Genetics of Suscptibility to Anthrax Toxin in vivo

描述(申请人提供)：炭疽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)、水肿性因子(EF)和致死性因子(LF)。在二元组合中，PA与EF结合形成水肿性毒素，而PA与LF结合形成致死毒素(LT)，后者是介导致死性炭疽病的主要毒素。使用近交系小鼠的活体研究表明，死亡率是由基因控制的，由于巨噬细胞耗尽的小鼠对LT攻击具有抵抗力，因此巨噬细胞介导了这一反应。利用体外对LT诱导的巨噬细胞溶解的不同敏感性进行的遗传学研究将控制这一表型的基因Ltxs1定位在小鼠中央11号染色体上。最近，一种定位候选基因克隆方法将Ltxs1定位为Kif1c。然而，关于直接感染LT或感染后死亡率的遗传学研究受到的关注有限。为了评价Ltxs1/Kif1c在LT攻击后死亡率的遗传控制中的作用，我们利用一组携带LT抗性DBA/2J小鼠中央11号染色体不同片段的区间特异性重组同源基因系进行了同源定位研究，这些重组同源基因系通过分子标记辅助选择导入到LT敏感的BALB/cByJ背景上。研究结果表明，LT攻击引起的死亡率受位于中央11号染色体上的3个连锁数量性状基因座(QTL)控制，它们分别是：Litxs1/Kif1c、Litxs2和Litxs3。重要的是，为了概括CD2F1杂种对死亡的显性抗性，在所有三个QTL上都需要DBA/2J等位基因。在这一应用中，我们建议采用位置候选基因克隆的方法来识别与Ltxs2和Ltxs3相关的基因。为此，我们将：1)使用高分辨率的同源图谱将候选区间缩小到1.0厘米或更小的分辨率，2)建立跨区间的DBA/2J和BALB/cByJ BAC重叠群，以及3)产生BALB/c-TGN(D2-BAC Kif1c)、DBA/2-TGN(C-BAC Kif1c)、BALB/c-TGN(CD11b-Kif1cd)和DBA/2-TGN(CD11b-Kif1cc)转基因株系，并评估它们对LT和巨噬细胞溶解的敏感性。这将作为BAC和单基因转基因定位可用于鉴定Ltxs2和Ltxs3基因的基本证据，并证实Kif1c是DBA/2J Ltxs1区间内控制死亡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.