Pathogenesis of Burkholderia mallei and pseudomallei

鼻疽伯克霍尔德氏菌和假鼻疽伯克霍尔德氏菌的发病机制

• 批准号: 8336186
• 负责人: Frank Gherardini
• 金额: $ 81.15万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8336186
• 关键词: Actins; Acute; Animals; Antigens; Asses; Attenuated; Bacillus (bacterium); Bacteria; Biological Assay; Bioterrorism; Blood; Burkholderia; Burkholderia mallei; Burkholderia pseudomallei; Categories; Cell Communication; Cell Line; Cells; Centers for Disease Control and Prevention (U.S.); Characteristics; Cytosol; Data; Defect; Disease; Disease Outcome; Dose; Environment; Equine mule; Equus caballus; Exhibits; Fluorescence Microscopy; Gentamicins; Giant Cells; Glanders; Gram-Negative Bacteria; Granulocyte-Macrophage Colony-Stimulating Factor; Growth; Hamsters; Human; Immune response; Immunoblotting; In Vitro; Infection; Infection Control; Interferons; Interleukin-10; Interleukin-6; Invaded; Kanamycin; LAMP-1; Laboratories; Licensing; Liver; Lung; Melioidosis; Membrane Proteins; Microscopy; Modeling; Mus; Nature; Nitric Oxide; North America; O Antigens; Organ; Organism; Outcome; Pathogenesis; Pathology; Peripheral; Phagocytes; Phenotype; Play; Polysaccharides; RANTES; Research; Respiratory Tract Infections; Role; Serum; Spleen; System; TNF gene; Testing; Tissues; Vaccines; Vacuole; Virulence; Virulence Factors; aminoguanidine; base; capsule; cell motility; comparative; cytokine; human NOS2A protein; immunoprophylaxis; in vivo Model; inhibitor/antagonist; killings; macrophage; man; monolayer; mutant; pathogen; polymerization; respiratory; response; tool; uptake; vaccine candidate

Burkholderia pseudomallei, the etiological agent of melioidosis, is a Gram-negative, facultatively anaerobic, motile bacillus that is responsible for a broad spectrum of illnesses observed in both humans and animals. Burkholderia mallei, the etiological agent of glanders, is a Gram-negative bacterium that is responsible for disease in donkeys, mules, horses and occasionally humans. Unlike the environmental saprophyte B. pseudomallei, however, B. mallei does not persist in nature outside of its soliped hosts. While B. mallei and B. pseudomallei are genotypically similar, significant phenotypic differences do exist between the two pathogenic species. Although glanders is one of the oldest diseases known to man, relatively little is known about the pathogenesis of disease caused by B. mallei. This phenomenon is primarily due to the lack of disease in North America along with the fact that B. mallei can be a particularly dangerous organism to study even in a controlled laboratory environment. Both bacteria are considered BSL3 select agents by the CDC. Burkholderia - macrophage interactions: The study of pathogen host cell interactions in vitro is an important tool to define and characterize virulence factors of intracellular bacterial pathogens. The major species of Burkholderia include B. pseudomallei; B. mallei and an avirulent environmentally stable isolate B. thailandensis. B. pseudomallei macrophage interactions have been extensively studied but there is little known about the interactions of B. mallei with macrophages. We have performed a comparative analysis of B. mallei and B. pseudomallei macrophage interactions using the murine macrophage cell line (RAW 264.7). Our findings show that although B. mallei is capable of invading and replicating in RAW cells it is less efficiently internalized and grows more slowly. The optimal multiplicity of infection is critical for permissive B. mallei intracellular growth. