摘要
Tularemia is a potentially fatally illness caused by the facultative intracellular Gram-negative bacterium Francisella tularensis. Virulent strains of F. tularensis can cause a fatal disease after inhalation of a few as ten organisms. Due to the highly pathogenic features of Francisella, it has been designated as a Tier 1 select agent, meaning that its possession and handling is highly restricted. Macrophages are phagocytes that play a central role in the innate immune response to infection that can be used by certain pathogens, including Francisella, as a niche for bacterial replication and dissemination during infection. After infection of macrophages Francisella escapes from the phagosome and replicates in the cytosol, however the bacterial factors required for these aspects of virulence are incompletely defined.
Here we describe the isolation and characterization of F. tularensis subspecies tularensis strain Schu S4 mutants in iglI, iglJ, and pdpC, three genes located in the Francisella Pathogenicity Island. Our data demonstrate that these mutants were unable to replicate in macrophages due to a defect in phagosome escape. However, a small percentage of pdpC mutants were able to reach the cytosol and replicate moderately. Both iglJ and pdpC mutants were highly attenuated for virulence in a mouse intranasal infection model, however pdpC but not iglJ mutants, were able to disseminate from the lung before eventual clearance. These data demonstrated that the FPI genes tested were essential for F. tularensis Schu S4 virulence, but suggest that they may have different functions due to the unique phenotype observed for pdpC mutants.
Our studies also characterized the role of F. tularensis O-antigen and capsule to facilitate interactions with components of the serum complement system; demonstrating that the O-antigen is required for binding of IgM to the bacteria in order to initiate complement opsonization. IgM dependent complement opsonization of both F. tularensis Schu S4 and LVS strains facilitated enhanced phagocytosis of the bacteria by complement receptors 3 and 4 of human macrophages. In addition, we examined the mechanisms of macrophage cytotoxicity and proinflammatory cytokine secretion that was induced after infection with a Schu S4 LPS O-antigen and capsule mutant. The response to the mutant was dependent on phagosome escapes, suggesting a cytosolic pattern recognition receptor was involved in recognition of the bacteria. We found that the cytotoxic and proinflammatory responses had both similar and distinct requirements between human and murine macrophages. Infection with the O-antigen mutant induced robust proinflammatory cytokine secretion that was dependent on caspase-1, cathepsin B, and ASC while cytotoxicity was partially dependent on these molecules. Importantly, we demonstrated that wild-type Schu S4 predominately activated apoptotic caspases, and not inflammatory caspases, during infection and had a blunted cytotoxic response. This was in contrast to the robust cytotoxicity and activation of inflammatory caspases after infection with the non-virulent strain LVS. Together, these studies demonstrated that the Schu S4 LPS O-antigen and capsule are required for evasion of macrophage cytosolic host defense mechanisms.
摘要译文
Tularemia是一种潜在致命的疾病，由兼性细胞内革兰氏阴性细菌弗兰西斯拉氏杆菌吸入几十种微生物后，土拉氏菌可引起致命疾病。由于Francisella的高致病性特征，已被指定为第1级选择剂，意味着其拥有和处理受到高度限制。巨噬细胞是吞噬细胞，其在对特定病原体可以使用的感染的先天免疫应答中起核心作用，包括Francisella，作为感染期间细菌复制和传播的利基。在从噬菌体感染巨噬细胞弗朗西斯菌逃逸并在细胞质中复制之后，然而这些毒性方面所需的细菌因子是不完全确定的。这里我们描述F the的分离和表征iglI，iglJ和pdpC中的土拉木亚种土拉氏菌菌株Schu S4突变体，位于Francisella PathogenicityIsland 中的三个基因我们的数据表明，这些突变体由于吞噬体逃逸的缺陷而无法在巨噬细胞中复制。然而，小百分比的pdpC突变体能够到达细胞溶质并中等重复。在小鼠鼻内感染模型中，两种iglJ和pdpC突变体对于毒力高度减毒，然而pdpC但不是iglJ突变体，能够在最终清除之前从肺传播。这些数据表明，测试的FPI基因对于F 是必需的但是，由于对pdpC突变体的独特表型，它们可能具有不同的功能。我们的研究还表征了F the土拉硫醚O抗原和胶囊，以促进与血清补体系统的成分的相互作用;证明O抗原是IgM与细菌结合所必需的，以启动补体调理作用.IgM依赖性补体调理作用土拉氏菌Schu S4和LVS菌株通过人巨噬细胞的补体受体3和4促进了细菌对细菌的吞噬作用。此外，我们检测了用Schu S4 LPS O抗原和胶囊突变体感染后诱导的巨噬细胞细胞毒性和促炎细胞因子分泌的机制对突变体的反应依赖于吞噬体逃逸，表明细胞溶质模式识别受体参与细菌的识别我们发现细胞毒性和促炎反应在人和鼠巨噬细胞之间具有相似和不同的要求与O抗原突变体的感染诱导依赖于caspase-1，组织蛋白酶B的强烈促炎细胞因子分泌，ASC，而细胞毒性部分依赖于这些分子。重要的是，我们证明野生型Schu S4主要激活凋亡的半胱天冬酶，而不是炎性半胱天冬酶，在感染期间具有钝化的细胞毒性反应。这与用非毒性菌株LVS after感染后的炎症半胱天冬酶的强烈的细胞毒性和活化相反总之，这些研究表明，Schu S4 LPS O抗原和胶囊是逃避巨噬细胞胞质宿主防御机制所必需的
Long, Matthew Eugene. Manipulation of the innate immune response and evasion of macrophage host defense mechanisms by Francisella tularensis[D]. US: The University of Iowa, 2014