Iron Uptake and Virulence of Burkholderia pseudomallei

点击翻译按钮获取中文摘要

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.