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that MOIs significantly influence the outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells. We further tested differences in intracellular survival and multiplication among wild type and various mutants of B. mallei and B. pseudomallei. Eighteen mutants produced in each background of B. mallei and B. pseudomallei were tested in the RAW cell infection model. A type III secretion mutant of B. pseudomallei (strain 26bT3) showed marked differences in internalization and growth in RAW cells. An identical B. mallei type III secretion mutant (BMT3) and a B. mallei LPS mutant (GMrmlD) were incapable of growth in RAW cells. The capsular polysaccharide of B. pseudomallei is an essential virulence determinant that is required for replication in murine macrophages, as well as protection from host serum cidal activity and opsonophagocytosis. In a recent study, the immune response directed against a B. pseudomallei capsule mutant (JW270) was investigated in an acute respiratory murine model. JW270 was significantly attenuated in this model (2 log), to levels resembling those of avirulent B. thailandensis. At lethal doses, JW270 colonized lung, liver, and spleen at levels similar to the wild type strain, and was found to trigger a reduced pathology in the liver and spleen. Several cytokine responses were altered in these tissues, and importantly, the levels of IFN- were reduced in the liver and spleen of JW270-infected mice, but not in the lung. These results suggest that the capsular polysaccharide of B. pseudomallei is a critical virulence determinant in respiratory tract infections and that it is an important antigen for generating the Th1 immune response commonly observed in systemic melioidosis. Furthermore, these data suggest that host recognition of B. pseudomallei capsular polysaccharide in the lungs may not be as important to the disease outcome as the innate immune response in the peripheral organs. The results indicated that in vitro and in vivo modeling of virulence using RAW macrophages and mice are a simple and credible approach to screen Burkholderia mutants to better understand the pathogenesis of glanders and melioidosis. B. mallei is a facultative intracellular pathogen that can cause fatal disease in animals and humans. To better understand the role of phagocytic cells in the control of infections caused by this organism, studies were initiated to examine the interactions of B. mallei with RAW 264.7 murine macrophages. Utilizing modified kanamycin-protection assays, B. mallei was shown to survive and replicate in RAW 264.7 cells infected at multiplicities of infection (moi) of less than 1. In contrast, the organism was efficiently cleared by the macrophages when infected at an moi of 10. Interestingly, studies demonstrated that the monolayers only produced high levels of TNF-a, IL-6, IL-10, GM-CSF, RANTES and IFN-b when infected at an moi of 10. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that moi significantly influence the outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells. In humam primary macrophages the story seems to differ. B. mallei cells were effectively killed by human blood derived primary macrophages and by human primary aveolar macrophages derived from heathly subjects. This suggested that healthy humans are not a good host for this bacterium unlike B. pseudomallei which readily infects normal healthy humans. Recent studies have shown that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for survival in a hamster model of glanders. To better understand