Genetics of Suscptibility to Anthrax Toxin in vivo

描述（由申请人提供）：炭疽芽孢杆菌的主要毒力因子是一种外毒素，由三种不同的蛋白质组成：保护性抗原（PA）、水肿因子（EF）和致死因子（LF）。在二元组合中，与EF相关的PA形成水肿毒素，而与LF相关的PA形成致死毒素（LT），这是介导致命炭疽病理的主要毒素。使用近交系小鼠的体内研究表明，死亡率是遗传控制的，巨噬细胞介导了这种反应，因为巨噬细胞耗尽的小鼠对LT挑战有抵抗力。利用对lt诱导的巨噬细胞溶解的体外差异易感性进行的遗传学研究将控制这种表型的基因Ltxs1定位到中央小鼠11号染色体上。最近，一种定位候选基因克隆方法鉴定Ltxs1为Kif1c。然而，对肝移植直接攻击或感染后死亡率的遗传学研究受到的关注有限。为了评估Ltxs1/Kif1c在LT攻击后死亡率遗传控制中的作用，我们进行了一项基因定位研究，使用一组间隔特异性重组基因系，这些基因系携带来自LT抗性DBA/2J小鼠的11号中央染色体的不同片段，这些片段通过标记辅助选择渗入到LT易感的BALB/cByJ背景中。本研究结果表明，LT致死性受中央11号染色体Ltxs1/Kif1c、Ltxs2和Ltxs3三个连锁数量性状位点（QTL）控制。重要的是，为了重现在CD2 F1杂交种中看到的对死亡的显性抗性，DBA/2J等位基因在所有三个QTL上都是必需的。在这个应用中，我们建议采用位置候选基因克隆方法来鉴定Ltxs2和Ltxs3的基因。为此，我们将：1)使用高分辨率同源图谱将候选区间缩小到1.0 cM或更小的分辨率，2)在区间内建立DBA/2J和BALB/cByJ BAC组合，3)生成BALB/c-TgN（D2-BAC Kif1c）、DBA/2- tgn （C-BAC Kif1c）、BALB/c-TgN（CD11b-Kif1cd）和DBA/2- tgn （CD11b-Kif1cc）转基因系，并评估它们对LT和巨噬细胞溶解的敏感性。这将证明BAC和单基因转基因定位可以用于鉴定Ltxs2和Ltxs3基因，并验证Kif1c是DBA/2J Ltxs1区间内控制死亡率和巨噬细胞溶解的基因。

DESCRIPTION (provided by applicant): The major virulence factor of Bacillus anthracis is an exotoxin composed of three separate proteins: protective antigen (PA), edema factor (EF) and lethal factor (LF). In binary combinations, PA in association with EF forms edema toxin whereas PA in association with LF forms lethal toxin (LT), the principal toxin mediating lethal anthrax pathologies. In vivo studies using inbred strains of mice revealed that mortality is genetically controlled, and that macrophages mediate this response since macrophage-depleted mice are resistant to LT challenge. Genetic studies using differential in vitro susceptibility to LT-induced macrophage cytolysis mapped Ltxs1, the gene controlling this phenotype, to central mouse chromosome 11. Recently, a positional candidate gene cloning approach identified Ltxs1 as Kif1c. However, genetic studies on mortality following direct challenge with LT or infection have received limited attention. To assess the role of Ltxs1/Kif1c in the genetic control of mortality following LT challenge, we carried out a congenic mapping study using a panel of interval-specific recombinant congenic lines carrying various segments of central chromosome 11 derived from LT resistant DBA/2J mice which were introgressed by marker assisted selection onto the LT susceptible BALB/cByJ background. The results of this study revealed that mortality elicited by LT challenge is controlled by three linked quantitative trait loci (QTL) on central chromosome 11: Ltxs1/Kif1c, Ltxs2 and Ltxs3. Importantly, in order to recapitulate dominant resistance to mortality as seen in CD2 F1 hybrids, DBA/2J alleles are required at all three QTL. In this application, we propose to undertake a positional candidate gene cloning approach to identify the genes underlying Ltxs2 and Ltxs3. Toward this end we will: 1) use high resolution congenic mapping to reduce the candidate intervals to a resolution of 1.0 cM or less, 2) establish DBA/2J and BALB/cByJ BAC contigs across the intervals, and 3) generate BALB/c-TgN(D2-BAC Kif1c), DBA/2-TgN(C-BAC Kif1c), BALB/c-TgN(CD11b-Kif1cd), and DBA/2-TgN(CD11b-Kif1cc) transgenic lines and assess their susceptibility to LT and macrophage cytolysis. This will serve as proof of principal that BAC and single gene transgenic mapping can be used to identify the Ltxs2 and Ltxs3 genes, and verify that Kif1c is the gene within the DBA/2J Ltxs1 interval controlling mortality and macrophage cytolysis.