The SCID Mouse Model for Identifying Virulence Determinants in Coxiella burnetii

Schaik, Erin J. van; Case, Elizabeth Di Russo; Martínez, Eric; Bonazzi, Matteo; Samuel, James E.

doi:10.3389/fcimb.2017.00025

Cited by 37 publications

(61 citation statements)

References 38 publications

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“…In Galleria mellonella , injection of WT Coxiella results in efficient killing of larvae and this is attenuated in the absence of cig2 [15,34]. Similarly, cig2 ::Tn bacteria were recovered from the spleen and lung of severe combined immunodeficiency (SCID) mice at significantly lower levels than WT Coxiella [35]. Hence, the CCV being maintained in an autolysosomal state, through Cig2 activity, is required for full virulence of Coxiella.…”

Section: Discussionmentioning

confidence: 99%

Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)

Latomanski

Newton

2018

Autophagy

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Coxiella burnetii is an intracellular bacterial pathogen which causes Q fever, a human infection with the ability to cause chronic disease with potentially life-threatening outcomes. In humans, Coxiella infects alveolar macrophages where it replicates to high numbers in a unique, pathogen-directed lysosome-derived vacuole. This compartment, termed the Coxiella-containing vacuole (CCV), has a low internal pH and contains markers both of lysosomes and autophagosomes. The CCV membrane is also enriched with CLTC (clathrin heavy chain) and this contributes to the success of the CCV. Here, we describe a role for CLTC, a scaffolding protein of clathrin-coated vesicles, in facilitating the fusion of autophagosomes with the CCV. During gene silencing of CLTC, CCVs are unable to fuse with each other, a phenotype also seen when silencing genes involved in macroautophagy/autophagy. MAP1LC3B/LC3B, which is normally observed inside the CCV, is excluded from CCVs in the absence of CLTC. Additionally, this study demonstrates that autophagosome fusion contributes to CCV size as cell starvation and subsequent autophagy induction leads to further CCV expansion. This is CLTC dependent, as the absence of CLTC renders autophagosomes no longer able to contribute to the expansion of the CCV. This investigation provides a functional link between CLTC and autophagy in the context of Coxiella infection and highlights the CCV as an important tool to explore the interactions between these vesicular trafficking pathways.

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Section: Discussionmentioning

confidence: 99%

Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)

Latomanski

Newton

2018

Autophagy

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“…The caveat of working with phase II C. burnetii is the lack of animal models for assessing in vivo pathogenesis. To promote the identification of virulence factors in phase II C. burnetii , an SCID mouse model was recently established (17). An alternative infection model using larvae of the greater wax moth Galleria mellonella was also used to identify virulence factors in phase II C. burnetii (18, 19).…”

Section: Introductionmentioning

confidence: 99%

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

Luo

Sun

et al. 2019

Preprint

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20Coxiella burnetii is a Gram-negative, facultative intracellular microorganism that can 21 cause acute or chronic Q fever in human. It was recognized as an obligate intracellular 22 organism until the revolutionary design of an axenic cystine culture medium (ACCM). 23Present axenic culture of C. burnetii strictly requires a hypoxic condition (<10% 24 oxygen). Here we investigated the normoxic growth of C. burnetii strains in ACCM-2 25 with or without tryptophan supplementation. Three C. burnetii strains -Henzerling 26 phase I, Nine Mile phase II and a Nine Mile phase II transformant, were included. The 27 transformant contains a pMMGK plasmid that is composed of a RSF1010 ori, a 28 repABC operon, an eGFP gene and a kanamycin resistance cassette. We found that, 29 under normoxia if staring from an appropriate concentration of fresh age inocula, 30 Nine Mile phase II can grow significantly in ACCM-2 with tryptophan, while the 31 transformant can grow robustly in ACCM-2 with or without tryptophan. In contrast, 32 long-term frozen stocks of phase II and its transformant, and Henzerling phase I of 33 different ages had no growth capability under normoxia under any circumstances. 34 Furthermore, frozen stocks of the transformant consistently caused large 35 splenomegaly in SCID mice, while wild type Nine Mile phase II induced a lesser 36 extent of splenomegaly. Taken together, our data show that normoxic cultivation of 37 phase II C. burnetii can be achieved under certain conditions. Our data suggests that 38 tryptophan and an unknown temperature sensitive signal are involved in the 39 expression of genes for normoxic growth regulated by quorum sensing in C. burnetii. 40 129 Mile phase II and NMIIpMMGK were used. In the second experiment, 9 five weeks 130 old mice were averaged into three infection groups: one month old Nine Mile phase II, 131 one-month old NMIIpMMGK and 19 months old NMIIpMMGK. At indicated days 132 post-infection, mice were weighted and sacrificed to harvest spleens to determine 133 splenomegaly (spleen weight/body weight). Each spleen was homogenized in 2 mL 134 PBS. Total DNAs from 20 μL of each tissue homogenate were purified with DNeasy 135

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“…The SCID mouse model was established for identifying virulence factors in C. burnetii phase II [41]. We next analyzed the plasmid’s potential role in C. burnetii pathogenesis by using this model (Figure 8).…”

Section: Resultsmentioning

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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The SCID Mouse Model for Identifying Virulence Determinants in Coxiella burnetii

Cited by 37 publications

References 38 publications

Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)

Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)

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

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

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