Pathogenesis of Burkholderia mallei and pseudomallei

类鼻疽伯克霍尔德菌和B.鼻疽分别是类鼻疽病和腺体病的病原体。 它们是影响人类和动物宿主的相关革兰氏阴性细菌病原体。 B。类鼻疽和B.鼻疽通过皮下接种和吸入途径都是传染性的，并且两种病原体都可以引起脓毒性感染，具有非常高的死亡率（>80%）。由于这两种病原体都可用作针对未感染人群的生物制剂，因此疾病控制中心（CDC）将其指定为B类生物制剂。 传染性菌株的广泛传播需要雾化递送以获得最大影响，因此，了解鼻疽和类鼻疽的肺部形式比研究这些疾病的皮下形式重要得多。 因此，确定导致肺部病理的细菌/宿主相互作用已成为我们实验室的主要重点。B。类鼻疽和B.鼻疽是兼性细胞内病原体，可以在上皮细胞和吞噬细胞系中侵入、存活和复制。为了避免被吞噬细胞清除，B。类鼻疽和B.鼻疽具有广泛的氧化/亚硝化应激蛋白（例如，SOD、过氧化氢酶、谷胱甘肽过氧化物酶等）其减少/消除由宿主产生的有毒氧/氮自由基。在大肠在大肠杆菌中，类似防御酶的表达受转录激活因子OxyR的调控。 B中的突变体。鼻疽ATCC 23344和B.已通过接合转移质粒pGEV的插入失活构建了类鼻疽杆菌DD 503。与野生型亲本菌株相比，所得的delta-oxyR突变体对过氧化氢更敏感。使用改良的卡那霉素保护试验，B.假鼻疽oxyR突变体不能在小鼠或人巨噬细胞中存活，并且在小鼠或人上皮细胞中具有降低的存活性。 此外，B. mallei oxyR突变体不能在原代和永生化的小鼠巨噬细胞中存活。 为了进一步评估OxyR在发病机制中的作用，用野生型（WT）B攻击小鼠。类鼻疽，WT B。鼻疽或oxyR突变体。 如巨噬细胞测定所提示的，B.鼻疽和B.假鼻疽是非感染性的，表明OxyR在这些病原体的毒力中起关键作用。 这些实验为氧化应激反应在伯克霍尔德氏菌存活和发病机制中的作用提供了有价值的见解。 几种动物模型已被用于B的研究。类鼻疽和B.鼻疽 目前世界各地的实验室用于研究类鼻疽和鼻疽的动物模型是鼠模型。 由于小鼠可用作这些疾病的慢性和急性形式的模型，具有成本效益，并且具有优良的遗传工具，因此它是评估伯克霍尔德氏菌发病机制和宿主对定植，感染和疾病反应的关键方面的理想选择。我们实验室的一个重点是发展急性鼠呼吸道类鼻疽和鼻疽感染模型。 通过典型的鼻内感染途径感染的小鼠会出现肺部多灶性病变以及气管支气管淋巴结、上呼吸道炎症和血脑屏障炎症（脑膜炎）。 在目前使用的小鼠模型中，与脑膜炎相关的神经系统症状是一种常见的并发症，需要在急性呼吸性类鼻疽发生之前对动物实施安乐死。 为了克服这个问题，我们的实验室已经改进了一种肺内接种技术，该技术允许将细菌直接插入小鼠的肺部，同时保持BSL-3的遏制。此外，我们已经将光杆状菌lux操纵子引入致病性B。在活动物感染过程中，假鼻疽菌的感染顺序为： 诊断成像，用于监测小鼠靶器官的细菌定植（即，肺、肝和脾），显示使用改进的肠内技术接种的小鼠在48小时后出现肺损伤。然而，这些受感染的动物未发生使用鼻内感染方法时观察到的典型上呼吸道和脑膜感染。此外，该技术使我们能够表征突变体并鉴定参与呼吸道类鼻疽病的重要基因。使用这种技术，我们已经证明，B。鼻疽和B.缺乏功能性OxyR的类鼻疽菌株是无毒的。此外，缺乏替代σ因子RpoN或亚硝化应激反应调节因子NsrR的菌株具有减弱的毒力表型。基于这些数据，我们开发的肺内感染技术似乎可以将病原体直接输送到肺部，更接近于模拟肺类鼻疽。这将是一个非常有帮助的工具，用于鉴定编码蛋白质的基因，这些蛋白质参与急性肺类鼻疽和腺体的发病机制。

Burkholderia pseudomallei and B. mallei are the etiological agents of the diseases melioidosis and Glanders, respectively. They are related Gram-negative bacterial pathogens affecting both human and animal hosts. B. pseudomallei and B. mallei are infectious through both subcutaneous inoculation and inhalation routes of entry and both pathogens can elicit septic infections with very high mortality rates (>80%). Since both pathogens could be used as biological agents against naive human populations, they have been designated as Category B Biological Agents by the Centers for Disease Control (CDC). Widespread dissemination of infectious strains would require an aerosolized delivery for maximum impact, and therefore, understanding the pulmonary forms of glanders and melioidosis is much more important than studying the subcutaneous forms of these diseases. As such, defining the bacteria/host interactions that lead to pulmonary pathology has become a major focus of our laboratory. B. pseudomallei and B. mallei are facultative intracellular pathogens that can invade, survive and replicate in epithelial and phagocytic cell lines. In order to avoid clearance by phagocytic cells, B. pseudomallei and B. mallei possess an extensive repertoire of oxidative/nitrositive stress proteins (e.g., SOD, catalase, glutathione peroxidase etc.) that reduce/eliminate toxic oxygen/nitrogen radicals generated by the host. In E. coli, expression of similar defense enzymes is regulated by the transcriptional activator, OxyR. Mutants in B. mallei ATCC23344 and B. pseudomallei DD503 have been constructed by insertional inactivation via conjugative transfer of the plasmid pZSV. The resulting delta-oxyR mutants were more sensitive to hydrogen peroxide compared to the wild-type, parent strains. Using