Coxiella burnetii Sterol-Modifying Protein Stmp1 Regulates Cholesterol in the Intracellular Niche

Coxiella burnetii replicates in a phagolysosome-like vacuole called the Coxiella-containing vacuole (CCV). While host cholesterol readily traffics to the CCV, cholesterol accumulation leads to CCV acidification and bacterial death. Thus, bacterial regulation of CCV cholesterol content is essential for Coxiella pathogenesis. Coxiella expresses a sterol-modifying protein, Stmp1, that may function to lower CCV cholesterol through enzymatic modification. Using an Stmp1 knockout (Δstmp1), we determined that Stmp1 is not essential for axenic growth. Inside host cells, however, Δstmp1 mutant bacteria form smaller CCVs which accumulate cholesterol, preferentially fuse with lysosomes, and become more acidic, correlating with a significant growth defect. However, in cholesterol-free cells, Δstmp1 mutant bacteria grow similarly to wild-type bacteria but are hypersensitive to cholesterol supplementation. To better understand the underlying mechanism behind the Δstmp1 mutant phenotype, we performed sterol profiling. Surprisingly, we found that Δstmp1 mutant-infected macrophages accumulated the potent cholesterol homeostasis regulator 25-hydroxycholesterol (25-HC). We next determined whether dysregulated 25-HC alters Coxiella infection by treating wild-type Coxiella-infected cells with 25-HC. Similar to the Δstmp1 mutant phenotype, 25-HC increased CCV proteolytic activity and inhibited bacterial growth. Collectively, these data indicate that Stmp1 alters host cholesterol metabolism and is essential to establish a mature CCV which supports Coxiella growth. IMPORTANCE Coxiella burnetii is the causative agent of human Q fever, an emerging infectious disease and significant cause of culture-negative endocarditis. Acute infections are often undiagnosed, there are no licensed vaccines in the United States, and chronic Q fever requires a prolonged antibiotic treatment. Therefore, new treatment and preventive options are critically needed. Coxiella is an obligate intracellular bacterium that replicates within a large acidic phagolysosome-like compartment, the Coxiella-containing vacuole (CCV). We previously discovered that cholesterol accumulation in the CCV increases its acidification, leading to bacterial death. Therefore, in order to survive in this harsh environment, Coxiella likely regulates CCV cholesterol levels. Here, we found that Coxiella sterol modifying protein (Stmp1) facilitates bacterial growth by reducing CCV cholesterol and host cell 25-hydroxycholesterol (25-HC) levels, which prevents excessive CCV fusion with host lysosomes and CCV acidification. This study establishes that Stmp1-mediated regulation of host cholesterol homeostasis is essential for Coxiella intracellular survival.