Acquiring of photosensitivity by Mycobacterium tuberculosis in vitro and inside infected macrophages is associated with accumulation of endogenous Zn-porphyrins.

Mycobacterium tuberculosis (Mtb) is able to transition into a dormant state, causing the latent state of tuberculosis. Dormant mycobacteria acquire resistance to all known antibacterial drugs and can survive in the human body for decades before becoming active. In the dormant forms of M. tuberculosis, the synthesis of porphyrins and its Zn-complexes significantly increased when 5-aminolevulinic acid (ALA) was added to the growth medium. Transcriptome analysis revealed an activation of 8 genes involved in the metabolism of tetrapyrroles during the Mtb transition into a dormant state, which may lead to the observed accumulation of free porphyrins. Dormant Mtb viability was reduced by more than 99.99% under illumination for 30 min (300 J/cm2) with 565 nm light that correspond for Zn-porphyrin and coproporphyrin absorptions. We did not observe any PDI effect in vitro using active bacteria grown without ALA. However, after accumulation of active cells in lung macrophages and their persistence within macrophages for several days in the presence of ALA, a significant sensitivity of active Mtb cells (ca. 99.99%) to light exposure was developed. These findings create a perspective for the treatment of latent and multidrug-resistant tuberculosis by the eradication of the pathogen in order to prevent recurrence of this disease.

The accumulation of methylated porphyrins in dormant cells of Mycolicibacterium smegmatis is accompanied by a decrease in membrane fluidity and an impede of the functioning of the respiratory chain.

Transition of Mycolicibacterium smegmatis (Msm) and Mycobacterium tuberculosis to dormancy in vitro is accompanied by an accumulation of free methylated forms of porphyrins (tetramethyl coproporphyrin - TMC) localized in the cell wall of dormant bacteria. A study of the fluorescence anisotropy of BODIPY based fluorescent probes on individual cell level using confocal microscope revealed significant changes in this parameter for BODIPY FL C16 from 0.05 to 0.22 for vegetative and dormant Msm cells correspondingly. Similarly, the increase of TMC concentration in vegetative Msm cells grown in the presence of 5-aminolevulinic acid (a known inducer of porphyrin synthesis) resulted in an increase of BODIPY FL C16 anisotropy. These changes in TMC concentration and membrane fluidity were accompanied by an inhibition of the activity of the respiratory chain measured by oxygen consumption and a reduction of the DCPIP redox acceptor. During the first 8 h of the reactivation of the dormant Msm cells, the porphyrin content and probe fluorescent anisotropy returned to the level for vegetative bacteria. We suggested that upon transition to dormancy, an accumulation of TMC in membranes leads to a decrease in membrane fluidity, resulting in an inhibition of the respiratory chain activity. However, direct interactions of TMC with membrane bound enzymes cannot also be excluded. This, in turn, may result in the down regulation of many metabolic energy-dependent reactions as a part of mechanisms accompanying the transition to a hypometabolic state of mycobacteria.

The Effect of Antimicrobial Photodynamic Inactivation on the Protein Profile of Dormant Mycolicibacterium smegmatis Containing Endogenous Porphyrins.

During transition into a dormant state, Mycolicibacterium (Mycobacterium) smegmatis cells are able to accumulate free porphyrins that makes them sensitive to photodynamic inactivation (PDI). The formation of dormant cells in a liquid medium with an increased concentration of magnesium (up to 25 mM) and zinc (up to 62 µM) resulted in an increase in the total amount of endogenous porphyrins in dormant M. smegmatis cells and their photosensitivity, especially for bacteria phagocytosed by macrophages. To gain insight into possible targets for PDI in bacterial dormant mycobacterial cells, a proteomic profiling with SDS gel electrophoresis and mass spectrometry analysis were conducted. Illumination of dormant forms of M. smegmatis resulted in the disappearance of proteins in the separating SDS gel. Dormant cells obtained under an elevated concentration of metal ions were more sensitive to PDI. Differential analysis of proteins with their identification with MALDI-TOF revealed that 45.2% and 63.9% of individual proteins disappeared from the separating gel after illumination for 5 and 15 min, respectively. Light-sensitive proteins include enzymes belonging to the glycolytic pathway, TCA cycle, pentose phosphate pathway, oxidative phosphorylation and energy production. Several proteins involved in protecting against oxygen stress and protein aggregation were found to be sensitive to light. This makes dormant cells highly vulnerable to harmful factors during a long stay in a non-replicative state. PDI caused inhibition of the respiratory chain activity and destroyed enzymes involved in the synthesis of proteins and nucleic acids, the processes which are necessary for dormant cell reactivation and their transition to multiplying bacteria. Because of such multiple targeting, PDI action via endogenous porphyrins could be considered as an effective approach for killing dormant bacteria and a perspective to inactivate dormant mycobacteria and combat the latent form of mycobacteriosis, first of all, with surface localization.

