Culture under normoxic conditions and enhanced virulence of phase II<i>Coxiella burnetii</i>transformed with a RSF1010-based shuttle vector

Luo, Shengdong; He, Zemin; Sun, Zhihui; Yu, Yonghui; Jiang, Yongqiang; Tong, Yigang; Song, Lihua

doi:10.1101/747220

Cited by 2 publications

(4 citation statements)

References 30 publications

(41 reference statements)

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“…This enhanced virulence of NMIIpMMGK was likely associated with this strain's ability of normoxic growth. Characterization of C. burnetii normoxic growth has been described in a separate manuscript (46). Furthermore, the sizes of splenomegaly were in accordance with the C. burnetii genome equivalents in all infected mice (Fig.…”

Section: Resultssupporting

confidence: 67%

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

Luo

Fan

et al. 2021

J Bacteriol

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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.

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Section: Resultssupporting

confidence: 67%

“…Though similar to other RSF1010-ori-based shuttle vectors, the pMMGK vector conferred an unexpected capability of normoxic growth on C. burnetii, which is likely to be related to the transformant's enhanced virulence. The normoxic growth of C. burnetii was characterized in a separate manuscript (46).…”

Section: Discussionmentioning

confidence: 99%

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

Luo

Fan

et al. 2021

J Bacteriol

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“…This enhanced virulence of NMII pMMGK is likely associated with this strain’s ability of normoxic growth. Characterization of C. burnetii normoxic growth was described in a separate manuscript [42]. Moreover, the sizes of splenomegaly are in accordance with C. burnetii genome equivalents in all infected mice (Figure 8C).…”

Section: Resultssupporting

confidence: 64%

“…Though similar with other RSF1010-ori based shuttle vectors, the pMMGK vector confers an unexpected capability of normoxic growth on C. burnetii , which likely relates to the transformant’s enhanced virulence. The normoxic growth of C. burnetii was characterized in a separate manuscript [42].…”

Section: Discussionmentioning

confidence: 99%

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

Luo

Sun

Fan

et al. 2020

Preprint

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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...

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Culture under normoxic conditions and enhanced virulence of phase IICoxiella burnetiitransformed with a RSF1010-based shuttle vector

Cited by 2 publications

References 30 publications

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

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

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

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