The obligate intracellular bacterial pathogen C. burnetii burnetii causes the zoonosis Q fever. The intracellular lifestyle of C. burnetii has led to the assumption that cell-mediated immunity is the most important immune component for protection against this pathogen. However, passive immunization with immune serum can protect nave animals from challenge with virulent C. burnetii, indicating a role for antibody (Ab) in protection. The mechanism of this Ab-mediated protection is unknown. Therefore, we conducted a study to determine whether Fc receptors (FcR) or complement contribute to Ab-mediated immunity (AMI) to C. burnetii. Infection of DC with Ab-opsonized C. burnetii resulted in increased expression of maturation markers and inflammatory cytokine production. The effect of Ab opsonized C. burnetii on DC was FcR dependent as evidenced by a reduced response of DC from FcR knockout (FcR k/o) compared to C57Bl/6 (B6) mice. To address the potential role of FcR in Ab-mediated protection in vivo, we compared the response of passively immunized FcR k/o mice to the B6 controls. Interestingly, we found that FcR are not essential for AMI to C. burnetii in vivo. We subsequently examined the role of complement in AMI by passively immunizing and challenging several different strains of complement-deficient mice and found that AMI to C. burnetii is also complement-independent. C. burnetii has a tropism for mononuclear phagocytes where it directs biogenesis of a phagolysosome-like niche termed the parasitophorous vacuole (PV). The degradative properties of the PV are unknown. Moreover, there are conflicting reports on the nature and growth permissiveness of PV harboring virulent phase I or avirulent phase II C. burnetii in human macrophages. Employing infection of primary human monocyte-derived macrophages (HMDM) and THP-1 cells as host cell models, we directly compared the replication of phase I and phase II variants and the characteristics of their respective PV. In both cell types, phase variants replicate with similar kinetics and induce secretion of similar amounts of pro-inflammatory cytokines (Il-6 and TNF). PV containing phase I or phase II organisms decorate similarly at early (8 h) and late (72 h) time points post-infection with the early endosomal marker Rab5, the late endosomal/lysosomal markers Rab7 and CD63, and lysosomal marker cathepsin D. PV harboring phase I or phase II C. burnetii quickly degrade E. coli and contain proteolytically active cathepsin D. Thus, the phagolysosome-like PVs of virulent and avirulent C. burnetii are phenotypically-indistinguishable, degradative compartments that are equally permissive for pathogen growth. These data indicate infection of human macrophages with biosafety level 2 phase II C. burnetii is an accurate model to investigate host-pathogen interactions. We have previously reported that cholesterol is important for C. burnetii PV biogenesis. Interestingly, C. burnetii encodes homologs of two eucaryotic-like sterol reductases CBU1158 (sterol delta-7 reductase) and CBU1206 (sterol delta-24 reductase) that in mammalian cells catalyze reduction reactions in the final steps of cholesterol biosynthesis. We have preliminary data showing that CBU1206 is a functional reductase that rescues a Saccharomyces cerevisiae delta-24 reductase mutant (erg4) from brefeldin A sensitivity. We hypothesize that these orphan enzymes modify host cholesterol intermediates that serve critical structural and/or signaling roles in PV development. In all cell types examined, C. burnetii carries out a lengthy infectious cycle with minimal cytopathic effect. We recently demonstrated that C. burnetii inhibits apoptotic cell death during infection of human macrophages, presumably as a pathogenic strategy to ensure a stable, intracellular niche. A multiplex analysis of 15 host signaling proteins involved in stress response, cytokine production, and cell survival reveals sustained activation (at least 72 h post-infection) of the pro-survival kinases Akt and Erk1/2. Activation is abolished if cells are treated with rifampin to inhibit C. burnetii RNA synthesis, thereby indicating activation is a pathogen directed event. Pharmacological inhibition of Akt or Erk1/2 significantly decreased C. burnetiis anti-apoptotic activity. Collectively, these results indicate the importance of C. burnetii modulation of host signaling and demonstrate a vital role for Akt and Erk1/2 in successful intracellular parasitism and maintenance of host viability. Protein effectors of C. burnetii PV biogenesis and anti-apoptosis/pro-survival signalling are likely translocated into the host cytosol via a Dot/Icm type IV secretion system (T4SS). Using L. pneumophila producing C. burnetii proteins fused to adenylate cyclase, we have identified 29 effectors that are translocated to the host cytosol in a Dot/Icm-dependent fashion. Six effectors (all hypothetical proteins) are encoded by the C. burnetii cryptic plasmid, thereby implicating a hitherto unknown role for this molecule in host cell manipulation. Eleven effectors contain eukaryotic-like ankyrin repeat domains (Anks). Interestingly, C. burnetii isolates with different virulence potential encode disparate numbers of intact Anks, suggesting Anks may modulate strain-specific virulence. The 12 remaining effectors are chromosomally-encoded hypothetical proteins. Further characterization of C. burnetii Dot/Icm effectors will provide critical insight into the pathogen/host interplay that results in PV establishment, host cell survival, and pathotype-specific virulence. A biphasic developmental cycle whereby highly resistant small cell variants (SCV) are generated from large cell variants (LCV) is considered fundamental to C. burnetii virulence. Previous work from our lab revealed that LCV are the replicative form of C. burnetii, and that SCV and LCV are compositionally and antigenically different. Further molecular and biochemical analyses of SCV and LCV morphogenesis is necessary to better understand the physiological relevance of C. burnetii biphasic development. However, the obligate intracellular nature of C. burnetii imposes considerable experimental constraints that would be circumvented by axenic (host cell free) growth. To this end, we developed a medium termed Acidified Cysteine Citrate Medium (ACCM) that supports robust axenic replication of C. burnetii. ACCM was derived from Complex Coxiella Medium (CCM), which we previously demonstrated to sustain C. burnetii metabolic activity, but not replication. Expression microarray analysis of C. burnetii incubated in CCM showed reduced pathogen expression of ribosomal genes, suggesting suboptimal protein synthesis. Subsequent supplementation of CCM with additional amino acids and peptides, to create ACCM, significantly increased C. burnetii de novo protein synthesis. Moreover, Phenotype Microarray analysis revealed that C. burnetii is a microaerophile. When incubated in ACCM in a 2.5% oxygen environment robust cell division of C. burnetii was observed with greater than 3 logs of bacterial replication over 6 days. Importantly, organisms cultivated in ACCM are as infectious for Vero cells as cell culture propagated organisms. Moreover, ACCM cultured organisms undergo SCV to LCV development indistinguishable from C. burnetii propagated in vivo. ACCM agar also supports colony growth, which will facilitate isolation of clonal populations C. burnetii.
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