Altering lipid droplet homeostasis affects Coxiella burnetii intracellular growth

Coxiella burnetii is an obligate intracellular bacterial pathogen and a causative agent of culture-negative endocarditis. While C. burnetii initially infects alveolar macrophages, it has also been found in lipid droplet (LD)-containing foamy macrophages in the cardiac valves of endocarditis patients. In addition, transcriptional studies of C. burnetii-infected macrophages reported differential regulation of the LD coat protein-encoding gene perilipin 2 (plin-2). To further investigate the relationship between LDs and C. burnetii, we compared LD numbers using fluorescence microscopy in mock-infected and C. burnetii-infected alveolar macrophages. On average, C. burnetii-infected macrophages contained twice as many LDs as mock-infected macrophages. LD numbers increased as early as 24 hours post-infection, an effect reversed by blocking C. burnetii protein synthesis. The observed LD accumulation was dependent on the C. burnetii Type 4B Secretion System (T4BSS), a major virulence factor that manipulates host cellular processes by secreting bacterial effector proteins into the host cell cytoplasm. To determine the importance of LDs during C. burnetii infection, we manipulated LD homeostasis and assessed C. burnetii intracellular growth. Surprisingly, blocking LD formation with the pharmacological inhibitors triacsin C or T863, or knocking out acyl-CoA transferase-1 (acat-1) in alveolar macrophages, increased C. burnetii growth at least 2-fold. Conversely, preventing LD lipolysis by inhibiting adipose triglyceride lipase (ATGL) with atglistatin almost completely blocked bacterial growth, suggesting LD breakdown is essential for C. burnetii. Together these data suggest that maintenance of LD homeostasis, possibly via the C. burnetii T4BSS, is critical for bacterial growth.

贝氏柯克斯体（Coxiella burnetii）是一种专性胞内细菌病原体，亦是培养阴性心内膜炎的致病菌。该菌最初可感染肺泡巨噬细胞，同时也可在心内膜炎患者心脏瓣膜内的含脂滴（lipid droplet，LD）泡沫状巨噬细胞中被检出。此外，针对贝氏柯克斯体感染巨噬细胞的转录研究显示，脂滴被膜蛋白编码基因perilipin 2（plin-2）的表达存在差异调控。为进一步探究脂滴与贝氏柯克斯体之间的关联，本研究通过荧光显微镜术对比了模拟感染组与贝氏柯克斯体感染组肺泡巨噬细胞的脂滴数量。结果显示，贝氏柯克斯体感染的巨噬细胞平均脂滴数量为模拟感染组的两倍。脂滴数量早在感染后24小时即出现升高，该效应可通过阻断贝氏柯克斯体的蛋白质合成予以逆转。所观察到的脂滴积累依赖于贝氏柯克斯体IV型分泌系统4B亚型（Type 4B Secretion System，T4BSS）——这是一类主要的毒力因子，可通过向宿主细胞质中分泌细菌效应蛋白来调控宿主细胞进程。为确定脂滴在贝氏柯克斯体感染过程中的重要性，本研究对脂滴稳态进行了干预，并评估了贝氏柯克斯体的胞内增殖情况。出乎意料的是，使用药理学抑制剂triacsin C或T863阻断脂滴形成，或在肺泡巨噬细胞中敲除酰基辅酶A转移酶1（acyl-CoA transferase-1，acat-1），均可使贝氏柯克斯体的增殖水平提升至少两倍。反之，以atglistatin抑制脂肪甘油三酯脂酶（adipose triglyceride lipase，ATGL）以阻断脂解作用，则几乎完全抑制了细菌增殖，这表明脂滴的降解对于贝氏柯克斯体而言是必需的。综上，本研究数据表明，脂滴稳态的维持（可能依赖于贝氏柯克斯体的IV型分泌系统4B亚型）对于该细菌的增殖至关重要。