a modified kanamycin-protection assay, B. pseudomallei oxyR mutants were unable to survive in mouse or human macrophages and had decreased survivability in mouse or human epithelial cells. In addition, the B. mallei oxyR mutant was unable to survive in primary and immortal mouse macrophage cells. To further assess the role of OxyR in pathogenesis, mice were challenged with wild-type (WT) B. pseudomallei, WT B. mallei or oxyR mutants. As suggested by the macrophage assays, oxyR mutants of both B. mallei and B. pseudomallei were non-infectious, suggesting a critical role for OxyR in virulence of these pathogens. These experiments are providing valuable insight into the role of the oxidative stress response in the survival and pathogenesis of Burkholderia sp. Several animal models have been used for the study of B. pseudomallei and B. mallei. The current animal model used for studying both melioidosis and glanders by laboratories worldwide is the murine model. Since the mouse can be used as a model for both chronic and acute forms of these diseases, is cost effective and has excellent genetic tools available, it is ideal for assesing critical aspects of burkholderia pathogenesis and the host response to colonization, infection and disease. A focus of our laboratory has been the development of acute murine respiratory melioidosis and glanders models of infection. Mice infected by the typical intranasal route of infection develop multifocal lesions of the lung as well as inflammation of the tracheobronchial lymph node, upper respiratory tract, and inflammation blood-brain barrier (meningitis). In currently used mouse models, neurological symptoms associated with meningitis are a common complication necessitating the euthanization of animals before acute respiratory melioidosis occurs. To overcome this problem, our lab has refined a technique for intratracheal inoculation which allows for direct insertion of the bacteria into the lungs of the mouse while maintaining BSL-3 containment. Further, we have introduced the Photorhabdus lux operon into pathogenic B. pseudomallei in order of visulalize bacteria during the infection of live animals. Diagnostic imaging, used to monitor bacterial colonization of mouse target organs (i.e., lungs, liver, and spleen), showed that mice inoculated using the improved intratracheal technique developed pulmonary lesions after 48h. However, these infected animals did not develop the upper respiratory and meningial infections typical observed when intranasal infection methods are used. Additionally, this technique has allowed us to characterize mutants and identify important genes involved in respiratory melioidosis. Using this technique, we have demonstrated that B. mallei and B. pseudomallei strains lacking a functional OxyR are avirulent. In addition, strains lacking the alternative sigma factor, RpoN or the nitrositive stress response regulator, NsrR, have an attenuated virulence phenotype. Based on these data, it appears that the intratracheal infection technique that we have developed allows for the direct delivery of the pathogen to the lungs and more closely mimics pulmonary melioidosis. This will be a very helpful tool for idenifying genes encoding proteins involved in the pathogenesis of acute pulmonary melioidosis and Glanders.