Proteomic insights into the adaptation of Mycobacterium bovis to hypoxic conditions

Bovine tuberculosis (bTB) is an important cattle disease with major public health and economic impacts. Mycobacterium bovis, its causative agent, is thought to persist in non-replicative forms within the host, similar to Mycobacterium tuberculosis, leading to chronic or latent infection. In this study, we used the Wayne model-an in vitro system that gradually depletes oxygen-to mimic the hypoxic conditions M. bovis may encounter during latency. Growth analysis showed that part of the bacterial culture remained viable but non-replicative under hypoxia, while another fraction likely lysed, as indicated by declining optical density during late hypoxia and reduced colony-forming units. Secreted proteome analysis identified 36 proteins detected exclusively in culture supernatants, with Cut3, SapM, and Cdh accumulating more under hypoxia (p < 0.05, FDR = 0.25). In the cellular proteome, 288 proteins showed differential accumulation (p < 0.05, FDR = 0.25), with 172 more abundant under hypoxia. Under oxygen depletion, M. bovis increased proteins related to nitrogen and lipid metabolism, purine biosynthesis, carbon metabolism, anaplerotic pathways, and several DosR regulon proteins. Aerated cultures showed higher levels of proteins involved in transcription, translation, DNA replication, and virulence. Protein secretion decreased under hypoxia. Overall, M. bovis remodels its proteome to persist in a viable, non-replicative state.