TheCoxiella burnetiiQpH1 plasmid is a virulence factor for colonizing bone marrow-derived murine macrophages

Abstract

23Coxiella burnetii carries a large conserved plasmid or plasmid-like chromosomally 24 integrated sequence of unknown function. Here we report the curing of QpH1 plasmid 25 from C. burnetii Nine Mile phase II, the characterization of QpH1-deficient C. 26 burnetii in in vitro and in vivo infection models, and the characterization of plasmid 27 biology. A shuttle vector pQGK, which is composed of the CBUA0036-0039a region 28 (predicted for QpH1 maintenance), an E. coli plasmid ori, eGFP and kanamycin 29 resistance genes was constructed. The pQGK vector can be stably transformed into 30 Nine Mile II and maintained at a similar low copy like QpH1. Importantly, 31 transformation with pQGK cured the endogenous QpH1 due to plasmid 32 incompatibility. Compared to a Nine Mile II transformant of a RSF1010-based vector, 33 the pQGK transformant shows an identical one-step growth curve in axenic media, a 34 similar growth curve in Buffalo green monkey kidney cells, an evident growth defect 35 in macrophage-like THP-1 cells, and dramatically reduced ability of colonizing bone 36 marrow-derived murine macrophages. In the SCID mouse infection model, the pQGK 37 transformants caused a lesser extent of splenomegaly. Moreover, the plasmid biology 38 was investigated by mutagenesis. We found CBUA0037-0039 are essential for 39 plasmid maintenance, and CBUA0037-0038 account for plasmid compatibility. Taken 40 together, our data suggest that QpH1 encodes factor(s) essential for colonizing murine 41 macrophages, and to a lesser extent for colonizing human macrophages. This study 42 highlights a critical role of QpH1 for C. burnetii persistence in rodents, and expands 43 the toolkit for genetic studies in C. burnetii. 45Author summary 46 It is postulated that C. burnetii recently evolved from an inherited symbiont of ticks 47 by the acquisition of novel virulence factors. All C. burnetii isolates carry a large 48 plasmid or have a chromosomally integrated plasmid-like sequence. The plasmid is a 49 candidate virulence factor that contributes to C. burnetii evolution. Here we describe 50 the construction of novel shuttle vectors that allow to make plasmid-deficient C. 51 burnetii mutants. With this plasmid-curing approach, we characterized the role of the 52 QpH1 plasmid in in vitro and in vivo C. burnetii infection models. We found that the 53 plasmid plays a critical role for C. burnetii growth in macrophages, especially in 54 murine macrophages, but not in axenic media and BGMK cells. Our work highlights 55 an essential role of the plasmid for the acquisition of colonizing capability in rodents 56 by C. burnetii. This study represents a major step toward unravelling the mystery of 57 the C. burnetii cryptic plasmids. 58 59 Introduction 60 C. burnetii is a Gram-negative intracellular bacterium that causes Q fever, a world 61 widely distributed zoonosis [1]. It is highly infectious and is classified as a potential 62 biowarfare agent [2]. Its infections in humans are mostly asymptomatic but may 63 manifest as an acut...

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.…”

“…Despite the apparent vertical inheritance of xerL given its restriction within the order Legionellales, the Coxiella burnetii orthologs of xerL (hereafter xerL Cb ) reside not on the circular chromosome, but on a large ubiquitous plasmid that is critical for virulence (42)(43)(44)(45). While Xer/dif systems have established roles in resolving aberrant plasmid dimers, these are the only Xer sequences within the C. burnetii genome.…”

A toxin-antitoxin system ensures plasmid stability in Coxiella burnetii

“…Based on the known QpH1 ORF content the resulting strain would grow normally in axenic media. Plasmid incompatibility was recently used as a method to cure NMII of the QpH1 plasmid (Luo et al, 2021). Growth analysis of the ΔQpH1 strain found no growth defect in axenic media but the presence of QpH1 was essential for growth in Vero cells.…”

“…Using this technique, Colonne et_al (Colonne et al, 2016) created gene deletions of cbuA0015 and cbuA0016 and showed these mutants had a small atypical CCV in infected THP-1 macrophages (Colonne et al, 2016). Recently a new shuttle vector (pQGK) was developed and was used to cure C. burnetii of the QpH1 plasmid (Luo et al, 2021). This QpH1-deficient strain of C. burnetii had a significant growth defect in bone marrow derived macrophages (Luo et al, 2021).…”

“…Recently a new shuttle vector (pQGK) was developed and was used to cure C. burnetii of the QpH1 plasmid (Luo et al, 2021). This QpH1-deficient strain of C. burnetii had a significant growth defect in bone marrow derived macrophages (Luo et al, 2021). Together these studies indicate that the ORF content of the C. burnetii plasmid is important for virulence.…”