Genetics of Coxiella burnetii

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Coxiella burnetii is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness. Coxiella's former obligate intracellular nature significantly impeded genetic characterization of putative virulence factors. However, our seminal advance of host cell-free (axenic) growth of Coxiella in acidified citrate cysteine medium (ACCM) enabled us to quickly develop a a complete genetics tool box. Most recently, we developed a defined medium that supports robust growth of Coxiella called ACCM-D that contains amino acids as sole carbon and energy sources. Coxiella is auxotrophic for Arg, Lys, Pro and Tyr by lacking the final enzymes in biosynthesis. Heterologous expression by Coxiella of Legionella pneumophila argGH, lysA and proAB and E.coli tyrB rescues growth in Arg, Lys, Pro and Tyr ACCM-D dropout media, respectively, thus providing four methods for nutritional selection of Coxiella transformants. Strong, non-antibiotic-based selection of genetic transformants is an important advance considering selectable markers based on antibiotic resistance are limited for this select agent. All C. burnetii isolates sequenced to date harbor an autonomous replicating plasmid (QpH1, QpDG,, QPRS QpRS, or QpDV) or have QpRS-like plasmid sequences integrated into the chromosome. The roles of the genes present on these sequences are unknown but some encode Dot/Icm type IVB secretion substrates. The role of the genes encoded on QpH1 were investigated. Using a new C. burnetii shuttle vector (pBR322-TyrB-QpH1ori) containing the C. burnetii QpH1 origin of replication and new nutritional-based selection marker (tyrosine) in C. burnetii, we were able to cure the QpH1 plasmid from the C. burnetii Nine Mile Phase II strain. The QpH1 strain grew normally in axenic media but had a significant growth defect in Vero cells, suggesting the plasmid is critical for C. burnetii virulence. We developed an inducible CRISPR interference system using a second nutritional selection marker (proline) to examine the role of individual QpH1 plasmid genes. Individual knockdown of the 8 Dot/Icm substrates encoded by QpH1 did not affect growth in axenic media or THP1 cells, while knockdown of cbuA0027 resulted in significant growth defects in axenic media and THP1 cells. The cbuA0027 gene is the downstream component of a two-gene operon with cbuA0028. CBUA0028 and CBUA0027 are homologous to the HigB toxin and HigA anti-toxin, respectively, from Vibrio cholerae that are part of the RelE-like family of toxin-antitoxin systems. Overexpression of cbuA0028 resulted in a severe defect in intracellular growth that could be rescued by coexpression with cbuA0027. Production of CBUA0028 in a cell free system inhibited production of a control protein in a dose-dependent manner. Consistent with other toxin-antitoxin systems, CBUA0027 bound directly to CBUA0028 and the resulting complex could bind the cbuA0028 promoter (PcbuA0028), CBUA0027 could also bind PcbuA0028. Two cbuA0027 promoters internal to cbuA0028 were also identified. In summary, our data indicate that the reason C. burnetii isolates maintain an autonomously replicating plasmid is due to the presence of a plasmid-based toxin-antitoxin system. C. burnetii transitions between a replicative, metabolically-active large-cell variant (LCV), and a spore-like quiescent small-cell variant (SCV) in order to ensure survival between host cells and mammalian hosts. C. burnetii encodes three canonical two-component systems, four orphan hybrid histidine kinases, five orphan response regulators, and a histidine phosphotransfer protein that have been speculated to play roles in the signaling required for C. burnetii morphogenesis and virulence, but very few of these systems have been characterized. By employing a CRISPR interference system for genetic manipulation of C. burnetii, we have created single and multi-gene transcriptional knock-down strains involving most of these signaling genes. Through this, we have determined that the CBU_0780/CBU_0955/CBU_1043 orphan response regulators coordinately and disparately regulate expression of SCV-associated genes in C. burnetii LCVs. We also revealed a potential role for the C. burnetii PhoBR canonical two component system in virulence, regulation of Pi maintenance, and Pi transport. We also outline a novel mechanism by which PhoBR function may be regulated by an atypical PhoQ-like protein. These foundational results will inform future studies on the role of C. burnetii two component regulators in virulence and morphogenesis. The current human Q fever vaccine, Q-VAX, is a fixed, whole cell vaccine (WCV) and is licensed solely for use in Australia. While highly efficacious, Coxiella WCVs are associated with a potentially severe postvaccination dermal hypersensitivity reaction in people with pre-existing immunity to Coxiella, which limits their wider use. Consequently, a less reactogenic vaccine is needed. We investigated contributions of the Coxiella Dot/Icm type IVB secretion system (T4BSS) and lipopolysaccharide (LPS) in protection and reactogenicity of fixed WCVs. A 32.5 kb region containing 23 dot/icm genes was deleted in the virulent Nine Mile phase I (NMI) strain and the resulting mutant was evaluated in guinea pig models of Q fever infection, vaccination and challenge, and post-vaccination hypersensitivity. The NMI dot/icm strain was avirulent, protective as a WCV against a robust Coxiella challenge, and displayed potentially altered reactogenicity compared to wild type Coxiella.