Upon host cell infection, the obligate intracellular bacterium Coxiella burnetii resides and multiplies within the Coxiella-Containing Vacuole (CCV). The nascent CCV progresses through the endosomal maturation pathway into a phagolysosome, acquiring endosomal and lysosomal markers, as well as acidic pH and active proteases and hydrolases. Approximately 24-48 hours post infection, heterotypic fusion between the CCV and host endosomes/lysosomes leads to CCV expansion and bacterial replication in the mature CCV. Initial CCV acidification is required to activate C. burnetii metabolism and the Type 4B Secretion System (T4BSS), which secretes effector proteins required for CCV maturation. However, we found that the mature CCV is less acidic (pH~5.2) than lysosomes (pH~4.8). Further, inducing CCV acidification to pH~4.8 causes C. burnetii lysis, suggesting C. burnetii actively regulates pH of the mature CCV. Because heterotypic fusion with host endosomes/lysosomes may influence CCV pH, we investigated endosomal maturation in cells infected with wildtype (WT) or T4BSS mutant (ΔdotA) C. burnetii. In WT-infected cells, we observed a significant decrease in proteolytically active, LAMP1-positive endolysosomal vesicles, compared to mock or ΔdotA-infected cells. Using a ratiometric assay to measure endosomal pH, we determined that the average pH of terminal endosomes in WT-infected cells was pH~5.8, compared to pH~4.75 in mock and ΔdotA-infected cells. While endosomes progressively acidified from the periphery (pH~5.5) to the perinuclear area (pH~4.7) in both mock and ΔdotA-infected cells, endosomes did not acidify beyond pH~5.2 in WT-infected cells. Finally, increasing lysosomal biogenesis by overexpressing the transcription factor EB resulted in smaller, more proteolytically active CCVs and a significant decrease in C. burnetii growth, indicating host lysosomes are detrimental to C. burnetii. Overall, our data suggest that C. burnetii inhibits endosomal maturation to reduce the number of proteolytically active lysosomes available for heterotypic fusion with the CCV, possibly as a mechanism to regulate CCV pH.

中文翻译：

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细菌生长需要荚膜柯氏杆菌4B型分泌系统依赖的溶酶体成熟过程。

宿主细胞感染后，专性细胞内细菌伯氏柯氏杆菌在含Coxiella的液泡（CCV）中驻留并繁殖。新生的CCV通过内体成熟途径进入吞噬体，获得内体和溶酶体标记，以及酸性pH值，活性蛋白酶和水解酶。感染后约24-48小时，CCV与宿主内体/溶酶体之间的异型融合导致CCV扩增和成熟CCV中的细菌复制。需要初始CCV酸化来激活伯氏梭菌代谢和4B型分泌系统（T4BSS），后者分泌CCV成熟所需的效应蛋白。但是，我们发现成熟的CCV的酸性（pH〜5.2）低于溶酶体（pH〜4.8）。此外，将CCV酸化至pH〜4.8会导致烧伤梭状芽胞杆菌溶解，提示伯氏梭菌可主动调节成熟CCV的pH。因为与宿主内体/溶酶体的异型融合可能影响CCV pH，所以我们研究了野生型（WT）或T4BSS突变体（ΔdotA）伯氏梭菌（C. burnetii）感染的细胞中的内体成熟。在WT感染的细胞中，我们观察到与模拟或ΔdotA感染的细胞相比，蛋白水解活性，LAMP1阳性的溶酶体囊泡显着减少。使用比例测定法测量内体pH，我们确定WT感染的细胞中末端内体的平均pH为pH〜5.8，相比之下，模拟和ΔdotA感染的细胞中pH〜4.75。在模拟和ΔdotA感染的细胞中，内体从周围（pH〜5.5）逐渐酸化至核周区域（pH〜4.7），而在WT感染的细胞中，内体并未酸化至pH〜5.2以上。最后，通过过度表达转录因子EB增加溶酶体生物发生，导致较小，更具蛋白水解活性的CCV以及Burnetii的生长显着减少，表明宿主溶酶体对Burnetii有害。总体而言，我们的数据表明，伯氏梭菌可抑制内体成熟，从而减少可与CCV进行异型融合的蛋白水解活性溶酶体的数量，这可能是调节CCV pH的机制。