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Abstract The protein profile of dormant Mtb obtained after the gradual acidification of Mtb culture was studied to find antigenic proteins for humans that are expressed by M. tuberculosis (Mtb) cells in vitro under conditions close to the situation of persistence in vivo. According to 2D electrophoresis, a significant diversity of proteins in dormant cells was found. However, the representation of individual proteins in dormant versus active cells differed substantially. Immunoblotting in different protein fractions of dormant cells revealed ten proteins that are able to bind antibodies in pooled sera of TB patients. Two proteins (Rv2018 and Rv0341) are new immunogenics that were not previously found in other studies. Four proteins (Rv0341, Rv2018, Rv1509, Rv2986) with the maximal structural specificity for Mtb due to their unique extended domains were selected for further analysis. These proteins were expressed in E. coli cells and studied via enzyme-linked immunosorbent assay (ELISA) for the immunogenicity of individual sera of TB patients and healthy donors. All proteins were found to have the ability to react with individual sera of TB patients. In TB patients, 5–45% (depending on the particulate protein) have a titer that is higher than the average titers of healthy donors +SD; the most immunogenic was protein Rv2986. Thus, the application of phenotypically changed (dormant) Mtb cells makes it possible to identify a specific repertoire of immunodominant proteins that could be used in the construction of polypeptides that are useful for the serodiagnosis of active/latent TB.

Under gradual acidification of the culture medium mycobacterial cells transit into a specific state characterized by low level of metabolic activity and morphological alterations. This state of non-replicative persistence (dormancy) is directly linked to physiological drug resistance, which complicates the efforts to eradicate the latent forms of TB. In order to find new anti-latent TB compounds, the metabolic processes which may occur in the state of dormancy and during the transition into the active state (reactivation) should be characterized.In the current study we analyzed the untargeted metabolomic profiles of dormant and reactivating Mycolicibacterium smegmatis cells (a model microorganism, bearing many common physiological traits of MTB), on the global scale level, since the characterization and analysis of the metabolites' dynamics would provide a comprehensive overview on global biochemical responses of the bacteria to stress conditions.The reactivation process was tracked by measuring the value of membrane potential, applying a ratio-metric approach, by the method of flow-cytometry. The crucial timepoints were selected and the bacteria were sampled to LC-MS metabolic profiling.Reactivation of these cells after 60 days of storage revealed that this process proceeds in two stages: (I) a period, which lasts for 10 h and is characterized by a constant CFU number, unchangeable cell size, a minuscule increase of respiratory activity and a noticeable increase in membrane potential value, indicating the onset of the first metabolic processes during this time interval; the second phase (10-26 h) is characterized by acceleration of endogenous respiration, changes in the size of the cells and it finishes with the beginning of cells division. Analysis of the changes in the relative abundances of KEGG-annotated metabolites revealed that a significant number of metabolites, such as stearic acid, glycerol, D-glucose, trehalose-6-phosphate decrease their concentrations over the reactivation time, whereas in contrast, such metabolites as dodecanoic acid, mycobactin S, and other compounds of PG/AG biosynthesis are synthesized during reactivation. Differential analysis of metabolic profiles disclosed the activation of a number of metabolic pathways at the early reactivation stage: biosynthesis of secondary metabolites, purine and pyrimidine metabolism, glycerophospholipid and fatty acids metabolism etc. CONCLUSION: The data obtained indicate, despite the long-term storage of dormant cells in a state of minimal metabolic activity, according to metabolic profiling, they still retained a large number of metabolites. In the process of reactivation, the incremental stochastic assembly of the complete metabolic pathways occurs.