Bacillus licheniformis: A Producer of Antimicrobial Substances, including Antimycobacterials, Which Are Feasible for Medical Applications.

Bacillus licheniformis produces several classes of antimicrobial substances, including bacteriocins, which are peptides or proteins with different structural composition and molecular mass: ribosomally synthesized by bacteria (1.4-20 kDa), non-ribosomally synthesized peptides and cyclic lipopeptides (0.8-42 kDa) and exopolysaccharides (>1000 kDa). Different bacteriocins act against Gram-positive or Gram-negative bacteria, fungal pathogens and amoeba cells. The main mechanisms of bacteriocin lytic activity include interaction of peptides with membranes of target cells resulting in structural alterations, pore-forming, and inhibition of cell wall biosynthesis. DNase and RNase activity for some bacteriocines are also postulated. Non-ribosomal peptides are synthesized by special non-ribosomal multimodular peptide synthetases and contain unnatural amino acids or fatty acids. Their harmful effect is due to their ability to form pores in biological membranes, destabilize lipid packaging, and disrupt the peptidoglycan layer. Lipopeptides, as biosurfactants, are able to destroy bacterial biofilms. Secreted polysaccharides are high molecular weight compounds, composed of repeated units of sugar moieties attached to a carrier lipid. Their antagonistic action was revealed in relation to bacteria, viruses, and fungi. Exopolysaccharides also inhibit the formation of biofilms by pathogenic bacteria and prevent their colonization on various surfaces. However, mechanism of the harmful effect for many secreted antibacterial substances remains unknown. The antimicrobial activity for most substances has been studied in vitro only, but some substances have been characterized in vivo and they have found practical applications in medicine and veterinary. The cyclic lipopeptides that have surfactant properties are used in some industries. In this review, special attention is paid to the antimycobacterials produced by B. licheniformis as a possible approach to combat multidrug-resistant and latent tuberculosis. In particular, licheniformins and bacitracins have shown strong antimycobacterial activity. However, the medical application of some antibacterials with promising in vitro antimycobacterial activity has been limited by their toxicity to animals and humans. As such, similar to the enhancement in the antimycobacterial activity of natural bacteriocins achieved using genetic engineering, the reduction in toxicity using the same approach appears feasible. The unique capability of B. licheniformis to synthesize and produce a range of different antibacterial compounds means that this organism can act as a natural universal vehicle for antibiotic substances in the form of probiotic cultures and strains to combat various types of pathogens, including mycobacteria.

Hypobiosis of Mycobacteria: Biochemical Aspects.

Under suboptimal growth conditions, bacteria can transit to the dormant forms characterized by a significantly reduced metabolic activity, resistance to various stress factors, and absence of cell proliferation. Traditionally, the dormant state is associated with the formation of highly differentiated cysts and spores. However, non-spore-forming bacteria can transfer to the dormant-like hypobiotic state with the generation of less differentiated cyst-like forms (which are different from spores). This review focuses on morphological and biochemical changes occurred during formation of dormant forms of mycobacteria in particular pathogenic M. tuberculosis (Mtb) caused latent forms of tuberculosis. These forms are characterized by the low metabolic activity, the absence of cell division, resistance to some antibiotics, marked morphological changes, and loss of ability to grow on standard solid media ("non-culturable" state). Being produced in vitro, dormant Mtb retained ability to maintain latent infection in mice. After a long period of dormancy, mycobacteria retain a number of stable proteins with a potential enzymatic activity which could participate in maintaining of low-level metabolic activity in period of dormancy. Indeed, the metabolomic analysis showed significant levels of metabolites in the dormant cells even after a long period of dormancy, which may be indicative of residual metabolism in dormant mycobacteria. Special role may play intracellularly accumulated trehalose in dormant mycobacteria. Trehalose appears to stabilize dormant cells, as evidenced by the direct correlation between the trehalose content and cell viability during the long-term dormancy. In addition, trehalose can be considered as a reserve energy substrate consumed during reactivation of dormant mycobacteria due to the ATP-dependent conversion of trehalase from the latent to the active state. Another feature of dormant mycobacteria is a high representation of proteins participating in the enzymatic defense against stress factors and of low-molecular-weight compounds protecting cells in the absence of replication. Dormant mycobacteria contain a large number of hydrolyzing enzymes, which, on the one hand, ensure inactivation of biomolecules damaged by stress. On the other hand, the products of these enzymatic reactions can be used for the maintenance of energy state and vital activity of bacterial cells during their long-term survival in the dormant state, i.e., for creating a situation that we propose to refer to as the "catabolic survival". In general, dormant non-replicating mycobacterial cells can be described as morphologically altered forms that contain principal macromolecules and are stabilized and protected from the damaging factors by an arsenal of proteins and low-molecular-weight compounds. Because of the presumable occurrence of metabolic reactions in such cells, this form of survival should be referred to as hypobiosis.

