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.