For adaptation to stressful conditions, Mycobacterium tuberculosis (Mtb) is prone to transit to a dormant, non-replicative state, which is believed to be the basis of the latent form of tuberculosis infection. Dormant bacteria persist in the host for a long period without multiplication, cannot be detected from biological samples by microbiological methods, however, their “non-culturable” state is reversible. Mechanisms supporting very long capacity of mycobacteria for resuscitation and further multiplication after prolonged survival in a dormant phase remain unclear. Using methods of 2D electrophoresis and MALDI-TOF analysis, in this study we characterized changes in the proteomic profile of Mtb stored for more than a year as dormant, non-replicating cells with a negligible metabolic activity, full resistance to antibiotics, and altered morphology (ovoid forms). Despite some protein degradation, the proteome of 1-year-old dormant mycobacteria retained numerous intact proteins. Their protein profile differed profoundly from that of metabolically active cells, but was similar to the proteome of the 4-month-old dormant bacteria. Such protein stability is likely to be due to the presence of a significant number of enzymes involved in the protection from oxidative stress (katG/Rv1908, sodA/Rv3846, sodC/Rv0432, bpoC/Rv0554), as well as chaperones (dnaJ1/Rv0352, htpG/Rv2299, groEL2/Rv0440, dnaK/Rv0350, groES/Rv3418, groEL1/Rv3417, HtpG/Rv2299c, hspX/Rv2031), and DNA-stabilizing proteins. In addition, dormant cells proteome contains enzymes involved in specific metabolic pathways (glycolytic reactions, shortened TCA cycle, degradative processes) potentially providing a low-level metabolism, or these proteins could be “frozen” for usage in the reactivation process before biosynthetic processes start. The observed stability of proteins in a dormant state could be a basis for the long-term preservation of Mtb cell vitality and hence for latent tuberculosis.

Mycobacteria are able to form dormant cells, which survive for a long time without multiplication. The molecular mechanisms behind prolonged survival of dormant cells are not fully described. In particular, little information is known on biochemical processes which might take place in cells under dormancy. To gain insight into this problem, Mycobacterium smegmatis cells in deep dormant state were obtained after gradual acidification of the growth medium in prolonged stationary phase followed by 1 month of storage at room temperature. Such cells were characterized by low metabolic activity, including respiration, resistance to antibiotics, and altered morphology. The protein composition of cytoplasm and membrane fractions obtained from active and dormant cells were compared by 2D electrophoresis. Almost half of the proteins found in the proteome of dormant cells were absent in that of active cells. This result differs significantly from published results obtained in other studies employing different models of mycobacterium dormancy. This discrepancy could be explained by a deeper dormancy developed in the present model. A feature of a “dormant proteome” is high representation of enzymes involved in glycolysis and defense systems that inactivate or detoxify reactive oxygen and nitrogen species, aldehydes, and oxidized lipids. Dormant mycobacteria are enriched by degradative enzymes, which could eliminate damaged molecules, or the products of such degradation could be reutilized by the cell during prolonged storage. We suggest that some enzymes in dormant cells are inactive, having been used upon transition to the dormant state, or proteins stored in dormant cells for further cell reactivation. At the same time, some proteins could be functional and play roles in maintenance of cell metabolism, albeit at a very slow rate. This study provides a clue as to which biochemical processes could be active under dormancy to ensure long-term viability of dormant mycobacteria.

Under gradual acidification of growth medium resulting in the formation of dormant Mycobacterium smegmatis, a significant accumulation of free trehalose in dormant cells was observed. According to 1H- and 13C-NMR spectroscopy up to 64% of total organic substances in the dormant cell extract was represented by trehalose whilst the trehalose content in an extract of active cells taken from early stationary phase was not more than 15%. Trehalose biosynthesis during transition to the dormant state is provided by activation of genes involved in the OtsA-OtsB and TreY-TreZ pathways (according to RT-PCR). Varying the concentration of free trehalose in dormant cells by expression of MSMEG_4535 coding for trehalase we found that cell viability depends on trehalose level: cells with a high amount of trehalose survive much better than cells with a low amount. Upon resuscitation of dormant M. smegmatis, a decrease of free trehalose and an increase in glucose concentration occurred in the early period of resuscitation (after 2 h). Evidently, breakdown of trehalose by trehalase takes place at this time as a transient increase in trehalase activity was observed between 1 and 3 h of resuscitation. Activation of trehalase was not due to de novo biosynthesis but because of self-activation of the enzyme from the inactive state in dormant cells. Because, even a low concentration of ATP (2 mM) prevents self-activation of trehalase in vitro and after activation the enzyme is still sensitive to ATP we suggest that the transient character of trehalase activation in cells is due to variation in intracellular ATP concentration found in the early resuscitation period. The negative influence of the trehalase inhibitor validamycin A on the resuscitation of dormant cells proves the importance of trehalase for resuscitation. These experiments demonstrate the significance of free trehalose accumulation for the maintenance of dormant mycobacterial viability and the involvement of trehalose breakdown in early events leading to cell reactivation similar to yeast and fungal spores.

