Rpf Proteins Are the Factors of Reactivation of the Dormant Forms of Actinobacteria.

As the response to unfavorable growth conditions, nonsporulating mycobacteria transform into the dormant state with the concomitant formation of the specialized dormant forms characterized by low metabolic activity and resistance to antibiotics. Such dormant cells can be reactivated under the influence of several factors including proteins of Rpf (Resuscitation promoting factor) family, which possess peptidoglycan hydrolase activity and were considered to belong to the group of the autocrine growth factors of the bacteria. Remarkable interest toward Rpf family is determined by its participation in resuscitation of the dormant forms of Mycobacterium tuberculosis, what in turn is the key element in resuscitation of the latent tuberculosis - an infectious disease that affects one third of the World's population. Experiments with Rpf mutant forms and with strains deleted in these proteins revealed a relationship between the enzymatic activity of this protein and its ability to resuscitate mycobacteria both in vitro and in vivo. This review discusses possible mechanisms of Rpf action including those related to possible participation of the products of mycobacterial Rpf-mediated cell wall hydrolysis (muropeptides) as signaling molecules. The unique ability of Rpf proteins to resuscitate the dormant forms of mycobacteria and to stimulate their proliferation would allow these proteins to occupy their niche in medicine - in diagnostics and in creation of antituberculosis subunit vaccines.

Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases.

The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed.

[Free radicals in mercury-resistant bacteria indicate a novel metabolic pathway]. / Svobodnye radikaly u rtut'-rezistentnykh bakterii kak indikatory novogo metabolicheskogo puti.

A mercury resistant-soil bacterium P.10.15, identified as a close relative of Pseudomonas veronii, was shown to accumulate a specific compound in the stationary phase of growth. This compound is converted to a long-lived free radical under oxidizing conditions, as registered by its EPR signal at room temperature. The compound was purified by ion-exchange and gel-filtration chromatography and identified by mass spectroscopy, 2D NMR, and EPR as a trisaccharide beta-D-GlcpNOH,CH3-(1-->6)-alpha-D-Glcp-(1-->1)-alpha-D-Glcp, or, in other words, as 6-O-(2-deoxy-2-[N-methyl]hydroxylamino-beta-D- glucopyranosyl)-alpha-alpha-trehalose, previously discovered in Micrococcus luteus (lysodeikticus) and named lysodektose. The compound is suggested to be a novel intermediate of a previously unknown basic metabolic pathway of trehalose transformation in bacteria, a potential target for antibacterial drug development.

[Immunomodulatory properties of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate and search for its new derivatives]. / Immunomoduliatornye svoistva i poisk novykh proizvodnykh 2-C-Metil-D-éritriol-2,4-tsiklopirofosfata.

A strong immunomodulatory effect of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC) responsible for the survival of bacteria was shown on isolated macrophages and in experimental infections in mice (typhoid and tularemia). Derivatives of MEC were found by 1H-NMR spectroscopy under stress conditions in colorless mutants of the bacteria and isolated to be subsequently purified and used for modulation of the immune system of animals.

[Interaction of bacteria with mercuric compounds]. / Vzaimodeistvie bakterii s rtutnymi soedineniiami.

We investigated the interaction of mercuric compounds with the bacteria Corynebacterium ammoniagenes, Micrococcus luteus, and Mycobacterium smegmatus capable of producing hydroxylamines (R-NOH) and 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MECP), which are prone to form free radicals. The interaction of these substances with Hg2+ ions and their dynamics during the mercuric poisoning of bacteria was studied by EPR and NMR. Under stress conditions induced by lowering pH or generation of active oxygen species, the bacteria and, especially, their mutants with enhanced sensitivity to oxidative stress, were found to respond to exposure to 1-3 micrograms/ml HgCl2 and p-chloromercuribenzoate by a several-fold increase in their viability. The data obtained were interpreted in terms of the involvement of the sulfhydryl groups of bacterial surface proteins in this phenomenon. The interaction of bacteria with mercuric compounds may affect the pathogenesis of tuberculosis and other diseases.

[Stability of a new product of oxidative stress in bacterial cells]. / Stabil'nost' novogo produkta okislitel'nogo stress v kletkakh bakterii.

Data on 32P-label incorporation with subsequent addition of non-radiolabelled o-phosphate suggest that the new phosphorus compound, 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC), accumulated in the cells of some bacterial species in response to oxidative stress does not rapidly exchange phosphorus with external o-phosphate 3 hours after the introduction of its synthesis inducers into the Corynebacterium ammoniagenes culture. The accumulated MEC is retained in the cells despite the action of the cell wall synthesis inhibitor, chloramphenicol, or the energetic poisons, KCN and iodoacetate and also under anaerobic conditions. It has been shown that incubation of the cell-free lysate of a non-induced culture, Micrococcus luteus, with MEC does not result in MEC hydrolysis; therefore, MEC accumulation after the redox-mediator addition is hardly due to the hydrolase inactivation but, rather, is due to the activation of the MEC-synthesizing enzyme. The cells of C. ammoniagenes incorporate 32P from [32P]MEC but not 14C from [14C]MEC. This points to MEC hydrolysis prior to the uptake of its phosphoryl fragment by the cells. In this case 32P is found in the fractions differing by their position from MEC fractions. Experiments with sheep erythrocytes and mouse splenocytes revealed that MEC (10-100 micrograms per 1,000,000 splenocytes) does not influence the antibody production by these cells, whereas used at concentrations of 200-550 micrograms per 1,000,000 cells, MEC enhances the antibody production. However, while doing so, MEC causes the destruction of a considerable portion of splenocytes and sheep erythrocytes.