Identifying Coxiella burnetii Genes Essential for Subversion of the Host Immune System

Coxiella burnetii, the causative agent of Q fever in humans, is a Gram-negative intracellular bacterium. Although the organism was first isolated in the 1930s, little is known about the specific mechanisms underlying its virulence. This is largely due to its obligate intracellular lifestyle. Recent advances in both axenic growth and genetic manipulation of C. burnetii allowed efficient generation and isolation of random mutations and enabled more definitive studies of the genes essential for virulence. The goal of this project was to generate a large collection of specific isogenic C. burnetii mutants and employ in vitro and in vivo screens to determine the individual contributions of their affected genes to pathogenicity. We used a Himar1 transposon system to generate a library of 1) defined clonal mutants and 2) pools of random transposon mutants in order to approach saturation with non-lethal mutants of the low virulence C. burnetii isolate, Nine Mile, phase II (NMII), RSA 439, which is approved for use in a biosafety level two laboratory. Mutants from both libraries were compared in various growth conditions or infection models to identify differences in growth phenotype relative to wild-type C. burnetii. The libraries are also amenable to high-throughput analysis using transposon sequencing or transposon directed insertion site sequencing (TraDIS) to compare pooled mutants between input and output infection assays. In this study, we optimized methods to generate defined transposon mutants, resulting in mutations in nearly 20% of the predicted open reading frames (ORFs) and provide methodology to expand the library for future studies. Included in these mutants were a number of bioinformatically predicted virulence factors based on phenotypes in other bacterial pathogens that we further compared in cellular and animal models of infection. Our findings are consistent with previous studies that demonstrate the Dot/Icm T4BSS is essential for generating and replicating in a large parasitophorous vacuole (PV). We developed methods for high throughput screening of Tn mutants in vitro by imaging on either confocal microscope or BioTek Cytation3 imaging system. We generated a genome saturation transposon mutant pool by combining transposon mutant pools from 35 independent transformation reactions. These combined in vitro and in vivo screens dramatically improve our knowledge of specific virulence determinants for this pathogen and provide a substantial amount of data for future studies

The Type IV Secretion System Effector Protein CirA Stimulates the GTPase Activity of RhoA and Is Required for Virulence in a Mouse Model of Coxiella burnetii Infection

Coxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial for C. burnetii intracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment of Coxiella burnetii infection and virulence in mammalian cell culture and mouse models of infection

Identification of Coxiella burnetii Type IV Secretion Substrates Required for Intracellular Replication and Coxiella-Containing Vacuole Formation

Coxiella burnetii, the etiological agent of acute and chronic Q fever in humans, is a naturally intracellular pathogen that directs the formation of an acidic Coxiella-containing vacuole (CCV) derived from the host lysosomal network. Central to its pathogenesis is a specialized type IVB secretion system (T4SS) that delivers effectors essential for intracellular replication and CCV formation. Using a bioinformatics-guided approach, 234 T4SS candidate substrates were identified. Expression of each candidate as a TEM-1 β-lactamase fusion protein led to the identification of 53 substrates that were translocated in a Dot/Icm-dependent manner. Ectopic expression in HeLa cells revealed that these substrates trafficked to distinct subcellular sites, including the endoplasmic reticulum, mitochondrion, and nucleus. Expression in Saccharomyces cerevisiae identified several substrates that were capable of interfering with yeast growth, suggesting that these substrates target crucial host processes. To determine if any of these T4SS substrates are necessary for intracellular replication, we isolated 20 clonal T4SS substrate mutants using the Himar1 transposon and transposase. Among these, 10 mutants exhibited defects in intracellular growth and CCV formation in HeLa and J774A.1 cells but displayed normal growth in bacteriological medium. Collectively, these results indicate that C. burnetii encodes a large repertoire of T4SS substrates that play integral roles in host cell subversion and CCV formation and suggest less redundancy in effector function than has been found in the comparative Legionella Dot/Icm model