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: 7592304
• 负责人: Frank Gherardini
• 金额: $ 65.93万
• 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/7592304
• 关键词: Acids; Agonist; Amides; Animals; Antigens; Arabinose; Asses; Bacillus (bacterium); Bacteria; Biological; Biological Assay; Bioterrorism; Burkholderia; Burkholderia mallei; Burkholderia pseudomallei; Categories; Cell Communication; Cell Line; Cell surface; Cells; Centers for Disease Control and Prevention (U.S.); Chemicals; Clinical; Complex; DCNU; Dendritic Cells; Development; Disaccharides; Disease; Embryo; Endotoxic Shock; Endotoxins; Environment; Equine mule; Equus caballus; Escherichia coli; Esters; Glanders; Glucosamine; Gram-Negative Bacteria; Growth; Human; IL8 gene; Immune response; Immunoblotting; In Vitro; Infection; Inflammation; Inflammatory; Interleukin-6; Invaded; Kidney; Laboratories; Licensing; Link; Lipid A; Lipopolysaccharides; MALDI-TOF Mass Spectrometry; Mammals; Melioidosis; Membrane; Membrane Proteins; Modeling; Mus; Myristic Acids; Nature; Nitric Oxide; North America; O Antigens; Oligosaccharides; Organism; Outcome; Pathogenesis; Pentas; Play; Polysaccharides; Positioning Attribute; Production; Property; RANTES; Research; Resistance; Role; Serum; Shock; Structure; System; TNF gene; Testing; Vaccines; Vertebral column; Virulence; Virulence Factors; Virulent; aminoguanidine; base; beta-hydroxymyristic acid; capsule; cell envelope; comparative; cytokine; human NOS2A protein; human TLR4 protein; human disease; immunoprophylaxis; in vitro Model; inhibitor/antagonist; lipopolysaccharide B; macrophage; man; monocyte; monolayer; mutant; pathogen; programs; response; tool

Summary: 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 BL3 select agents by the CDC. Characterization of the lipid A and O-antigen moities of the LPS of B. pseudomallei and B. mallei: Lipopolysaccharides (LPS), also commonly referred to as endotoxins, are a major component of Gram-negative cell envelopes. The barrier functions provided by bacterial outer membranes are largely due to the presence of these molecules. LPS antigens expressed by smooth strains are composed of three covalently linked domains: an O-antigen, a core region and a lipid A moiety. O-antigens, consisting of oligosaccharide repeats, are the outermost domains of LPS molecules expressed on bacterial cell surfaces. Because of this, they are often a primary target of innate and acquired immune responses. Lipid A, the hydrophobic membrane-anchor component of LPS molecules, is the domain responsible for stimulating pathophysiological responses in mammals such as cytokine production, inflammation and shock. To better understand the role of lipopolysaccharide (LPS) in the pathogenesis of these diseases, studies were initiated to characterize the structural and biological properties of lipid A moieties expressed by this organism. Using a combination of chemical analyses and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, B. mallei was shown to express a heterogeneous mixture of tetra- and penta-acylated lipid A species that were non-stoichiometrically substituted with 4-amino-4-deoxy-arabinose residues. The major penta-acylated species consisted of bisphosphorylated D-glucosamine disaccharide backbones possessing two amide linked 3-hydroxyhexadecanoic acids, two ester linked 3-hydroxytetradecanoic acids (C14:0(3-OH)) and an acyloxyacyl linked tetradecanoic acid, whereas, the major tetra-acylated species possessed all but the 3-linked C14:0(3-OH) residues. In addition, studies demonstrated that B. mallei LPS was able to stimulate human embryonic kidney 293 cells expressing human Toll-like receptor 4 (hTLR4) complexes to secrete high levels of IL-8. Furthermore, when stimulated with B. mallei LPS, human macrophage-like cells (THP-1 and U-937) as well as monocyte-derived macrophages and dendritic cells secreted TNF-, IL-6 and RANTES at levels similar to those observed using Escherichia coli LPS as a control. Although devoid of hexa-acylated species, B. mallei LPS appears to be a potent hTLR4 agonist and is, therefore, likely to play a significant role in the pathogenesis of human disease. TNF-a and IL-1b produced by LPS stimulated macrophages are two of the major pro-inflammatory cytokines responsible for the clinical manifestations of endotoxic shock. Previous studies have demonstrated that the O-antigens expressed by these organisms are required for serum resistance. More importantly, the O-antigens have been also been identified as putative vaccine candidates for immunoprophylaxis against melioidosis and glanders. Studies have shown that the O-antigens expressed by B. mallei isolates are antigenically and structurally similar to those expressed by B. pseudomallei isolates. Structural analyses by Burtnick et al have demonstrated that like the predominant O-antigen expressed by B. pseudomallei isolates, the O-antigen expressed by B. mallei is an unbranched heteropolymer consisting of disaccharide repeats having the structure 3)-beta-D-glucopyranose-(1-3)-6-deoxy-alpha-L-talopyranose-(1- in which the 6-deoxy-a-L-talopyranose (L-6dTalp) residues are non-stoichiometrically modified by 2-O-acetyl and 2-O-methyl substitutions. Unlike B. pseudomallei, however, the L-6dTalp residues of the B. mallei O-antigen do not appear to be acetylated at the O-4 position. In contrast to many other Gram-negative pathogens, virulent isolates of B. mallei and B. pseudomallei appear to express a very limited repertoire of chemically distinct O-antigens. Based upon these findings, we have recently initiated studies to develop melioidosis and glanders vaccine candidates utilizing O-antigens purified from B. pseudomallei and B. mallei isolates. 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 results indicated that in vitro modeling of virulence using RAW macrophages is a simple and credible approach to screen Burkholderia mutants as a rational for analyses in animals.

