Metabolic profiling of dormant Mycolicibacterium smegmatis cells' reactivation reveals a gradual assembly of metabolic processes.

INTRODUCTION: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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.

Lectin-based detection of Escherichia coli and Staphylococcus aureus by flow cytometry.

This study develops a flow cytometry analysis of the bacterial pathogens Escherichia coli and Staphylococcus aureus based on a ligand-bioreceptor interaction. We used fluorescently labeled plant lectins as natural receptors that could specifically interact with the cell wall carbohydrates of bacteria. An epifluorescence microscopy was used as an additional approach to confirm and visualize lectin-carbohydrate interactions. The binding specificity of plant lectins to E. coli and S. aureus cells was studied, and wheat germ agglutinin, which provided high-affinity interactions, was selected as a receptor. Using this method, bacterial pathogens can be detected in concentrations of up to 106 cells/mL within 5 min. Their accessibility and universality make lectin reagents a promising tool to control a wide range of bacterial pathogens.

Photoinactivation of dormant Mycobacterium smegmatis due to its endogenous porphyrins.

Mycobacterium tuberculosis is able to transition into a dormant state, causing a latent state of tuberculosis. Dormant mycobacteria acquire phenotypic resistance to all known antibacterial drugs; they are also able to maintain vitality in the host for decades and become active, causing the active form of the disease. In order to cure latent tuberculosis, new approaches should be developed. Earlier, we discovered accumulation in significant concentrations of porphyrins in dormant Mycobacterium smegmatis, which is a close, fast-growing relative of the causative agent of tuberculosis. In this study, we explore a new possibility to kill dormant mycobacteria by photodynamic inactivation (PDI) using accumulated porphyrins as endogenous photosensitisers. The dormant M. smegmatis were obtained under gradual acidification in Sauton's medium, for 14 days. Cells were exposed to light with different wavelengths emitted by three Spectra X light-emitting diodes (395/25, 470/24, 575/25 nm) and one separated 634-nm LED for 15 min. An increase in the concentration of coproporphyrin in M. smegmatis after 6 days of growth correlated with the beginning of a decrease in metabolic activity and formation of ovoid dormant forms. Dormant bacteria were sensitive to PDI and killed after 15-30 min of illumination, in contrast to active cells. The greatest inactivation of dormant mycobacteria occurred at 395 and 575 nm, which coincides with the main maximum of the absorption spectrum of extracted porphyrins. We, for the first time, demonstrate a successful application of PDI for inactivation of dormant mycobacteria, due to significant accumulation of endogenous photosensitisers-porphyrins.

Benzoylphenyl thiocyanates are new, effective inhibitors of the mycobacterial resuscitation promoting factor B protein.

BACKGROUND: Resuscitation promoting factors (Rpfs) are the proteins involved in the process of reactivation of the dormant cells of mycobacteria. Recently a new class of nitrophenylthiocyanates (NPTs), capable of inhibiting the biological and enzymatic activities of Rpfs has been discovered. In the current study the inhibitory properties of the compounds containing both nitro and thiocyanate groups alongside with the compounds with the modified number and different spatial location of the substituents are compared. METHODS: New benzoylphenyl thiocyanates alongside with nitrophenylthiocyanates were tested in the enzymatic assay of bacterial peptidoglycan hydrolysis as well as against strains of several actinobacteria (Mycobacterium smegmatis, Mycobacterium tuberculosis) on in-lab developed models of resuscitation of the dormant forms. RESULTS: Introduction of the additional nitro and thiocyanate groups to the benzophenone scaffold did not influence the inhibitory activity of the compounds. Removal of the nitro groups analogously did not impair the functional properties of the molecules. Among the tested compounds two molecules without nitro group: 3-benzoylphenyl thiocyanate and 4-benzoylphenyl thiocyanate demonstrated the maximum activity in both enzymatic assay (inhibition of the Rpf-mediated peptidoglycan hydrolysis) and in the resuscitation assay of the dormant M. tuberculosis cells. CONCLUSIONS: The current study demonstrates dispensability of the nitro group in the NPT's structure for inhibition of the enzymatic and biological activities of the Rpf protein molecules. These findings provide new prospects in anti-TB drug discovery especially in finding of molecular scaffolds effective for the latent infection treatment.

Peptidoglycan fragments stimulate resuscitation of "non-culturable" mycobacteria.

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.

A product of RpfB and RipA joint enzymatic action promotes the resuscitation of dormant mycobacteria.

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 ß-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-ß(1 → 4)-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 Mycobacterium smegmatis model. It was found that the disaccharide-dipeptide was the minimal structure capable of resuscitating the dormant mycobacterial cells over the concentration range of 9-100 ng · mL(-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.

Finding of the low molecular weight inhibitors of resuscitation promoting factor enzymatic and resuscitation activity.

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.