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.

Under gradual acidification of growth medium resulting in the formation of dormant Mycobacterium smegmatis, a significant accumulation of free trehalose in dormant cells was observed. According to 1H- and 13C-NMR spectroscopy up to 64% of total organic substances in the dormant cell extract was represented by trehalose whilst the trehalose content in an extract of active cells taken from early stationary phase was not more than 15%. Trehalose biosynthesis during transition to the dormant state is provided by activation of genes involved in the OtsA-OtsB and TreY-TreZ pathways (according to RT-PCR). Varying the concentration of free trehalose in dormant cells by expression of MSMEG_4535 coding for trehalase we found that cell viability depends on trehalose level: cells with a high amount of trehalose survive much better than cells with a low amount. Upon resuscitation of dormant M. smegmatis, a decrease of free trehalose and an increase in glucose concentration occurred in the early period of resuscitation (after 2 h). Evidently, breakdown of trehalose by trehalase takes place at this time as a transient increase in trehalase activity was observed between 1 and 3 h of resuscitation. Activation of trehalase was not due to de novo biosynthesis but because of self-activation of the enzyme from the inactive state in dormant cells. Because, even a low concentration of ATP (2 mM) prevents self-activation of trehalase in vitro and after activation the enzyme is still sensitive to ATP we suggest that the transient character of trehalase activation in cells is due to variation in intracellular ATP concentration found in the early resuscitation period. The negative influence of the trehalase inhibitor validamycin A on the resuscitation of dormant cells proves the importance of trehalase for resuscitation. These experiments demonstrate the significance of free trehalose accumulation for the maintenance of dormant mycobacterial viability and the involvement of trehalose breakdown in early events leading to cell reactivation similar to yeast and fungal spores.

The effect of Resuscitation promoting factor (Rpf) on intercellular contacts in the cultures of Micrococcus luteus and Mycobacterium smegmatis was investigated using dynamic light scattering (DLS, photon correlation spectroscopy). During the stationary growth phase, the cells of the tested cultures formed extensive aggregates 100 and 300 μ in size for M. smegmatis and M. luteus, respectively. The number of solitary cells was insignificant. Addition of the recombinant Rpf protein (15 μg/ml) resulted dispersion of cell aggregates and emergence of solitary cells. This effect of Rpf decreased in the presence of nitrophenylthiocyanates (NPTs), specific Rpf inhibitors. Presumably, Rpf is involved in the regulation of intracellular interactions and in biofilm formation.

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.

The culturability of several actinobacteria is controlled by resuscitation-promoting factors (Rpfs). These are proteins containing a c. 70-residue domain that adopts a lysozyme-like fold. The invariant catalytic glutamate residue found in lysozyme and various bacterial lytic transglycosylases is also conserved in the Rpf proteins. Rpf from Micrococcus luteus, the founder member of this protein family, is indeed a muralytic enzyme, as revealed by its activity in zymograms containing M. luteus cell walls and its ability to (i) cause lysis of Escherichia coli when expressed and secreted into the periplasm; (ii) release fluorescent material from fluorescamine-labelled cell walls of M. luteus; and (iii) hydrolyse the artificial lysozyme substrate, 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside. Rpf activity was reduced but not completely abolished when the invariant glutamate residue was altered. Moreover, none of the other acidic residues in the Rpf domain was absolutely required for muralytic activity. Replacement of one or both of the cysteine residues that probably form a disulphide bridge within Rpf impaired but did not completely abolish muralytic activity. The muralytic activities of the Rpf mutants were correlated with their abilities to stimulate bacterial culturability and resuscitation, consistent with the view that the biological activity of Rpf results directly or indirectly from its ability to cleave bonds in bacterial peptidoglycan.

