Abstract
Resuscitation promoting factors (Rpfs), belonging to a family of secreted actinobacterial proteins with predicted peptidoglycan (PG) hydrolytic activities, participate in the reactivation of dormant cells. In the present study we demonstrate that a recombinant truncated form of Micrococcus luteus Rpf hydrolyzes isolated PG of Mycobacterium smegmatis and Mycobacterium tuberculosis liberating PG fragments of different size. These fragments possess stimulatory activity toward “non-culturable” dormant M. smegmatis and M. tuberculosis cells, similar to the activity of recombinant Rpf. Relatively large PG fragments (0.1–0.5 μm) obtained either by Rpf digestion or by PG ultrasonication revealed resuscitation activities when added in concentrations 0.1–0.2 μg/ml to the resuscitation medium. It is suggested that PG fragments could either directly activate the resuscitation pathway of dormant mycobacteria or serve as a substrate for endogenous Rpf, resulting in low molecular weight products with resuscitation activity. Whilst both suggestions are plausible, it was observed that PG-dependent resuscitation activity was suppressed by means of a specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate), which provides additional support for the second of these possibilities.
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Acknowledgments
ASK thanks Programme “Molecular and cellular Biology”, Russian Academy of Sciences; GRD and MOS thank the Federal Target Programme “Molecular and cellular Biology “Scientific and scientific-pedagogical personnel of innovative Russia”, contracts 14740.1056; 14.740.11.0246; 14.740.11.08.01 and RFBR, Grants (11-04-00713-a and 11-04-01440-a) for financial support. We also would like to thank Dr. Vadim Makarov for Rpf inhibitors.
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Nikitushkin, V.D., Demina, G.R., Shleeva, M.O. et al. Peptidoglycan fragments stimulate resuscitation of “non-culturable” mycobacteria. Antonie van Leeuwenhoek 103, 37–46 (2013). https://doi.org/10.1007/s10482-012-9784-1
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DOI: https://doi.org/10.1007/s10482-012-9784-1