Role of mycobacteria porins and efflux pumps on the intracellular survival within macrophages

The characteristic membrane of mycobacteria and its related components were already implicated in the enhanced persistence and success of pathogenic mycobacteria in their hosts. The porin and efflux pump-content of the membrane dictates the permeability and accumulation of bactericidal compounds, whether being antibiotics or other molecules released by the infected macrophages such as nitric oxide. In the first part of this thesis, the role of newly discovered putative porin MtpA, conserved in M. bovis BCG and M. tuberculosis was addressed. Several mutant clones of M. bovis BCG were tested to elucidate the role of MtpA on intracellular survival inside macrophages. It was determined that absence of the porin enhances intracellular survival in mouse macrophages yet reduces it in human macrophages. It was also observed that the MtpA mutant induced lower activation of pro-inflammatory response in all the macrophages tested and lower production of nitric oxide in mouse macrophages. The described activity of MtpA was found to require only one of its structural domains and could not be complemented using the non-pathogenic M. smegmatis porin MspA. These evidences point for a specific role of MtpA in membrane permeability to bactericidal molecules released by macrophages and also the possibility of this porin being involved in innate immunity recognition of the bacilli. In the second part, the antimicrobial activity of previously described POA-derivates was analyzed. In those prodrugs, POA is linked by an ester bond to a long linear carbon chain. Upon cleavage, POA and a long linear alcohol chain is released. Here is demonstrated that these LLCAs possess bactericidal activity and circumvent POA resistance mechanisms dependent on drug efflux-pumps. In those POA-resistant bacteria, such as M. avium, the antimicrobial activity displayed by the new POA-derivates is owed to the release of the LLACs and not to POA itself.