Cellular and Developmental Biology of Coxiella burnetii

高抗性小细胞变异（SCV）由大细胞变异（LCV）产生的双相发育周期被认为是伯纳氏杆菌毒力的基础。我们对burnetii发育形式进行了蛋白质组分析，以深入了解其独特的生物学和免疫学特性。通过二维凝胶电泳分离的SCV和LCV裂解物的银染色凝胶可分辨出675种发育形式的蛋白质。48种蛋白在LCV中的丰度是SCV的2倍以上，6种蛋白在SCV中的丰度是LCV的2倍以上。通过质谱分析分别鉴定了4个和15个SCV和LCV的上调蛋白，其预测的功能作用与代谢活跃的LCV和结构抗性的SCV一致。分别用感染/接种豚鼠血清和人急性Q热患者恢复期血清检测细胞形态裂解物的一维和二维免疫印迹，显示出独特的SCV/LCV抗原和常见的SCV/LCV抗原，这些抗原通常是差异合成的。通过质谱鉴定了人感染过程中识别的抗原，包括先前描述的伯纳蒂胞杆菌免疫优势蛋白以及可能在临床Q热的病理生理学和/或诱导保护性免疫中重要的新型免疫原性蛋白。

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. We conducted a proteome analysis of C. burnetii developmental forms to provide insight into their unique biological and immunological properties. Silver-stained gels of SCV and LCV lysates separated by two-dimensional (2-D) gel electrophoresis resolved over 675 proteins in both developmental forms. Forty-eight proteins were greater than two-fold more abundant in LCV than SCV with six proteins greater than two-fold more abundant in SCV than LCV. Four and 15 upregulated proteins of SCV and LCV, respectively, were identified by mass spectrometry and their predicted functional roles are consistent with a metabolically active LCV and a structurally resistant SCV. One dimensional and 2-D immunoblots of cell form lysates probed with sera from infected/vaccinated guinea pigs and convalescent serum from human acute Q fever patients, respectively, revealed both unique SCV/LCV antigens and common SCV/LCV antigens that were often differentially synthesized. Antigens recognized during human infection were identified by mass spectroscopy and included both previously described immunodominant proteins of C. burnetii as well as novel immunogenic proteins that may be important in the pathophysiology of clinical Q fever and/or the induction of protective immunity. C. burnetii actively participates in the biogenesis of its parasitophorous vacuole (PV) by synthesizing proteins that modulate vesicular interactions and other host processes. Functional characterization of these molecules will yield important insight into C. burnetii-host interactions, and we are currently using there complementary strategies to achieve this goal. The first strategy employs subcellular fractionation and proteomics. C. burnetii and host factors that regulate PV formation are likely localized to the PV membrane; therefore, their identification would be aided by an efficient method to isolate this vacuole. To this end, we developed a method to separate intact PV from host cell material that relies on fusion of the vacuole with latex bead-containing phagosomes (LBP). Transmission electron microscopy confirms the isolation of intact PV containing latex beads. Immunoblotting demonstrates that C. burnetii PV lysates are dramatically enriched for the late endosome/lysosome markers LAMP-1 and LAMP-2 when compared to total host cell lysates. Conversely, PV preparations are devoid of p62 and GM130, markers of the nucleus and Golgi apparatus, respectively, indicating effective separation of the vacuole from these host cell compartments. Two-dimensional gel electrophoresis and immunoblotting reveal distinct protein differences between C. burnetii PV and LBP that may refect specific recruitment of C. burnetii effectors to the PV membrane. The second approach exploits the presence of a specialized Coxiella type IV secretion system with close homology to that of its relative Legionella pneumophila. We have successfully used Legionella as a surrogate host and the calmodulin-activated adenylate cyclase (Cya) screen to identify candidate Coxiella type IV secretion substrates. The Cya screen relies on the fact that elevated adenylate cyclase activity is only observed when a Cya-Coxiella fusion protein is delivered to the host cytoplasm. Using this strategy, we have now identified 12 Coxiella proteins that are secreted by Legionella in a type IV-dependent fashion. These are currently being functionally characterized. The final strategy relies on secretion of effectors by Coxiella in a cell free medium. Axenic (host cell free) buffers have been described that activate C. burnetii metabolism in vitro, but metabolism is short-lived with bacterial protein synthesis halting after a few hours. By systematically evaluating complex nutrient mixtures, we developed an axenic medium termed Complex Coxiella Medium (CCM) that supports robust and sustained C. burnetii metabolic activity in the form of protein and ATP synthesis. Moreover, we have also shown that maximal respiratory chain activity occurs in an atmosphere of 2.5% oxygen, consistent with microaerophilic metabolism in this bacterium. Not only does the robust metabolic activity of C. burnetii in CCM provide a potential means to identify secreted Coxiella proteins, it also represents a powerful in vitro tool to study metabolic responses to stresses that are likely encountered by the pathogen within the host cell. A potential target of secreted C. burnetii effector molecules is the apoptotic cell death pathway. Indeed, in all cell types examined, C. burnetii carries out a lengthy infectious cycle with minimal cytopathic effects. We recently demonstrated that C. burnetii inhibits apoptotic cell death during infection of human THP-1 monocyte-derived macrophages and primary monkey alveolar macrophages. C. burnetii-infected cells show significant protection from death relative to uninfected cells following treatment with staurosporine, a potent inducer of mitochondrial-mediated intrinsic apoptosis. This protection correlates with reduced cleavage of caspase-9, caspase-3, and poly (ADP-ribose) polymerase, all proteolytic events that occur during apoptosis. In addition to intrinsic apoptosis, C. burnetii-infected cells are also protected from extrinsic apoptosis induced by TNF-alpha. Infected cells demonstrate sustained activation of the pro-survival serine/threonine kinase Akt that may provide protection from cell death via both intrinsic and extrinsic pathways. Infected cells display increased synthesis of the anti-apoptotic proteins A1/Bfl-1 and c-IAP2 and altered expression of multiple anti- and pro-apoptotic genes. Collectively, these data suggest that C. burnetii stimulates host cell survival signaling and modulates both extrinsic death receptor-mediated and intrinsic mitochondrial-mediated apoptotic pathways to inhibit host cell death. This is likely a pathogenic strategy that ensures a stable, intracellular niche for the course of the pathogens infectious cycle. The observation that inhibition of apoptosis requires C. burnetii RNA and protein synthesis strongly implicates a role for secreted pathogen effector molecules in this process. 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 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 increased the rate C. burnetii uptake by DC, but had no effect on pathogen replication. The effect of Ab opsonized C. burnetii on DC was 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 role of FcR in AMI in vivo, we compared the response of passively immunized FcR k/o mice to the B6 controls. Interestingly, AMI was unaltered in FcR k/o mice indicating that this phenomenon is not FcR dependent in vivo. Our results indicate that, despite the important role of FcR in the response of DC to Ab-opsonized C. burnetii in vitro, AMI in vivo is FcR independent.