Provision of Mycobacterial Determinants of Survival and Fitness within the Bovine Host

AIMS: The overall goal of this proposal is to use to Transposon Directed Insertion Sequencing (TraDIS) methodology to determine M. bovis gene fitness and essentiality in disease relevant models of natural infection. This approach will result in (a) identification of the metabolic and physiological adaptations that M. bovis makes in the bovine host (b) identification of the selective pressures within bovine alveolar macrophages and the bovine host (c) a list of attenuating mutations that will allow us in future projects to rationally design novel and improved vaccines for BTB (d) a saturated transposon library of M. bovis and an essentiality dataset for distribution and use by the mycobacterial research community. The overall aim of this proposal will be acheived through the following objectives (i) Construction and validation of a large transposon library of M. bovis with full coverage in non-essential genes and the derivation of an essential gene list for M. bovis (ii) Identification of essential genes in M. bovis in infection relevant in vitro conditions and ex vivo models (iii) Validation of the in vitro and ex vivo essential dataset obtained in objective (ii) (iv) Identification of essential genes in M. bovis in the bovine host (v) Validation of the in vivo essential dataset obtained above