Cellular and Developmental Biology of Coxiella burnetii

伯氏梭菌的细胞内生活方式使人们认为细胞介导的免疫是抵抗这种病原体的最重要的免疫成分。然而，用免疫血清被动免疫可以保护幼稚动物免受强毒伯纳蒂胞杆菌的攻击，这表明抗体（Ab）在保护中起作用。这种抗体介导免疫（AMI）的机制尚不清楚。我们之前已经证明，毒力强的伯氏梭菌在树突状细胞（DC）内感染和复制，而不诱导它们的成熟或激活。我们有新的数据表明，人类单核细胞来源和小鼠骨髓来源的DC摄取Ab活化的毒性伯纳氏梭菌会导致这些细胞成熟并产生炎症细胞因子。Ab活化增加了DC对伯氏梭菌的吸收率，但对病原体的复制没有影响。与C57Bl/6 （B6）小鼠相比，敲除Fc受体（FcR）对DC的反应（FcR k/o）降低，证明了Ab活化的伯氏梭菌对DC的影响是Fc受体（FcR）依赖性的。为了研究FcR在AMI中的潜在体内作用，我们比较了被动免疫的FcR k/o小鼠与B6对照组对感染的反应。有趣的是，在FcR k/o小鼠中AMI没有改变。因此，尽管体外实验中FcR在DC对Ab-opsonized burnetii的反应中发挥了重要作用，但在体内AMI是与FcR无关的。我们目前正在研究补体在AMI中的作用。

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 naive animals from challenge with virulent C. burnetii, indicating a role for antibody (Ab) in protection. The mechanism of this Ab-mediated immunity (AMI) is unknown. We have previously demonstrated that virulent C. burnetii infect and replicate within dendritic cells (DC) without inducing their maturation or activation. We have new data showing that uptake of Ab opsonized virulent C. burnetii by human monocyte-derived and murine bone marrow-derived DC causes these cells to mature and produce inflammatory cytokines. Ab opsonization increases the rate C. burnetii uptake by DC, but has no effect on pathogen replication. The effect of Ab opsonized C. burnetii on DC is Fc receptor (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 in vivo role of FcR in AMI, we compared the response to infection of passively immunized FcR k/o mice to the B6 controls. Interestingly, AMI is unaltered in FcR k/o mice. Thus, despite the important role of FcR in the response of DC to Ab-opsonized C. burnetii in vitro, AMI in vivo is FcR independent. We are currently investigating the role of complement in AMI. C. burnetii has a tropism for mononuclear phagocytes where it directs biogenesis of a replicative vacuolar niche termed the parasitophorous vacuole (PV). Owing to its lumenal acdic pH (5) and acquisition of multiple lysosomal proteins, the PV is considered lysosome-like. However, 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. Moreover, phase variants induce secretion of similar amounts of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor by HMDM. PV containing phase I or phase II organisms decorate similarly at early (8 h) and late (72 h) time points post-infection for 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 lysosome-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. 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 type IV secretion system with homology to the Dot/Icm system of Legionella pneumophila. Using L. pneumophila producing C. burnetii proteins fused to Bordetella pertussis adenylate cyclase, we have identified 22 effectors that are translocated to the host cytosol in a Dot/Icm-dependent fashion. Five 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 7 remaining effectors are chromosomally-encoded hypothetical proteins. Deletion analysis indicates the Dot/Icm secretion signal is located within the N-terminal 70 amino acids. Ectopic expression of effector-fluorescent protein fusions in mammalian cells reveals a variety of subcellular localizations including microtubules and the C. burnetii PV membrane. 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 results in robust cell division of C. burnetii was observed with greater than 2.5 logs of bacterial replication over 6 days. 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. Axenic growth of C. burnetii in ACCM will dramatically aid future studies of the organism aimed at elucidating the molecular biology and relevance of biphasic development, developing genetic tools, and identifying virulence factors important in the pathophysiology of clinical Q fever.