■伯氏柯西氏菌是一种革兰氏阴性的细胞内专性细菌,是一种引起人Q热的人畜共患病原体。在美国,缺乏有效的抗生素和获得许可的柯西氏菌疫苗,因此有必要对柯西氏菌的发病机理进行进一步研究。在宿主细胞内,柯西氏菌在酸性吞噬溶酶体样液泡中复制,称为含柯西氏菌液泡(CCV)。以前,我们已经证明CCV的pH对于柯西氏菌的存活是关键的,并且柯西氏菌4B型分泌系统通过抑制宿主内体成熟途径来调节CCV的pH。然而,柯西拉感染细胞中“未成熟”内体的运输模式仍不清楚。
■我们用GFP标记的Rab蛋白转染了HeLa细胞,随后用mCherry-Coxiella感染了它们,以可视化Rab蛋白定位。感染的细胞用抗Rab抗体免疫染色以确认Rab定位于CCV。定量Rab11a和Rab35阳性CCV,并定量感染细胞的总再循环内体含量。使用双命中siRNA介导的敲低结合免疫荧光测定或基于琼脂糖的集落形成单位测定来测量Rab11a和Rab35敲低对CCV面积和柯西氏菌细胞内生长的影响。
■用宿主Rab蛋白进行的CCV定位筛选显示,在感染过程中,循环内体相关蛋白Rab11a和Rab35定位于CCV,表明CCV在成熟过程中与宿主再循环内体相互作用。有趣的是,在任何给定时间点,只有一部分CCV为Rab11a或Rab35阳性.Rab11a/Rab35阳性CCV的定量显示,尽管Rab11a在3dpi时与CCV相互作用更多,Rab35在6dpi的CCV中更为普遍,这表明CCV根据感染阶段优先与Rab11a和Rab35相互作用。此外,我们观察到,与模拟相比,在柯西氏菌感染的细胞中,Rab11a和Rab35荧光强度显着增加,表明柯西氏菌增加了感染细胞中的再循环内体含量。最后,siRNA介导的Rab11a和Rab35的敲低导致CCV明显变小,并降低了柯西氏菌的细胞内生长。这表明循环内体Rab蛋白对于CCV扩增和细菌繁殖至关重要。
■我们的数据,第一次,表明CCV与宿主再循环内体动态相互作用,以促进柯西氏菌细胞内存活,并可能发现柯西氏菌发病机理所必需的新型宿主细胞因子。
UNASSIGNED: Coxiella burnetii is a gram-negative obligate intracellular bacterium and a zoonotic pathogen that causes human Q fever. The lack of effective antibiotics and a licensed vaccine for Coxiella in the U.S. warrants further research into Coxiella pathogenesis. Within the host cells, Coxiella replicates in an acidic phagolysosome-like vacuole termed Coxiella-containing vacuole (CCV). Previously, we have shown that the CCV pH is critical for Coxiella survival and that the Coxiella Type 4B secretion system regulates CCV pH by inhibiting the host endosomal maturation pathway. However, the trafficking pattern of the \'immature\' endosomes in Coxiella- infected cells remained unclear.
UNASSIGNED: We transfected HeLa cells with GFP-tagged Rab proteins and subsequently infected them with mCherry-Coxiella to visualize Rab protein localization. Infected cells were immunostained with anti-Rab antibodies to confirm the Rab localization to the CCV, to quantitate Rab11a and Rab35- positive CCVs, and to quantitate total recycling endosome content of infected cells. A dual-hit siRNA mediated knockdown combined with either immunofluorescent assay or an agarose-based colony-forming unit assay were used to measure the effects of Rab11a and Rab35 knockdown on CCV area and Coxiella intracellular growth.
UNASSIGNED: The CCV localization screen with host Rab proteins revealed that recycling endosome-associated proteins Rab11a and Rab35 localize to the CCV during infection, suggesting that CCV interacts with host recycling endosomes during maturation. Interestingly, only a subset of CCVs were Rab11a or Rab35-positive at any given time point. Quantitation of Rab11a/Rab35-positive CCVs revealed that while Rab11a interacts with the CCV more at 3 dpi, Rab35 is significantly more prevalent at CCVs at 6 dpi, suggesting that the CCV preferentially interacts with Rab11a and Rab35 depending on the stage of infection. Furthermore, we observed a significant increase in Rab11a and Rab35 fluorescent intensity in Coxiella-infected cells compared to mock, suggesting that Coxiella increases the recycling endosome content in infected cells. Finally, siRNA-mediated knockdown of Rab11a and Rab35 resulted in significantly smaller CCVs and reduced Coxiella intracellular growth, suggesting that recycling endosomal Rab proteins are essential for CCV expansion and bacterial multiplication.
UNASSIGNED: Our data, for the first time, show that the CCV dynamically interacts with host recycling endosomes for Coxiella intracellular survival and potentially uncovers novel host cell factors essential for Coxiella pathogenesis.