The Coxiella burnetii QpH1 Plasmid Is a Virulence Factor for Colonizing Bone Marrow-Derived Murine Macrophages

Abstract

Coxiella burnetii strains carry one of four large, conserved, autonomously replicating plasmids (QpH1, QpRS, QpDV, and QpDG) or a QpRS-like chromosomally integrated sequence of unknown function. Here we report the characterization of the QpH1 plasmid of C. burnetii Nine Mile phase II by making QpH1-deficient strains. A shuttle vector pQGK containing the CBUA0036-0039a region (predicted as being required for the QpH1 maintenance) was constructed. The pQGK vector can be stably transformed into the Nine Mile II and maintained at a similar low copy like QpH1. Importantly, transformation with pQGK cured the endogenous QpH1 due to plasmid incompatibility. Compared to a Nine Mile II transformant of a RSF1010-ori based vector, the pQGK transformant shows a similar growth curve in both axenic media and Buffalo green monkey kidney cells, a variable growth defect in macrophage-like THP-1 cells depending on the origin of inoculum, and dramatically reduced ability of colonizing wild-type bone marrow-derived murine macrophages. Furthermore, we found CBUA0037-0039 ORFs are essential for plasmid maintenance, and CBUA0037-0038 ORFs account for plasmid compatibility. And plasmid-deficient C. burnetii can be isolated by using CBUA0037 or -0038 deletion vectors. Furthermore, QpH1-deficient C. burnetii strains caused a lesser extent of splenomegaly in SCID mice but, intriguingly, they had significant growth in SCID mouse-sourced macrophages. Taken together, our data suggest that QpH1 encodes factor(s) essential for colonizing murine, not human, macrophages. This study suggests a critical role of QpH1 for C. burnetii persistence in rodents and expands the toolkit for the genetic studies in C. burnetii. Author summary All C. burnetii isolates carry one of four large, conserved, autonomously replicating plasmids or a plasmid-like chromosomally integrated sequence. The plasmid is a candidate virulence factor of unknown function. Here we describe the construction of novel shuttle vectors that allow making plasmid-deficient C. burnetii mutants. With this plasmid-curing approach, we characterized the role of the QpH1 plasmid in in vitro and in vivo C. burnetii infection models. We found that the plasmid plays a critical role for C. burnetii growth in murine macrophages. Our work suggests an essential role of the QpH1 plasmid for the acquisition of colonizing capability in rodents by C. burnetii. This study represents a major step toward unravelling the mystery of the C. burnetii cryptic plasmids.

Identification of essential genes in Coxiella burnetii

“…However, a recent report demonstrated curing C. burnetii NMII of this plasmid [88]. CBUA0037, CBUA0038 and CBUA0039 confer homologues of ParA, RepB and RepA – a putative origin of replication.…”

Mobile Element Integration Reveals a Chromosome Dimer Resolution System in Legionellales

“…One interpretation of these results is that integrants that do not maintain xerL chromosomally are rapidly selected against and not recovered. In light of our data, it will be important to distinguish which phenotypes previously associated with directed plasmid loss ( 44 ) reflect virulence-specific defects (associated with loss of specific virulence factors, such as effectors) and which phenotypes result from the loss of dif /XerL machinery.…”

Metabolic Plasticity Aids Amphotropism of Coxiella burnetii

“… C. burnetii is a zoonotic pathogen with an extensive host range, including humans, domestic animals, wildlife, and arthropods. While determinants for species-specific colonization are poorly defined, carriage of the plasmid QpH1 has been linked to C. burnetii colonization of murine macrophages ( 41 ). Upon infection, C. burnetii colonizes numerous tissues and cell types consistent with a requirement of the pathogen to adapt to nutritionally diverse environments.…”