The endogenous Coxiella burnetii plasmid encodes a functional toxin–antitoxin system

Abstract

Coxiella burnetii is the causative agent of Q fever. All C. burnetii isolates encode either an autonomously replicating plasmid (QpH1, QpDG, QpRS, or QpDV) or QpRS-like chromosomally integrated plasmid sequences. The role of the ORFs present in these sequences is unknown. Here, the role of the ORFs encoded on QpH1 was investigated.Using a new C. burnetii shuttle vector (pB-TyrB-QpH1ori), we cured the C. burnetii Nine Mile Phase II strain of QpH1. The ΔQpH1 strain grew normally in axenic media but had a significant growth defect in Vero cells, indicating QpH1 was important for C. burnetii virulence. We developed an inducible CRISPR interference system to examine the role of individual QpH1 plasmid genes. CRISPRi of cbuA0027 resulted in significant growth defects in axenic media and THP-1 cells. The cbuA0028/cbuA0027 operon encodes CBUA0028 (ToxP) and CBUA0027 (AntitoxP), which are homologous to the HigB2 toxin and HigA2 antitoxin, respectively, from Vibrio cholerae. Consistent with toxin-antitoxin systems, overexpression of toxP resulted in a severe intracellular growth defect that was rescued by co-expression of antitoxP. ToxP inhibited protein translation. AntitoxP bound the toxP promoter (PtoxP) and ToxP, with the resulting complex binding also PtoxP. In summary, our data indicate that C. burnetii maintains an autonomously replicating plasmid because of a plasmid-based toxin-antitoxin system.

Identification of Type 4B Secretion System Substrates That Are Conserved among Coxiella burnetii Genomes and Promote Intracellular Growth

“…S4B). These defects in bacterial replication and CCV fusion are consistent with phenotypes previously reported for C. burnetii cvpB or icmD mutant strains ( 9 , 23 , 28 , 29 ). Decreased CCV size in Vero cells correlated with reduced bacterial replication in THP-1 cells for CRISPRi cbu0122_1 and _3 , cbu1752_1 and _2 , cbu1825_1 , and cbu2007_1 and _2 strains.…”

“…Of the 32 proteins analyzed in this study, only CBU0122, CBU0410, CBU1198, CBU1752, CBU1819, CBU1825, and CBU2007 were validated here or previously as bona fide C. burnetii T4BSS substrates. C. burnetii intracellular growth requirements for these T4BSS substrates were investigated using an isopropyl-β- d -1-thiogalactopyranoside (IPTG)-inducible CRISPR interference (CRISPRi) system to suppress transcription of T4BSS substrate genes ( 28 ). C. burnetii CRISPRi strains induced with IPTG expressed a catalytically inactive form of the Cas9 protein (dCas9) along with a single guide RNA (sgRNA) for targeting of dCas9 to template DNA.…”

“…For each of the T4BSS substrates, three C. burnetii CRISPRi strains were generated that expressed different sgRNA sequences for the targeted gene (Table S2.) For positive controls of growth impairment, a C. burnetii CRISPRi strain was included that expressed sgRNA targeting the essential T4BSS structural gene icmD ( 28 ), along with three different CRISPRi strains that targeted cvpB , an effector required for intracellular growth ( 23 , 29 , 30 ). A C. burnetii strain that expressed dCas9 but did not target sgRNA was used as a negative control ( 28 ).…”

“…For positive controls of growth impairment, a C. burnetii CRISPRi strain was included that expressed sgRNA targeting the essential T4BSS structural gene icmD ( 28 ), along with three different CRISPRi strains that targeted cvpB , an effector required for intracellular growth ( 23 , 29 , 30 ). A C. burnetii strain that expressed dCas9 but did not target sgRNA was used as a negative control ( 28 ). In C. burnetii strains treated with IPTG, the expression of CRISPRi constructs was confirmed by immunoblotting of dCas9 (Fig.…”

An expanded genetic toolkit for inducible expression and targeted gene silencing inRickettsia parkeri

“…Different variants of dCas9 recognize different PAMs 23 , meaning that each dCas9 has a different repertoire of possible guide sequences that can be used to target a genome of interest. CRISPRi has been used for efficient and scalable gene knockdown in a wide variety of bacteria including Mycobacterium tuberculosis 24 , Caulobacter crescentus 25 , Chlamydia trachomatis 26 , and Coxiella burnetii 27,28 .…”