Dot/Icm type IVB secretion system requirements for Coxiella burnetii growth in human macrophages.
Dot/Icm type IVB secretion system requirements for Coxiella burnetii growth in human macrophages.
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作者：
Beare PA;Gilk SD;Larson CL;Hill J;Stead CM;Omsland A;Cockrell DC;Howe D;Voth DE;Heinzen RA
Q热发病机制的核心是病原体贝氏柯克斯体在单核吞噬细胞中的吞噬溶酶体样寄生虫空泡（PV）内的复制。贝氏梭菌调节PV生物发生和其他宿主细胞功能，如凋亡信号传导，推测是通过Dot/Icm型IVB分泌系统（T4 BSS）递送至宿主细胞溶质的蛋白质的活性。在这项研究中，我们利用携带IcmD的贝氏隐孢子虫菌株通过Himar 1转座子灭活，以研究Dot/Icm功能在贝氏隐孢子虫寄生人类THP-1巨噬细胞样细胞中的需求。的icmD：：Tn突变体未能分泌特征T4 BSS底物，缺陷与复制缺陷，PV发展，和凋亡保护。恢复IVB型分泌和细胞内生长的icmD：：Tn突变体需要互补与icmD，-J，和-B，表明极性效应的转座子插入下游点/icm基因。感染后1天icmDJB表达的诱导导致贝氏隐孢子虫复制和PV产生。总的来说，这些数据证明T4 BSS功能是贝氏梭菌对人巨噬细胞的生产性感染所必需的。然而，说明C. Burnetti等人，当在野生型细菌产生的PV中隔离时，icmD：：Tn突变体可以在细胞内复制，其中Dot/Icm功能以反式提供，并且在原生动物寄生虫亚马逊利什曼原虫产生的表型相似的PV中，其中宿主细胞缺乏Dot/Icm T4 BSS效应蛋白。 贝氏柯克斯体是引起人类Q热的病原体，它是唯一一种在类似吞噬溶酶体的空泡中复制的细菌病原体。该生物操纵宿主巨噬细胞以促进允许生长的空泡区室的生物发生。通过类比嗜肺军团菌的成熟细胞微生物学，贝氏隐球菌的Dot/Icm型IVB分泌系统被认为是宿主细胞修饰中的关键毒力因子，其将具有效应子功能的蛋白质直接递送到宿主细胞胞质溶胶中。使用新的遗传工具，我们验证了Dot/Icm功能对于贝氏隐孢子虫对人巨噬细胞的生产性感染是必不可少的。有趣的是，尽管产生同源分泌系统，嗜肺军团菌和贝氏隐球菌在其各自的感染周期中对Dot/Icm功能具有显著不同的时间要求。
Central to Q fever pathogenesis is replication of the causative agent, Coxiella burnetii, within a phagolysosome-like parasitophorous vacuole (PV) in mononuclear phagocytes. C. burnetii modulates PV biogenesis and other host cell functions, such as apoptotic signaling, presumably via the activity of proteins delivered to the host cytosol by a Dot/Icm type IVB secretion system (T4BSS). In this study, we utilized a C. burnetii strain carrying IcmD inactivated by the Himar1 transposon to investigate the requirements for Dot/Icm function in C. burnetii parasitism of human THP-1 macrophage-like cells. The icmD::Tn mutant failed to secrete characterized T4BSS substrates, a defect that correlated with deficient replication, PV development, and apoptosis protection. Restoration of type IVB secretion and intracellular growth of the icmD::Tn mutant required complementation with icmD, -J, and -B, indicating a polar effect of the transposon insertion on downstream dot/icm genes. Induction of icmDJB expression at 1 day postinfection resulted in C. burnetii replication and PV generation. Collectively, these data prove that T4BSS function is required for productive infection of human macrophages by C. burnetii. However, illustrating the metabolic flexibility of C. burnetti, the icmD::Tn mutant could replicate intracellularly when sequestered in a PV generated by wild-type bacteria, where Dot/Icm function is provided in trans, and within a phenotypically similar PV generated by the protozoan parasite Leishmania amazonensis, where host cells are devoid of Dot/Icm T4BSS effector proteins. Coxiella burnetii, the cause of human Q fever, is the only bacterial pathogen known to replicate in a vacuole resembling a phagolysosome. The organism manipulates host macrophages to promote the biogenesis of a vacuolar compartment permissive for growth. By analogy to the well-established cellular microbiology of Legionella pneumophila, the Dot/Icm type IVB secretion system of C. burnetii is implicated as a critical virulence factor in host cell modification that delivers proteins with effector functions directly into the host cell cytosol. Using new genetic tools, we verify that Dot/Icm function is essential for productive infection of human macrophages by C. burnetii. Interestingly, despite the production of homologous secretion systems, L. pneumophila and C. burnetii have strikingly different temporal requirements for Dot/Icm function during their respective infectious cycles.