Core E. Animal Model Core Project Leader: Clare Smith ABSTRACT Understanding the mechanisms through which Mycobacterium tuberculosis (Mtb) metabolites impact human tuberculosis (TB) disease requires implementation of model animal systems that are both tractable and faithfully replicate the TB disease states observed in humans. Here we propose to leverage the Collaborative Cross (CC), a genetically diverse panel of recombinant inbred mice that can be reproducibly and indefinitely regenerated. We have previously shown that the CC panel encompasses a broad spectrum of TB disease traits and infection microenvironments. CC mice provide a tractable model in which to study specific Mtb genetic-metabolite pairs, compared to standard inbred mice that show limited phenotypic variability. For example, in a genome-wide TnSeq experiment, we found that among 19 high value Mtb metabolic genes, only one controlled growth in the conventional C57BL/6J (BL6) mouse strain. However, more than half of mutants studied showed in vivo growth phenotypes when screened across CC mouse strains. Further, through study of the host genetic backgrounds in which individual bacterial genes do or do not control in vivo Mtb survival, the Smith laboratory can begin to study host factors in control of Mtb response. The Animal Core E will conduct experimental infection approaches to support Projects 1 and 3 that focus on virulence associated lipids and diagnostics, respectively. To support identification of Mtb metabolites as diagnostic tests in Project 3, we will characterize lungs and serum from mice infected with a high and low burden of Mtb. Supporting efforts to understand the role of host pressure on the mycobacterial envelope content in Project 1, we will produce Mtb strains passaged in vivo in mice. Extending our existing Tnseq approach to identify mycobacterial genes that control Mtb growth in vivo, we will select and test up 50 pooled Mtb CRISPR knockdown strains for pulmonary infection in CC strains. From these, five Mtb CRISPR knockdown strains with the strongest in vivo growth phenotypes will be further studied in detail as single gene knockdowns to determine their specific functions.