A toxin-antitoxin system ensures plasmid stability in Coxiella burnetii

Coxiella burnetii is the causative agent of Q fever. All C. burnetii isolates encode either an autonomous replicating plasmid (QpH1, QpDG, QpRS, or QpDV) or QpRS-like chromosomally integrated plasmid sequences. The role of the ORFs present on these sequences is unknown. Here, the role of the ORFs encoded on QpH1 was investigated. Using a new C. burnetii shuttle vector (pB-TyrB-QpH1ori) we cured Nine Mile Phase II of QpH1. The {Delta}QpH1 strain grew normally in axenic media but had a significant growth defect in Vero cells, indicating QpH1 was important for C. burnetii virulence. We developed an inducible CRISPR interference system to examine the role of individual QpH1 plasmid genes. CRISPRi of cbuA0027 resulted in significant growth defects in axenic media and THP-1 cells. The cbuA0028/cbuA0027 operon encodes CBUA0028 and CBUA0027, which are homologous to the HigB2 toxin and HigA2 anti-toxin, respectively, from Vibrio cholerae. Consistent with toxin-antitoxin systems, overexpression of cbuA0028 resulted in a severe intracellular growth defect that was rescued by co-expression of cbuA0027. CBUA0028 inhibited protein translation. CBUA0027 bound the cbuA0028 promoter (PcbuA0028) and CBUA0028, with the resulting complex binding also PcbuA0028. In summary, our data indicates C. burnetii maintains an autonomously replicating plasmid because of a plasmid-based toxin-antitoxin system.