MYCOBACTERIAL ENVIRONMENT WITHIN MACROPHAGES

鸟分枝杆菌和结核分枝杆菌是感染单核吞噬细胞并在组织巨噬细胞内存活的细胞内病原体。结核病每年导致数百万人死亡，而鸟支原体是导致艾滋病患者全身性疾病的最常见病原体。这两种细菌都在巨噬细胞的膜结合液泡内存活。这些生物的细胞内定位表明它们很适合在宿主中生存，并且它们可能可以在宿主细胞内获得足够的营养。已知环境中铁等元素的存在或缺失会影响细菌的基因表达。人们一直认为巨噬细胞液泡缺乏铁，作为宿主抵抗感染的防御机制。提高我们对分枝杆菌液泡内环境的理解以及分枝杆菌如何对液泡中存在的条件作出反应将为致病机制提供新的见解。我们建议：(1)通过两种方法(a)硬x射线显微镜和(b)细菌报告系统来确定巨噬细胞和树突细胞中M. avium液泡中铁、钾、磷、硫、钙、镁、锰、铜和锌等重要元素的存在和总浓度以及有效浓度。（2a）确定分枝杆菌液泡内环境对分枝杆菌推定毒力决定因素表达的作用；(b)确定这些决定因素在毒力中的重要性。这是我们第一次能够获得分枝杆菌吞噬体中单一元素浓度的数据。我们相信所提出的研究将产生有关吞噬体环境和分枝杆菌对其反应的重要信息。

Mycobacterium avium and Mycobacterium tuberculosis are intracellular pathogens that infect mononuclear phagocytes and survive within tissue macrophages. Tuberculosis is responsible for millions of deaths annually, and M. avium is the most common pathogen causing systemic disease in AIDS patients. Both bacteria survive inside membrane-bound vacuoles in macrophages. The intracellular localization of these organisms suggest that they are well adapted for survival in the host and that they probably can obtain sufficient amounts of nutrients inside the host cells. The presence or absence of elements, such as iron, in the environment is known to influence gene expression in bacteria. It has been assumed that macrophage vacuoles are deficient in iron, as a host defense mechanism against infection. Improving our understanding about the environment within the mycobacterial vacuole and how mycobacteria respond to the conditions present in the vacuoles will provide new insights into pathogenic mechanisms. We propose to: (1) Determine the presence and total concentration as well as the available concentration of important elements such as iron, potassium, phosphorus, sulphur, calcium, magnesium, manganese, copper and zinc in M. avium vacuole in macrophages and dentritic cells compared to M. smegmatis vacuoles by using two methods: (a) hard X-ray microscopy, and (b) bacterial reporte systems. (2a) Establishing the role of the environment within mycobacterial vacuoles on the expression of mycobacterial putative virulence determinants; (b) determine the importance of these determinants in virulence. This is the first time that we will be able to obtain data on the concentrations of single elements in mycobacterial phagosomes. We believe that the proposed studies will generate important information about the phagosome environment and the mycobacterial response to it.