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The muralitic activity of Rpf proteins and their ability to resuscitate dormant mycobacterial cells

Abstract number: 1733_1496

Demina G., Mukamolova G., Kaprelyants A.

Objectives:Micrococcus luteus secretes a small protein Rpf, which plays an important role in the resuscitation of dormant cells of several species of Gram-positive bacteria, e.g. M. luteus and pathogenic bacteria Mycobacteriumtuberculosis. Latent tuberculosis infection is believed to connect with the dormant state of M. tuberculosis in the human host. Rpf-like proteins contain specific domain which adopts a lysozime-like fold however enzymatic activity of Rpf proteins is poorly understood.

The purpose of this study is to elucidate an enzymatic activity of Rpf proteins and its relation to biological activity.

Methods: The muramidase activity was measured fluorimetrically, 4-methylumbelliferyl-beta-D-N,N'N"-triacetylchitotrioside was used as a substrate. The efficacy of resuscitated ``non-culturable'' M. smegmatis cells in liquid medium was estimated by MPN assay.

Results: Recombinant M. luteus Rpf is able to hydrolise the artifical lysozyme substrate which is structurally similar to peptidoglycan of bacterial cell wall. The invariant catalytic glutamate residue found in lysozyme and lytic transglycosylases is also conserved in the Rpf proteins. Replacement of glutamate residue by amino acids with less structural similarity resulted in a decrease in enzymatic activity of Rpf and its ability to resuscitate dormant Mycobacterium smegmatis cells. Rpf possible cleaves glycosidic bonds with inversion of configuration, but in contrast to lytic transglycosylase, Rpf doesn't form 1,6-anhydromuro derivatives (Keep et al., 2006). The effect of the some metal ions on the enzymatic activity was studied; the results show that Mg2+ and Ca2+ slightly activate the enzyme, while Zn2+ inhibits enzymatic activity.

Conclusions: Rpf proteins are evidently peptidoglycan hydrolase enzymes. Rpf's muralitic activity correlates with its ability to stimulate resuscitation of dormant bacteria, consistent with the view that biological activity of Rpf molecule is connected directly or indirectly with its ability to cleave glycosidic bonds in bacterial peptidoglycan.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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