概括： 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BL3选择剂。 假单胞菌和马利芽孢杆菌的LPS的脂质A和O-抗原的表征： 脂多糖（LPS）（通常称为内毒素）是革兰氏阴性细胞信封的主要组成部分。细菌外膜提供的屏障功能很大程度上是由于这些分子的存在。平滑菌株表达的LPS抗原由三个共价连接的结构域组成：O抗原，核心区域和脂质A部分。由寡糖重复序列组成的O-抗原是在细菌细胞表面表达的LPS分子的最外部结构域。因此，它们通常是先天和获得免疫反应的主要目标。 LPID A是LPS分子的疏水膜锚固成分，是负责刺激细胞因子产生，炎症和休克等哺乳动物的病理生理反应的领域。 为了更好地理解脂多糖（LPS）在这些疾病的发病机理中的作用，启动了研究以表征该生物体表达的脂质A部分的结构和生物学特性。使用化学分析和基质辅助激光解吸/电离飞行时间的质谱法，Mallei被证明表达了四酰和五酰化脂质A种的异质混合物，这些混合物用4-氨基-4-氨基-4-二氧化物 - 二氧基-4-二氧化脱氧基蛋白 - Arabinose-Arabinose betterueb。主要的五体囊化物种由双磷酸化的D-葡萄糖脱糖尿氨基还是具有两个酰胺连接的3-羟基二甲基甲酸，两个酯连接的3-羟基二甲基苯二甲酸酯链接二囊化物种除3连接的C14：0（3-OH）残基都具有所有物种。此外，研究表明，Mallei LPS能够刺激表达人Toll样受体4（HTLR4）复合物的人类胚胎肾脏293个细胞，以分泌高水平的IL-8。此外，当用Mallei LPS刺激时，人类巨噬细胞样细胞（THP-1和U-937）以及单核细胞衍生的巨噬细胞和树突状细胞分泌TNF-，IL-6和在类似于使用Escherichia coli LPS的tnf-，IL-6和rantes分泌的水平。尽管没有六酰化的物种，但Mallei B. mallei LPS似乎是一种有效的HTLR4激动剂，因此，在人类疾病的发病机理中可能发挥重要作用。 LPS刺激的巨噬细胞产生的TNF-A和IL-1B是负责内毒性休克临床表现的主要促炎细胞因子。 先前的研究表明，这些生物表达的O-抗原是血清耐药性所必需的。更重要的是，O-抗原也已被确定为针对黑胶病和腺体的免疫预防的推定疫苗候选者。研究表明，麦芽芽孢杆菌分离株表达的O-抗原在抗原和结构上与假单胞菌分离株表达的O抗原在结构上相似。 Burtnick等人进行的结构分析已经证明，像假单胞菌分离菌所表达的主要O-抗原一样，Mallei表达的O-抗原是一种无分支的杂聚物，由具有结构3的二糖重复序列组成3）其中6-脱氧 - l-talopyranose（L-6DTALP）残基是通过2-O-乙酰基和2-O-甲基取代的非岩化修饰的，与B. pseudomallei不同。革兰氏阴性病原体，马利芽孢杆菌和假单胞菌的有毒分离物似乎表达了基于这些发现的化学上不同的O-抗原的曲目，我们最近启动了用于使用O-Antigens pefipied o-antigens peceens pecudomale b. pseudomallei niparatie salio oferiosisoiss和腺体疫苗的研究。 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。 结果表明，使用原始巨噬细胞对毒力进行体外建模是一种简单且可信的方法，用于筛选Burkholderia突变体，作为动物分析的合理方法。