Elucidation of the mechanisms of host resistance against Mycobacterium tuberculosis infection and of TB pathogenesis is a high objective. Identification of genes and their alleles that confer resistance versus susceptibility to TB provides deep insight into basic mechanisms of immunity and pathology. Limitations to identifying human TB susceptibility genes are the polygenic control of susceptibility and the absence of clearly delineated phenotypes required for genetic analysis. Animal models of TB proved to be extremely valuable in elucidating immunity to mycobacteria and genetic control of susceptibility/resistance. Recently we and others accomplished a provisional genome-wide linkage analysis of TB susceptibility in mice, and mapped several quantitative trait loci (QTLs) in the genome control the course of the disease. Despite the fact that chromosome regions surrounding all QTLs contain genes that regulate the function of cells of the immune system (candidate genes), the physiologic basis for the difference in susceptibility to TB remains unknown and is a subject of this research project. To determine the genes that are differentially expressed in lung macrophages of susceptible and resistant mice following mycobacterial infection we have established a culture model of lung macrophages infected with mycobacteria that exactly follows genetic pattern of TB susceptibility control. We propose to compare gene expression in normal and M. tuberculosis-infected macrophages from susceptible and resistant mice using a DNA chip technology that allows the monitoring of more than 11,000 mouse genes simultaneously. To genetically dissect susceptibility to TB at the organism level, we will establish two independent pairs of congenic mouse strains. In each pair, genetic difference will be restricted to a small chromosome segment surrounding a particular QTL, one on distal chromosome 3 and the other on proximal chromosome 9. We will study gene expression in macrophages in these novel mouse strains and thus link shifts in gene expression with the alleles of particular QTLs. We will perform a new genome screening experiments, employing combination of strain in which an unusual inheritance of resistance with the strong heterosis effect was observed, in order to identify the novel chromosomal regions participating in TB control. We will define the chromosome 3 and 9 QTLs map location to approximately 1 cM intervals by a sequential 2-stage interval-specific congenic strains approach, and we will clone corresponding QTLs relying on testing of candidate genes available from complete gene map of the mouse.
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