the role of T6SS-1 in the pathogenesis of disease, studies were initiated to examine the interactions of B. mallei tssE mutants with RAW 264.7 murine macrophages. Utilizing modified gentamicin protection assays, results indicated that although the tssE mutants were able to survive within RAW 264.7 cells, significant replication defects were observed in comparison to controls. In addition, analysis of infected monolayers by DIC microscopy demonstrated that the tssE mutants lacked the ability to induce multinucleated giant cell formation. Via the use of fluorescence microscopy, tssE mutants were shown to undergo escape from LAMP-1 associated vacuoles. Curiously, however, following entry into the cytosol the mutants exhibited actin polymerization defects resulting in inefficient intra- and intercellular spread characteristics. Importantly, all mutant phenotypes observed in this study could be restored by complementation. Based upon these findings, it appears that T6SS-1 plays a critical role in replication and actin-based motility following uptake of B. mallei by RAW 264.7 cells.

类鼻疽伯克霍尔德氏菌是类鼻疽的病原体，是一种革兰氏阴性、兼性厌氧、能动的芽孢杆菌，导致人类和动物中观察到的多种疾病。鼻疽伯克霍尔德氏菌（Burkholderia mallei）是鼻疽病的病原体，是一种革兰氏阴性细菌，会导致驴、骡、马，偶尔也会导致人类疾病。然而，与环境腐生菌类鼻疽杆菌不同的是，鼻疽杆菌在其单足宿主之外的自然界中不会持续存在。虽然鼻疽和伪鼻疽在基因型上相似，但两种致病菌之间确实存在显着的表型差异。尽管鼻疽是人类已知的最古老的疾病之一，但人们对鼻疽引起的疾病的发病机制知之甚少。这种现象主要是由于北美缺乏疾病，而且即使在受控的实验室环境中，鼻疽杆菌也可能是一种特别危险的生物体。 CDC 认为这两种细菌都是 BSL3 选择菌。 伯克霍尔德杆菌-巨噬细胞相互作用： 体外病原体宿主细胞相互作用的研究是定义和表征细胞内细菌病原体毒力因子的重要工具。 伯克霍尔德杆菌的主要种类包括类鼻疽伯克霍尔德杆菌（B.pseudomallei）； B. mallei 和无毒环境稳定的分离株 B. thailandensis。 伪鼻疽杆菌巨噬细胞的相互作用已被广泛研究，但人们对鼻疽杆菌与巨噬细胞的相互作用知之甚少。 我们使用鼠巨噬细胞系 (RAW 264.7) 对鼻疽伯克霍尔德氏菌和伪鼻疽伯克氏菌巨噬细胞相互作用进行了比较分析。 我们的研究结果表明，虽然鼻疽杆菌能够在原始细胞中入侵和复制，但其内化效率较低且生长速度较慢。 最佳的感染复数对于鼻疽杆菌的细胞内生长至关重要。此外，一氧化氮测定和诱导型一氧化氮合酶 (iNOS) 免疫印迹分析揭示了 iNOS 活性与 RAW 264.7 细胞中鼻疽杆菌的清除之间存在很强的相关性。此外，用 iNOS 抑制剂氨基胍处理活化的巨噬细胞，可抑制鼻疽杆菌从受感染的单层细胞中的清除。基于这些结果，MOIs 似乎显着影响鼻疽杆菌和鼠巨噬细胞之间相互作用的结果，并且 iNOS 活性对于从活化的 RAW 264.7 细胞中清除鼻疽杆菌至关重要。我们进一步测试了野生型和鼻疽伯克霍尔德氏菌和伪鼻疽伯克氏菌的各种突变体之间细胞内存活和增殖的差异。 在 RAW 细胞感染模型中测试了鼻疽伯克霍尔德氏菌和假鼻疽伯克氏菌每个背景中产生的 18 个突变体。 类鼻疽杆菌（菌株 26bT3）的 III 型分泌突变体在 RAW 细胞中的内化和生长方面表现出显着差异。 相同的鼻疽杆菌 III 型分泌突变体 (BMT3) 和鼻疽杆菌 LPS 突变体 (GMrmlD) 不能在 RAW 细胞中生长。 类鼻疽杆菌的荚膜多糖是小鼠巨噬细胞中复制以及防止宿主血清杀伤活性和调理吞噬作用所需的重要毒力决定因素。 在最近的一项研究中，在急性呼吸道小鼠模型中研究了针对类鼻疽杆菌荚膜突变体（JW270）的免疫反应。 JW270 在该模型中显着减弱（2 个对数），达到与无毒力的泰国芽孢杆菌类似的水平。 在致死剂量下，JW270 在肺、肝脏和脾脏中的定殖水平与野生型菌株相似，并且被发现可以减轻肝脏和脾脏的病理变化。 这些组织中的几种细胞因子反应发生了改变，重要的是，JW270 感染小鼠的肝脏和脾脏中的 IFN-水平降低，但肺中却没有降低。 这些结果表明，类鼻疽杆菌的荚膜多糖是呼吸道感染的关键毒力决定因素，并且是产生系统性类鼻疽中常见的 Th1 免疫反应的重要抗原。 此外，这些数据表明，宿主对肺中类鼻疽杆菌荚膜多糖的识别对于疾病结果可能不如外周器官中的先天免疫反应那么重要。结果表明，使用 RAW 巨噬细胞和小鼠进行体外和体内毒力建模是筛选伯克霍尔德杆菌突变体的一种简单可靠的方法，可以更好地了解鼻疽和类鼻疽的发病机制。 鼻疽杆菌是一种兼性细胞内病原体，可在动物和动物中引起致命疾病。 人类。为了更好地了解吞噬细胞在控制该生物体引起的感染中的作用，开始研究鼻疽杆菌与 RAW 264.7 鼠巨噬细胞的相互作用。 利用改进的卡那霉素保护测定，鼻疽杆菌在感染复数 (moi) 小于 1 的 RAW 264.7 细胞中存活并复制。相比之下，当感染复数 (moi) 为10. 有趣的是，研究表明单层细胞只产生高水平的 TNF-a、IL-6、IL-10、GM-CSF、RANTES 和感染复数为 10 时的 IFN-b。此外，一氧化氮测定和诱导型一氧化氮合酶 (iNOS) 免疫印迹分析揭示了 iNOS 活性与 RAW 264.7 细胞中鼻疽杆菌的清除之间存在很强的相关性。此外，用 iNOS 抑制剂氨基胍处理活化的巨噬细胞，可抑制鼻疽杆菌从受感染的单层细胞中的清除。基于这些结果，moi 似乎显着影响鼻疽杆菌和鼠巨噬细胞之间相互作用的结果，并且 iNOS 活性对于从活化的 RAW 264.7 细胞中清除鼻疽杆菌至关重要。在人类原代巨噬细胞中，情况似乎有所不同。鼻疽细胞被人类血液来源的初级巨噬细胞和来自健康受试者的人类初级肺泡巨噬细胞有效杀死。 这表明健康人并不是这种细菌的良好宿主，不像鼻疽伯克氏菌，它很容易感染正常健康人。 最近的研究表明，该生物体表达的 Cluster 1 VI 型分泌系统 (T6SS-1) 对于鼻疽仓鼠模型的生存至关重要。为了更好地了解 T6SS-1 在疾病发病机制中的作用，开始研究鼻疽 tssE 突变体与 RAW 264.7 鼠巨噬细胞的相互作用。利用改良的庆大霉素保护测定，结果表明，尽管 tssE 突变体能够在 RAW 264.7 细胞中存活，但与对照相比，观察到显着的复制缺陷。此外，通过 DIC 显微镜对感染的单层细胞进行分析表明，tssE 突变体缺乏诱导多核巨细胞形成的能力。通过使用荧光显微镜，tssE 突变体显示出从 LAMP-1 相关液泡中逃逸。然而，奇怪的是，进入细胞质后，突变体表现出肌动蛋白聚合缺陷，导致细胞内和细胞间传播特性低效。重要的是，本研究中观察到的所有突变表型都可以通过互补来恢复。基于这些发现，T6SS-1 似乎在 RAW 264.7 细胞摄取鼻疽杆菌后的复制和基于肌动蛋白的运动中发挥着关键作用。