INNATE RESISTANCE TO MYCOBACTERIA--ROLE OF NRAMP GENE

结核病是北美令人担忧的一个主要来源，因为 这种感染在艾滋病中的流行增加和破坏性影响 患者，以及出现高毒力和多药耐药 结核分枝杆菌菌株。针对这种情况的宿主防御机制 感染，以及长期存活的细菌机制 在宿主体内，吞噬细胞仍然知之甚少。澄清和 了解自然界遗传差异的分子基础 在人类中观察到对分枝杆菌感染的易感性 实验动物，应该提供新的洞察力的机制 针对这些感染的宿主防御。在小鼠身上，自然抵抗力 对分枝杆菌的感染受BCG/ity/LSH基因控制。 我们已经克隆了一个卡介苗候选基因，命名为nramp(Natural 抗性相关巨噬细胞蛋白)，编码一种新的 巨噬细胞特异性膜转运蛋白。这两个主要目标是 这一提议正式证明了NRAMP和BCG是 同一基因，并对其进行了全面的遗传和生化分析 Nramp蛋白，目的是了解其在体内的生物学作用。 巨噬细胞。产生携带零等位基因的突变小鼠品系 在胚胎中同源重组产生的nramp基因座 干细胞将被用来确定1)NRAMP和BCG是否相同 2)进一步体内分析nramp在巨噬细胞中的作用 功能。作为替代方法，我们将使用转基因动物来 介绍nramp的BCG‘等位基因的克隆拷贝(129/SV，显性) 在BCGS小鼠背景(C57BL/6J，隐性)上，并确定它是否 能逆转C57BL/6J小鼠对感染的易感性。一种结构 并对nramp基因进行了功能鉴定。 特别强调顺式作用序列和反式作用序列的鉴定 构成或诱导巨噬细胞的作用因素 该基因的特定表达。我们还将确定小说是否 Nramp中与疾病易感性相关的突变可能是 在其他近亲交配的小鼠品系中发现，但在人类中也发现 NRAMP同源基因在分离的家系个体中的分布 易患肺结核。NRAMP的生化分析 蛋白质，包括产生特定的抗体，以及 其细胞和亚细胞定位的鉴定，推测 翻译后修饰，膜相关转运 功能，特别是与活性氮中间体有关的功能， 也提出了一些建议。总之，这些研究应该有助于理解 Nramp在巨噬细胞介导的分枝杆菌耐药中的作用 感染，也许为治疗干预提供了一个新的靶点 在调节宿主对结核病的防御方面。

Tuberculosis is a major source of concern in North America due to the increased prevalence and devastating effect of this infection in AIDS patients, and the emergence of highly virulent and multidrug resistant strains of M. tuberculosis. The host mechanisms of defense against this infection, and the bacterial mechanisms underlying long-term survival within the host phagocytes remain poorly understood. Elucidating and understanding the molecular basis of genetic differences in natural susceptibility to mycobacterial infections observed in humans and in experimental animals, should provide new insight into the mechanisms of host defense against these infections. In the mouse, natural resistance to infection with mycobacteria is controlled by the Bcg/Ity/Lsh gene. We have cloned a candidate gene for Bcg designated nramp (natural resistance associated macrophage protein) which codes for a novel macrophage specific membrane transport protein. The two major goals if this proposal are the formal demonstration that nramp and Bcg are the same gene, and the comprehensive genetic and biochemical analysis of the Nramp protein, with the aim of understanding its biological role in the macrophage. The creation of a mutant mouse strain carrying a null allele at the nramp locus generated by homologous recombination in embryonal stem cells will be used to determine 1) if nramp and Bcg are the same gene, and 2) to further analyze in vivo the role of nramp in macrophage function. As an alternative approach, we will use transgenic animals to introduce a cloned copy of the Bcg' allele of nramp (129/sv, dominant) onto a Bcgs mouse background (C57BL/6J, recessive), and determine if it can reverse susceptibility to infection in C57BL/6J mice. A structural and functional characterization of the nramp gene is proposed, with special emphasis on the identification of cis-acting sequences and trans- acting factors responsible for constitutive or inducible macrophage specific expression of this gene. We will also determine if novel mutations in nramp and associated with disease susceptibility can be identified in additional inbred mouse strains, but also in the human NRAMP homolog in individuals from familial pedigrees segregating for susceptibility to tuberculosis. A biochemical analysis of the Nramp protein, including the production of specific antibodies, and the identification of its cellular and subcellular localization, putative post-translational modifications, membrane associated transport functions, in particular with respect to reactive nitrogen intermediates, are also proposed. Together, these studies should help understand the role of nramp in macrophage mediated resistance to mycobacterial infections, perhaps providing a new target for therapeutic intervention in the modulation of host defenses against tuberculosis.