Abstract
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.
Highlights
Coxiella burnetii is a gram-negative obligate intracellular bacterium which causes human Q fever
The 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
Our study suggests that C. burnetii regulates vacuole acidity and blocks endosomal maturation in order to produce a permissive intracellular niche
Summary
Coxiella burnetii is a gram-negative obligate intracellular bacterium which causes human Q fever. Q fever manifests as a flu-like illness in acute disease and can develop into culture-negative endocarditis in chronic cases. The current treatment regimen for chronic C. burnetii infection requires a daily antibiotic combination therapy for at least 18 months [1], highlighting the need for more efficient therapeutics. 2–6 hours post infection, the phagosome fuses with lysosomes [3, 4], delivering lysosomal membrane proteins, including LAMP1 (Lysosome-associated membrane glycoprotein-1) and v-ATPase [5], and lysosomal enzymes such as acid phosphatases [4, 5] and cathepsins [2, 5, 6] to the phagosome. 24–48 hours post infection, phagosome expansion, presumably through heterotypic fusion with the endocytic pathway, gives rise to a large acidic phagolysosomal-like compartment termed the Coxiellacontaining vacuole (CCV) [5]
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