Characterising resuscitation promoting factor fluorescent-fusions in mycobacteria

Iria Uhía, Nitya Krishnan, Brian D. Robertson

Resuscitation promoting factor proteins (Rpfs) are peptidoglycan glycosidases capable of resuscitating dormant mycobacteria, and have been found to play a role in the pathogenesis of tuberculosis. However, the specific roles and localisation of each of the 5 Rpfs in Mycobacterium tuberculosis remain mostly unknown. In this work our aim was to construct fluorescent fusions of M. tuberculosis Rpf proteins as tools to investigate their function. We found that Rpf-fusions to the fluorescent protein mCherry are functional and able to promote cell growth under different conditions. However, fusions to Enhanced Green Fluorescent Protein (EGFP) were non-functional in the assays used and none were secreted into the extracellular medium, which suggests Rpfs may be secreted via the Sec pathway. No specific cellular localization was observed for either set of fusions using time-lapse video microscopy. We present the validation and testing of five M. tuberculosis Rpfs fused to mCherry, which are functional in resuscitation assays, but do not show any specific cellular localisation under the conditions tested. Our results suggest that Rpfs are likely to be secreted via the Sec pathway. We propose that such mCherry fusions will be useful tools for the further study of Rpf localisation, individual expression, and function.