Studies into the role of two mycobacterial proteins in stress response and survival inside macrophages

The success of M. tuberculosis lies in its ability to stay alive and persist in a potentially hostile environment represented by the macrophage phagosome. Hence there is a desperate need to identify the molecular mechanisms and associated proteins enabling mycobacterial survival and replication inside macrophages. Recent studies have shown that several mycobacterial proteins may play distinct roles during different stages of infection. This thesis was focused on investigation of the biological function of two mycobacterial proteins, Rv1219c and Rv3136 (PPE51 protein) during macrophage infection and stress response. A recent study has established that RaaS (Rv1219c in M. tuberculosis and BCG_1279c in M. bovis BCG) mediates mycobacterial survival in stationary phase and during mouse infection. RaaS (for regulator of antimicrobial-assisted survival) controls expression of ATP-dependent efflux pumps Bcg_1278c/1277c and DrrC and mediates survival of M. bovis BCG in growth non-permissive conditions. One of the aims of this project was investigating the role of RaaS in mycobacterial replication and persistence in macrophages. The result showed that ΔraaS mutant of M. bovis BCG was significantly impaired in initial survival in macrophages. Moreover, the mutant was extremely sensitive to H2O2 and low pH, the stress factors, which probably influence mycobacterial viability upon their uptake into macrophages. Treatment with reserpine, an inhibitor of ATP-dependent efflux pumps, prior to stress exposure had managed to improve the survival of the mutant suggesting that the impaired stress response of the raaS mutant was due to dysregulation of efflux pumps. While BCG_1279c and its orthologous in M. tuberculosis, Rv1219c, are important for survival inside macrophages and in stationary phase, published data has shown that Rv3136 is up-regulated during mycobacterial replication in macrophages. To establish the role of Rv3136 in macrophage infection and virulence, an rv3136 deletion mutant was generated in M. tuberculosis H37Rv. The mutant did not show any growth defect in laboratory medium. Although macrophage infection experiments did not link rv3136 to survival in macrophages, preliminary data from mouse infection indicated that Rv3136 could potentially play a role in survival inside the host.