Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

Abstract

Upon host cell infection, the obligate intracellular bacterium C. burnetii resides and multiplies within the Coxiella-Containing Vacuole (CCV). The nascent CCV progresses through the endosomal maturation pathway into a phagolysosome, acquiring 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 subsequent 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 recently found that the mature CCV is less acidic (pH~5.2) than lysosomes (pH~4.8). Further, CCV acidification to pH~4.8 causes C. burnetii lysis, suggesting C. burnetii actively regulates CCV pH. Because heterotypic fusion with host endosomes/lysosomes may influence CCV pH, we investigated endosomal maturation in cells infected with wildtype (WT) or T4BSS mutant ({Delta}dotA) C. burnetii. We observed significantly fewer LAMP1-positive lysosomes, along with less acidic \"mature\" endosomes (pH~5.8), in WT-infected cells, compared to mock or {Delta}dotA-infected cells. Further, while endosomes progressively acidified from the periphery (pH~5.5) to the perinuclear area (pH~4.7) in both mock and {Delta}dotA-infected cells, endosomes did not acidify beyond pH~5.2 in WT-infected cells, indicating that the C. burnetii T4BSS inhibits endosomal maturation. Finally, increasing the number of acidic lysosomes by overexpressing the transcription factor EB inhibited C. burnetii growth, indicating lysosomes are detrimental to C. burnetii. Overall, our data suggest that C. burnetii regulates CCV pH, possibly by reducing the number of host lysosomes available for heterotypic fusion.\n\nAuthor summaryThe obligate intracellular bacterium Coxiella burnetii causes human Q fever, which manifests as a flu-like illness but can develop into a life-threatening and difficult to treat endocarditis. C. burnetii, in contrast to many other intracellular bacteria, thrives within a lysosome-like vacuole in host cells. However, we previously found that the C. burnetii vacuole is not as acidic as lysosomes and increased acidification kills the bacteria, suggesting that C. burnetii regulates the pH of its vacuole. Here, we discovered that C. burnetii blocks endosomal maturation and acidification during host cell infection, resulting in fewer lysosomes in the host cell. Moreover, increasing lysosomes in the host cells blocked C. burnetii growth. Together, our study suggests that C. burnetii regulates vacuole acidity and blocks endosomal acidification in order to produce a permissive intracellular niche.