Resuscitation-promoting factor proteins (Rpfs) are known to participate in reactivating the dormant forms of actinobacteria. Structural analysis of the Rpf catalytic domain demonstrates its similarity to lysozyme and to lytic transglycosylases - the groups of enzymes that cleave the -1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and GlcNAc, and concomitantly form a 1,6-anhydro ring at the MurNAc residue. Analysis of the products formed from mycobacterial peptidoglycan hydrolysis reactions containing a mixture of RpfB and resuscitation-promoting factor interacting protein (RipA) allowed us to identify the suggested product of their action - N-acetylglucosaminyl-(14)-N-glycolyl-1,6-anhydromuramyl-l-alanyl-d-isoglutamate. To identify the role of this resulting product in resuscitation, we used a synthetic 1,6-anhydrodisaccharide-dipeptide, and tested its ability to stimulate resuscitation by using the dormant Mycobacteriumsmegmatis model. It was found that the disaccharide-dipeptide was the minimal structure capable of resuscitating the dormant mycobacterial cells over the concentration range of 9-100ngmL(-1). The current study therefore provides the first insights into the molecular mechanism of resuscitation from dormancy involving a product of RpfB/RipA-mediated peptidoglycan cleavage.

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.

Resuscitation promoting factors (Rpf) are a family of proteins secreted by actively growing actinobacteria, including Mycobacterium tuberculosis. Experimental evidence suggests that Rpfs play a distinct role in bacterial resuscitation and re-growth as well as reactivation of chronic tuberculosis in mice. The striking similarity of the Rpfs structure to cell wall hydrolysing enzymes has provided a basis for the development of novel low molecular weight inhibitors of Rpfs activity. In particular, recently characterised nitrophenylthiocyanate compounds could be considered as a promising scaffold for generation of therapeutic agents targeting reactivation of latent tuberculosis. This review describes recent progress in understanding of molecular mechanisms of Rpf biological activity.

The role of histone-like protein (Hlp) in the development of a dormant state in long-incubated stationary-phase Mycobacterium smegmatis cells was studied in two models: (1) adoption of 'nonculturable' (NC) state, which is reversible due to resuscitation with proteinaceous resuscitation-promoting factor (Rpf) and (2) the formation of morphologically distinct, ovoid resting forms. In the first model, inactivation of the hlp gene resulted in prolongation of culturability of starved cells followed by irreversible nonculturability when mycobacterial cells were unresponsive to resuscitation with Rpf. In the second model, M. smegmatis strain with the inactivated hlp gene was able to form dormant ovoid cells, but they were less resistant to heating and UV radiation than those of wild-type strain. The susceptibility of ovoid cells produced by Delta hlp mutant to these damaging factors was probably due to a less condensed state of DNA, as revealed by fluorescent microscopy and DAPI staining. Evidently, Hlp is essential for cell viability at a later stage of NC dormancy or provides a greater stability of specialized dormant forms.

The role of histone-like protein (Hlp) in the development of a dormant state in long-incubated stationary-phase Mycobacterium smegmatis cells was studied in two models: (1) adoption of ‘nonculturable’ (NC) state, which is reversible due to resuscitation with proteinaceous resuscitation-promoting factor (Rpf) and (2) the formation of morphologically distinct, ovoid resting forms. In the first model, inactivation of the hlp gene resulted in prolongation of culturability of starved cells followed by irreversible nonculturability when mycobacterial cells were unresponsive to resuscitation with Rpf. In the second model, M. smegmatis strain with the inactivated hlp gene was able to form dormant ovoid cells, but they were less resistant to heating and UV radiation than those of wild-type strain. The susceptibility of ovoid cells produced by Δhlp mutant to these damaging factors was probably due to a less condensed state of DNA, as revealed by fluorescent microscopy and DAPI staining. Evidently, Hlp is essential for cell viability at a later stage of NC dormancy or provides a greater stability of specialized dormant forms.

BACKGROUND: Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC(50) 1-7 microg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC(50). The candidate compounds suppressed resuscitation of dormant ("non-culturable") cells of M. smegmatis at 1 microg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 microg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations. CONCLUSIONS/SIGNIFICANCE: NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins.