Genetics of Suscptibility to Anthrax Toxin in vivo

性状（由申请方提供）：炭疽芽孢杆菌的主要毒力因子是一种外毒素，由三种单独的蛋白质组成：保护性抗原（PA）、水肿因子（EF）和致死因子（LF）。在二元组合中，PA与EF结合形成水肿毒素，而PA与LF结合形成致死毒素（LT），其是介导致死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，并且巨噬细胞介导这种反应，因为巨噬细胞耗尽的小鼠对LT攻击具有抗性。遗传研究使用差异在体外易感性LT诱导的巨噬细胞溶解映射Ltxs 1，控制这种表型的基因，中央小鼠染色体11。最近，一种位置候选基因克隆方法将Ltxs 1鉴定为Kif 1c。然而，对LT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Ltxs 1/Kif 1c在LT攻击后死亡率的遗传控制中的作用，我们使用一组间隔特异性重组同源系进行了同源作图研究，这些同源系携带来自LT抗性DBA/2 J小鼠的中央11号染色体的各个片段，这些片段通过标记辅助选择渗入到LT易感的BALB/cByJ背景中。本研究的结果表明，LT攻击引起的死亡率是由中央染色体11上的三个连锁的数量性状位点（QTL）：Ltxs 1/Kif 1c，Ltxs 2和Ltxs 3控制。重要的是，为了重现CD 2 F1杂种中所见的对死亡率的显性抗性，DBA/2 J等位基因在所有三个QTL上都是必需的。在本申请中，我们建议进行定位候选基因克隆方法，以确定潜在的Ltxs 2和Ltxs 3的基因。为此，我们将：1）使用高分辨率同源作图以将候选间隔减少到1.0 cM或更小的分辨率，2）跨间隔建立DBA/2 J和BALB/cByJ BAC重叠群，以及3）产生BALB/c-TgN（D2-BAC Kif1c）、DBA/2-TgN（C-BAC Kif 1c）、BALB/c-TgN（CD 11b-Kif 1cd）和DBA/2-TgN（CD 11b-Kif 1cc）转基因系，并评估它们对LT和巨噬细胞溶解的易感性。这将作为BAC和单基因转基因作图可用于鉴定Ltxs 2和Ltxs 3基因的原理证明，并验证Kif 1c是DBA/2 J Ltxs 1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.