Abstract
BackgroundResuscitation 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.ResultsWe 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.ConclusionsWe 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.
Highlights
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
We transformed M. smegmatis mc2155, a fast growing nonpathogenic mycobacteria used as a surrogate for M. tuberculosis, with plasmids harbouring each of the five constructs
Using static microscopy the five fusion proteins appeared distributed along the cell body, but in a fraction of cells RpfB-Enhanced Green Fluorescent Protein (EGFP), RpfC-EGFP and RpfE-EGFP localised at the tips and septum, the only areas of peptidoglycan production and growth in mycobacteria (Fig. 1)
Summary
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. After the first description in Micrococcus luteus [1], five homologs were found in Mycobacterium tuberculosis, which can reactivate growth of dormant mycobacteria [2, 3]. They share a conserved Rpf domain, a 70 amino-acid region responsible for biological activity [4]. Knockouts of these proteins have been extensively studied [5,6,7]. All Rpfs are expressed in early exponential and resuscitation phases, but are differentially expressed under stress [17], possibly due to differential regulation [18,19,20,21,22], suggesting their roles do not completely overlap
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