Iron Uptake and Virulence of Burkholderia pseudomallei

描述（由申请人提供）：类鼻疽病和腺腺病是由假马利氏伯克氏菌和马利氏杆菌两种特定病原体分别引起的具有相似病理生理学的威胁生命的传染病，在生物防御领域引起了巨大反响。尽管最近取得了重大进展，包括假马利氏杆菌K96243全基因组序列的阐明和正在进行的马利氏杆菌基因组测序，但对它们的毒力机制知之甚少。我们的长期目标是阐明这些疾病的发病机制。虽然许多因素都可能导致细菌的毒力，但病原细菌必须克服一个重要的非特异性防御机制才能建立感染，这就是人类和动物宿主保留铁的能力。因此，微生物的竞争优势是拥有编码产品的遗传决定因子，使它们能够利用其他不可用的铁。我们研究的直接目标是使用遗传和生化方法的结合来揭示这些病原体在疾病的败血症和细胞内阶段所采用的铁摄取的特定机制。为实现这些目标，我们将采取以下措施：微阵列和突变分析。我们将使用微阵列芯片检测假假芽孢杆菌K96243在体外补铁和限铁条件下的差异基因表达。我们还将研究巨噬细胞感染期间假芽孢杆菌诱导的基因表达模式。结合微阵列分析，我们将从现有的基因组测序项目中鉴定出假芽孢杆菌中重要的铁摄取基因的敲除突变体。选定的铁摄取突变体将被评估其随后在巨噬细胞内侵入和存活的能力以及动物模型中的毒力。2. 假马蹄莲和马蹄莲中铁载体的性质及结构分析。我们将对野生型和突变型假麦氏芽孢杆菌（K96243）和麦氏芽孢杆菌（ATCC 23344）产生的铁载体结构进行表征。这些化合物将通过高效液相色谱纯化，并通过核磁共振和质谱测定其结构细节。从我们的调查中获得的知识将有助于制定控制这些病原体引起的疾病的措施。

DESCRIPTION (provided by applicant): Melioidosis and glanders are life threatening infectious diseases with similar patho-physiology caused respectively by the select agents Burkholderia pseudomallei and B. mallei, with dramatic repercussions in the field of bio-defense. Despite major recent advances including the elucidation of the entire genome sequence of B. pseudomallei K96243 and the ongoing sequencing of the B. mallei genome, little is known about their mechanisms of virulence. Our long-range goal is to elucidate the pathogenesis of these diseases. Although many factors can contribute to bacterial virulence, one important nonspecific mechanism of defense pathogenic bacteria must overcome to establish infection, is the ability of human and animal hosts to withhold iron. Thus, a competitive advantage for microorganisms is the possession of genetic determinants encoding products that allow them to utilize otherwise unavailable iron. The immediate goal of our research is to use a combination of genetic and biochemical approaches to unveil specific mechanisms of iron uptake employed by these pathogens during both the septicemic and the intracellular phases of disease. To accomplish these goals we will perform: 1. Microarray and mutational analysis. We will use microarray chips to examine the differential gene expression of B. pseudomallei K96243, from cells grown in vitro under conditions of iron repletion and limitation. We will also examine patterns of gene expression induced in B. pseudomallei during infection of macrophages. In conjunction with microarray analysis we will generate knock-out mutants of important iron uptake genes in B. pseudomallei, identified from the available genome sequencing project. Selected iron uptake mutants will be assessed for their subsequent ability to invade and survive intracellularly in macrophages and for virulence in animal models. 2. The characterization and structural analysis of siderophores from B. pseudomallei and B. mallei. We will characterize the structure of siderophores produced by wild type and mutant B. pseudomallei (K96243) and B. mallei (ATCC 23344). These compounds will be purified by high performance liquid chromatography and their structural details determined by nuclear magnetic resonance and mass spectrometry. Knowledge gained from our investigation will contribute to the development of measures to control the diseases caused by these pathogens.