Dot/Icm Type IVB Secretion System Requirements for Coxiella burnetii Growth in Human Macrophages

ABSTRACT 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. IMPORTANCE 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. 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.

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