Department of Microbiology and Immunology, University of Arkansas for Medical Sciences Little Rock, AR, USA.
Front Microbiol. 2010 Dec 23;1:137. doi: 10.3389/fmicb.2010.00137. eCollection 2010.
Coxiella burnetii is the etiologic agent of human Q fever and targets alveolar phagocytic cells in vivo wherein the pathogen generates a phagolysosome-like parasitophorous vacuole (PV) for replication. C. burnetii displays a prolonged growth cycle, making PV maintenance critical for bacterial survival. Previous studies showed that C. burnetii mediates activation of eukaryotic kinases to inhibit cell death, indicating the importance of host signaling during infection. In the current study, we examined the role of eukaryotic kinase signaling in PV establishment. A panel of 113 inhibitors was analyzed for their impact on C. burnetii infection of human THP-1 macrophage-like cells and HeLa cells. Inhibition of 11 kinases or two phosphatases altered PV formation and prevented pathogen growth, with most inhibitor-treated cells harboring organisms in tight-fitting phagosomes, indicating kinase/phosphatase activation is required for PV maturation. Five inhibitors targeted protein kinase C (PKC), suggesting a critical role for this protein during intracellular growth. The PKC-specific substrate MARCKS was phosphorylated at 24 h post-infection and remained phosphorylated through 5 days post-infection, indicating prolonged regulation of the PKC pathway by C. burnetii. Infection also altered the activation status of p38, myosin light chain kinase, and cAMP-dependent protein kinase, suggesting C. burnetii subverts numerous phosphorylation cascades. These results underscore the importance of intracellular host signaling for C. burnetii PV biogenesis.
贝氏考克斯体是人类 Q 热的病原体，其在体内靶向肺泡吞噬细胞，在此过程中病原体产生类似于吞噬溶酶体的寄生性空泡（PV）以进行复制。贝氏考克斯体具有较长的生长周期，因此 PV 的维持对于细菌的存活至关重要。先前的研究表明，贝氏考克斯体介导了真核激酶的激活，以抑制细胞死亡，这表明在感染过程中宿主信号的重要性。在本研究中，我们研究了真核激酶信号在 PV 建立中的作用。分析了 113 种抑制剂对人类 THP-1 巨噬样细胞和 HeLa 细胞中贝氏考克斯体感染的影响。11 种激酶或两种磷酸酶的抑制改变了 PV 的形成并阻止了病原体的生长，大多数用抑制剂处理的细胞中含有紧密结合的吞噬体中的生物体，表明激酶/磷酸酶的激活是 PV 成熟所必需的。五种抑制剂靶向蛋白激酶 C（PKC），表明该蛋白在细胞内生长过程中起着关键作用。PKC 特异性底物 MARCKS 在感染后 24 小时被磷酸化，并在感染后 5 天保持磷酸化，表明贝氏考克斯体对 PKC 途径进行了长时间的调控。感染还改变了 p38、肌球蛋白轻链激酶和 cAMP 依赖性蛋白激酶的激活状态，表明贝氏考克斯体颠覆了许多磷酸化级联反应。这些结果强调了细胞内宿主信号对贝氏考克斯体 PV 生物发生的重要性。
