Abstract
Tuberculosis remains a major threat to human health accounting for 2 million annual deaths worldwide. M. bovis causes TB in cattle which is a serious issue in the UK. Mycobacteria are widely distributed in the environments that are also colonized by free living amoebae. In the present study, free-living amoeba (A. castellanii) has been used to study the genetic factors required for the intracellular survival of M. bovis. Role of region of difference 1 (RD1), isocitrate lyase (Rv0467), ClgR (Rv2745) and the VapC (Rv2548) toxin-antitoxin system was examined for survival in amoebae. While the role of RD1 in mycobacterial survival in amoebae could not be observed, isocitrate lyase and a transcriptional regulator (ClgR) might play some part in survival of M. bovis in A. castellanii. It was found that although the mycobacteria were able to remain inside the amoebae vacuoles, they were unable to control the pH as the vacuoles remained acidic. This is very interesting as it is in contrast to macrophages where the mycobacteria are controlling and keeping the phagosomal pH only weakly acidic. A library of ~2500 M. bovis mutants was also created and TraSH mutagenesis was performed to provide a systematic assessment of the importance of mycobacterial genes for intra-amoebic survival. The results indicate that perhaps Rv3087 plays some role in the ability of M. bovis to grow inside amoebae. Rv3087 is considered to be essential for growth in murine infection model and induced when tubercle bacilli are exposed to acidic conditions in vitro and in macrophages. However, these are preliminary findings that need to be confirmed by further research.