Genetics of Coxiella burnetii

National Institute Of Allergy And Infectious Diseases

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Abstract

Coxiella burnetii is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness. Coxiella's former obligate intracellular nature significantly impeded genetic characterization of putative virulence factors. However, our seminal advance of host cell-free (axenic) growth of Coxiella in acidified citrate cysteine medium (ACCM) enabled us to quickly develop a a complete genetics tool box. Most recently, we developed a defined medium that supports robust growth of Coxiella called ACCM-D that contains amino acids as sole carbon and energy sources. Coxiella is auxotrophic for Arg, Lys, Pro and Tyr by lacking the final enzymes in biosynthesis. Heterologous expression by Coxiella of Legionella pneumophila argGH, lysA and proAB and E.coli tyrB rescues growth in Arg, Lys, Pro and Tyr ACCM-D dropout media, respectively, thus providing four methods for nutritional selection of Coxiella transformants. Strong, non-antibiotic-based selection of genetic transformants is an important advance considering selectable markers based on antibiotic resistance are limited for this select agent. Collectively, our repertoire of Coxiella genetic tools now allows traditional mutation and complementation strategies for virulence factor discovery. Indeed, we have constructed knockout strains in both virulent and avirulent Coxiella, including those with deletions in genes encoding components of the Dot/Icm type IVB secretion system (T4BSS) and secreted proteins. These studies have confirmed that T4BSS function is critical for Coxiella growth in macrophages. Moreover, using Cre-lox, we have created a 32.4 kb dot/icm mutant of the virulent Nine Mile phase I strain that lacks the entire dot/icm locus required for synthesis of the T4BSS. The mutant displays vigorous growth in synthetic medium but cannot grow intracellularly. Mutational analysis has also identified genetic mechanisms of LPS phase variation associated with virulence. All Coxiella strains sequenced to date carry a large (32-54 kb), autonomously replicating plasmid or have chromosomally integrated plasmid-like sequences, suggesting that plasmid genes are important for infection. Seven genes on the QpH1 plasmid, carried by the reference Nine Mile strain, encode type 4B secretion system effector proteins suspected in mediating virulence. Only two of these genes are conserved between Coxiella plasmids or IPS. We developed a new E. coli-Coxiella shuttle vector (pBR322-CAT-sacB-tyrB-QpH1ori) that contains the QpH1 origin of replication cloned into an E. coli plasmid containing the tyrB gene. Coxiella production of TyrB in the presence of the tyrosine precursor 4-hydroxyphenylpyruvic acid (4-HPA) rescues the bacterium's natural auxotrophy for tyrosine. A CRISPRi system for gene silencing was also developed for Coxiella that utilizes proBA genes from Legionella to complement Coxiella's natural auxotrophy for proline. Introduction of pBR322-CAT-sacB-tyrB-QpH1ori into C. burnetii Nine Mile (phase II), followed by growth in tyrosine-deficient ACCM-D supplemented with 4-HPA, resulted in expulsion of the native QpH1 plasmid. The mutant strain grew normally in axenic medium but had a severe growth defect in host cells. Complementation using large fragments of QpH1 identified general regions required for intracellular growth. CRISPRi gene silencing is being used to identify individual QpH1 genes essential for Coxiella growth in host cells. This study identified two novel methods of nutritional selection of genetic transformants of Coxiella and established a CRISPRi system for robust conditional knockdown of gene expression. Coxiella encodes a paucity of transcriptional regulators that are likely critical for intramacrophage survival and/or developmental transitions. The PhoBR two-component system (TCS) of Coxiella is especially intriguing as homologous systems in other bacteria regulate virulence gene expression. Using gene knockouts, reporter assays, RNAseg, and whole bacterial proteome mass spectrometry, we resolved the regulatory cascade of PhoBR. Unraveling the regulatory networks of PhoBR and other TCS's will identify important virulence determinants. Coxiella undergoes an intracellular biphasic developmental cycle that generates two distinct morphological variants that can be distinguished by ultrastructure and protein composition. Small cell variants (SCV) do not replicate, contain condensed chromatin, and are considered extracellular survival forms. SCV differentiate into replicative large cell variants (LCV) with dispersed chromatin. Transition of LCV back to SCV occurs coincident with Coxiella entry into stationary growth phase, with nearly homogeneous SCV present upon extended incubation (2 to 4 weeks) of infected cell cultures. As an amenable model to help better understand the biological relevance of Coxiella differentiation, we established that SCV/LCV transitions are recapitulated by organisms growing in the third-generation axenic media, ACCM-D. This discovery enables studies of Coxiella developmental biology without experimental difficulties encountered with host cell-propagated bacteria. Comparative transcriptomics and proteomics of LCV and SCV have now revealed molecular determinants of morphological differentiation that likely contribute to the unique biological characteristics of cell forms. Genes associated with differentiation are now being inactivated and mutants phenotyped. The current human Q fever vaccine, Q-VAX, is a fixed, whole cell vaccine (WCV) and is licensed solely for use in Australia. While highly efficacious, Coxiella WCVs are associated with a potentially severe postvaccination dermal hypersensitivity reaction in people with pre-existing immunity to Coxiella, which limits their wider use. Consequently, a less reactogenic vaccine is needed. We investigated contributions of the Coxiella Dot/Icm type IVB secretion system (T4BSS) and lipopolysaccharide (LPS) in protection and reactogenicity of fixed WCVs. A 32.5 kb region containing 23 dot/icm genes was deleted in the virulent Nine Mile phase I (NMI) strain and the resulting mutant was evaluated in guinea pig models of Q fever infection, vaccination and challenge, and post-vaccination hypersensitivity. The NMI dot/icm strain was avirulent, protective as a WCV against a robust Coxiella challenge, and displayed potentially altered reactogenicity compared to wild type Coxiella. NMI and isogenic Nine Mile phase II (NMII) strains of Coxiella that produce smooth and rough LPS respectively, were similarly tested. NMI was significantly more protective than NMII as a WCV; however, both vaccines exhibited similar reactogenicity. Collectively, our results indicate that, like phase I LPS, the T4BSS is required for full virulence by Coxiella. Conversely, unlike phase I LPS, the T4BSS is not required for vaccine-induced protection. LPS length also does not appear to contribute to the dermal hypersensitivity reaction while the T4BSS may contribute to this response. NMI dot/icm represents an avirulent phase I strain with full vaccine efficacy and illustrates the potential of using genetically modified Coxiella as improved WCVs. As part of NIAID/RMLs response to the COVID-19 pandemic, the CPS collaborated with the Sonja Best lab (LV) to generate lentiviral constructs for expressing the SARS-CoV-2 receptor ACE2, and associated serine protease TMPRSS2, in mammalian cells. The purpose was to generate cell lines that support robust infection of SARS-CoV-2 for production of high titer viral stocks. Sensitive cell culture systems to investigate viral interactions with the innate immune system were also generated using lentiviral systems.