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.

Burkholderia Pseudomallei是Melioidisois的病因，是一种革兰氏阴性症，师生，厌氧性的，通风的芽孢杆菌，负责在人类和动物中观察到的广泛疾病。腺体的病因学家Burkholderia Mallei是一种革兰氏阴性细菌，负责驴，mu子，马和偶尔人类的疾病。但是，与环境腐生B.伪菌相比，马利芽孢杆菌在其溶剂宿主之外并没有持续存在。尽管Mallei和B. pseudomallei在基因型上是相似的，但两种致病物种之间确实存在显着的表型差异。尽管腺体是人类已知的最古老的疾病之一，但对玛丽芽孢杆菌引起的疾病发病机理的了解相对较少。这种现象主要是由于北美缺乏疾病的原因，即即使在受控的实验室环境中，马利芽孢杆菌也可能是一个特别危险的生物体。两种细菌都被CDC视为BSL3选择剂。 Burkholderia-巨噬细胞相互作用： 病原体宿主细胞相互作用体外的研究是定义和表征细胞内细菌病原体的毒力因子的重要工具。 Burkholderia的主要物种包括假单胞菌； B. Mallei和一个无动于衷的环境稳定的分离株B.泰国人。 B.假巨噬细胞相互作用已经进行了广泛的研究，但对Mallei与巨噬细胞的相互作用鲜为人知。 我们已经使用鼠巨噬细胞系（RAW 264.7）对Mallei和B. pseudomallei巨噬细胞相互作用进行了比较分析。 我们的发现表明，尽管Mallei能够在RAW细胞中入侵和复制，但它的内在化效率较低，增长较慢。 感染的最佳多重性对于宽容的麦芽芽孢杆菌细胞内生长至关重要。此外，一氧化氮测定和可诱导的一氧化氮合酶（INOS）免疫印迹分析显示，iNOS活性与来自RAW 264.7细胞的Mallei的清除之间存在很强的相关性。此外，用iNOS抑制剂Aminoguanidine治疗活化的巨噬细胞，抑制了受感染单层的Mallei的清除。基于这些结果，MOI似乎显着影响麦芽芽孢杆菌与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Mallei至关重要。我们进一步测试了细胞内存活和野生型和伪芽孢杆菌的各种突变体之间的差异。 在原细胞感染模型中测试了在麦芽芽孢杆菌和假单胞菌的每个背景中产生的18个突变体。 假单胞菌芽孢杆菌的III型分泌突变体（菌株26BT3）在生细胞中表现出明显的内在化和生长差异。 相同的Mallei III型分泌突变体（BMT3）和B. mallei LPS突变体（GMRMLD）在生细胞中无法生长。 假单胞菌的囊囊多糖是在鼠巨噬细胞中复制所需的必不可少的毒力决定因素，也可以保护宿主血清cidal活性和肠球菌病。 在最近的一项研究中，在急性呼吸鼠模型中研究了针对假单胞菌囊囊突变体（JW270）的免疫反应。 JW270在该模型（2 log）中显着减弱，其水平类似于泰国芽孢杆菌的水平。 在致命剂量下，JW270定居于类似于野生型菌株的水平的肺，肝脏和脾脏，并被发现会触发肝脏和脾脏的病理减少。 在这些组织中，几种细胞因子反应改变了，重要的是，在JW270感染的小鼠的肝脏和脾脏中，IFN的水平降低，但在肺中没有降低。 这些结果表明，假单胞菌的囊囊多糖是呼吸道感染中的临界毒力决定因素，并且它是产生在全身性梅尔塞氏病中通常观察到的Th1免疫反应的重要抗原。 此外，这些数据表明，肺部假囊杆菌囊囊多糖对疾病结果可能不如外围器官中的先天免疫反应那样重要。结果表明，使用原始巨噬细胞和小鼠对毒力的体外和体内建模是一种简单且可信的方法，可以更好地了解Burkholderia突变体，以更好地理解腺体和梅尔里尼氏病的发病机理。 B. Mallei是一种细胞内病原体，可引起动物的致命疾病， 人类。为了更好地理解吞噬细胞在控制这种生物引起的感染中的作用，开始了研究以检查麦芽芽孢杆菌与RAW 264.7鼠巨噬细胞的相互作用。 利用修饰的卡纳霉素保护测定法，显示出在不到1的RAW 264.7细胞中存活并复制的264.7个细胞（MOI）。相反，相反，有机体有效地被巨噬细胞有效地清除，巨噬细胞在10的MOI中被巨噬细胞有效地清除。 GM-CSF，RANTES和IFN-B以10的MOI感染时。此外，一氧化氮测定和诱导型一氧化氮合酶（INOS）免疫印迹分析显示，INOS活性与RAW 264.7细胞的Mallei的清除之间存在很强的相关性。此外，用iNOS抑制剂Aminoguanidine治疗活化的巨噬细胞，抑制了受感染单层的Mallei的清除。基于这些结果，MOI似乎显着影响麦芽芽孢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Mallei至关重要。在Humam主要巨噬细胞中，故事似乎有所不同。麦芽芽孢杆菌细胞被人类血液衍生的原代巨噬细胞和源自源自Heathly受试者的人类原发性大麻巨噬细胞有效地杀死。 这表明健康的人类不是该细菌的好宿主，与假芽芽孢杆菌不同，伪虫很容易感染正常健康的人类。 最近的研究表明，该生物体表达的1型VI型分泌系统（T6SS-1）对于腺体仓鼠模型中的生存​​至关重要。为了更好地了解T6SS-1在疾病发病机理中的作用，开始了研究以检查麦芽芽孢杆菌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法，结果表明，尽管TSSE突变体能够在RAW 264.7细胞中生存，但与对照组相比，观察到了明显的复制缺陷。此外，通过DIC显微镜对感染单层的分析表明，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，显示TSSE突变体可以从LAMP-1相关的液泡中逃脱。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，导致效率低下的细胞内扩散特征。重要的是，在这项研究中观察到的所有突变表型都可以通过互补恢复。基于这些发现，T6SS-1在RAW 264.7细胞对Mallei摄取后，T6SS-1在复制和基于肌动蛋白的运动中起关键作用。