasp磷酸化。最佳的光伏形成和细胞内细菌复制需要Vasp活性，因为siRNA介导的Vasp的耗尽减少了光伏大小和细菌生长。有趣的是，异位表达模拟磷酸的Vasp(S239E)突变蛋白阻止了最佳PV的形成，而Vasp(S157E)突变表达没有影响。Vasp(S239E)的表达也阻止了含珠的吞噬小体向PV的运输，表明适当的Vasp活性对于控制巨噬细胞中PV扩张的异型融合事件至关重要。最后，显性负性Vasp(S157A)在伯氏梭菌感染细胞中的表达损害了PV的形成，证实了该蛋白对正确感染的重要性。这项研究首次提供了空泡内细菌通过激活人巨噬细胞中的PKA来操纵VASP的证据。Q热由细胞内的伯氏柯克斯体引起，是一种通过气溶胶传播的感染，可发展为危及生命的慢性感染，如心内膜炎。病原体优先生长在肺泡巨噬细胞内的吞噬酶体样隔室中，称为寄生虫性空泡(PV)。伯氏梭菌能主动操控宿主cAMP依赖的蛋白激酶(PKA)信号，促进PV的形成和细胞存活。识别操纵PKA及其下游靶蛋白的细菌效应蛋白对于全面了解病原体介导的信号通路和开发新的治疗策略至关重要。在这里，我们发现PKA控制血管扩张剂刺激的磷酸蛋白(VASP)的活性，以促进PV的形成和细菌的复制。VASP调节细胞内细菌亚群使用的基于肌动蛋白的运动，以推动其通过宿主细胞胞浆进入旁观者细胞。然而，伯氏梭菌不使用基于肌动蛋白的运动性，而是在其整个生命周期中在膜结合的液泡中进行复制。因此，这项研究提供了VASP被液泡内细菌病原体操纵的第一个证据。确定Vasp在PV形成中的功能，并识别促进感染的其他PKA底物，将为Q热病期间宿主与病原体的相互作用提供新的见解。
Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). C. burnetii manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages. Q fever, caused by the intracellular bacterial pathogen Coxiella burnetii, is an aerosol-transmitted infection that can develop into life-threatening chronic infections such as endocarditis. The pathogen preferentially grows within alveolar macrophages in a phagolysosome-like compartment termed the parasitophorous vacuole (PV). C. burnetii actively manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation and cell survival. Identification of bacterial effector proteins that manipulate PKA and downstream target proteins is critical to fully understand pathogen-mediated signaling circuits and develop new therapeutic strategies. Here, we found that PKA controls vasodilator-stimulated phosphoprotein (VASP) activity to promote PV formation and bacterial replication. VASP regulates actin-based motility used by a subset of intracellular bacteria for propulsion through the host cell cytosol and into bystander cells. However, C. burnetii does not use actin-based motility and replicates throughout its life cycle within a membrane bound vacuole. Thus, this study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen. Characterization of VASP function in PV formation and identification of additional PKA substrates that promote infection will provide new insight into host-pathogen interactions during Q fever.
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影响因子：
6.7
作者：
Carey KL;Newton HJ;Lührmann A;Roy CR
通讯作者：
Roy CR
影响因子：
6.4
作者：
Beare PA;Gilk SD;Larson CL;Hill J;Stead CM;Omsland A;Cockrell DC;Howe D;Voth DE;Heinzen RA
通讯作者：
Heinzen RA
影响因子：
4.8
作者：
Barzik, M;Kotova, TI;Schafer, DA
通讯作者：
Schafer, DA
影响因子：
3.1
作者：
AKPORIAYE, ET;ROWATT, JD;BACA, OG
通讯作者：
BACA, OG
影响因子：
3.4
作者：
Graham JG;MacDonald LJ;Hussain SK;Sharma UM;Kurten RC;Voth DE
通讯作者：
Voth DE