Shotgun proteomic profiling of dormant, 'non-culturable' Mycobacterium tuberculosis.

Dormant cells of Mycobacterium tuberculosis, in addition to low metabolic activity and a high level of drug resistance, are characterized by 'non-culturability'-a specific reversible state of the inability of the cells to grow on solid media. The biochemical characterization of this physiological state of the pathogen is only superficial, pending clarification of the metabolic processes that may exist in such cells. In this study, applying LC-MS proteomic profiling, we report the analysis of proteins accumulated in dormant, 'non-culturable' M. tuberculosis cells in an in vitro model of self-acidification of mycobacteria in the post-stationary phase, simulating the in vivo persistence conditions-the raw data are available via ProteomeXchange with identifier PXD028849. This approach revealed the preservation of 1379 proteins in cells after 5 months of storage in dormancy; among them, 468 proteins were statistically different from those in the actively growing cells and bore a positive fold change (FC). Differential analysis revealed the proteins of the pH-dependent regulatory system PhoP and allowed the reconstruction of the reactions of central carbon/glycerol metabolism, as well as revealing the salvaged pathways of mycothiol and UMP biosynthesis, establishing the cohort of survival enzymes of dormancy. The annotated pathways mirror the adaptation of the mycobacterial metabolic machinery to life within lipid-rich macrophages: especially the involvement of the methyl citrate and glyoxylate pathways. Thus, the current in vitro model of M. tuberculosis self-acidification reflects the biochemical adaptation of these bacteria to persistence in vivo. Comparative analysis with published proteins displaying antigenic properties makes it possible to distinguish immunoreactive proteins among the proteins bearing a positive FC in dormancy, which may include specific antigens of latent tuberculosis. Additionally, the biotransformatory enzymes (oxidoreductases and hydrolases) capable of prodrug activation and stored up in the dormant state were annotated. These findings may potentially lead to the discovery of immunodiagnostic tests for early latent tuberculosis and trigger the discovery of efficient drugs/prodrugs with potency against non-replicating, dormant populations of mycobacteria.

Photoinactivation of mycobacteria to combat infection diseases: current state and perspectives.

The spread of multi-drug-resistant bacterial strains causing serious infectious diseases dictates the development of new approaches to combat these diseases. In addition to drug resistance, the important causative agent of tuberculosis (Mycobacterium tuberculosis (Mtb)) is able to persist asymptomatically in individuals for many years, causing latent forms of tuberculosis. In such a dormant state, Mtb cells are also resistant to known antibiotics. In this regard, photodynamic inactivation (PDI) could be an effective alternative to antibiotics as its action is based on the generation of active forms of oxygen independently on the presence of specific antibiotic targets, thereby inactivating both drug-resistant and dormant bacteria. In this review, we summarise examples of the application of PDI for the elimination of representatives of the genus Mycobacteria, both in vitro and in vivo. According to published results, including photosensitisers in the PDI regime results in a significantly higher lethal effect. Such experiments were mainly performed using chemically synthesised photosensitisers, which need to be transported to the areas of bacterial infections, limiting PDI usage by surface (skin) diseases. In this regard, endogenous photosensitisers (mainly porphyrins) could be used to solve the problem of transportation. In vitro experiments demonstrate the effective application of PDI for mycobacteria, including Mtb, using endogenous porphyrins; the intracellular contents of these substances can be elevated by administration of 5-aminolevulenic acid, a precursor of porphyrin synthesis. Photodynamic inactivation can also be used for dormant mycobacteria, which are characterised by high levels of endogenous porphyrins. Thus, PDI can effectively eliminate drug-resistant mycobacteria. The exploitation of modern light-transmitting techniques opens new possibilities to use PDI in clinical settings. KEY POINTS: •The potential effects of photodynamic inactivation of mycobacteria are critically reviewed. •Approaches to photoinactivation of mycobacteria using exogenous and endogenous photosensitisers are described. •Prospects for the use of photodynamic inactivation in the treatment of tuberculosis are discussed.

Prolonged infection triggered by dormant Mycobacterium tuberculosis: Immune and inflammatory responses in lungs of genetically susceptible and resistant mice.

We developed an approach for substantial attenuation of Mycobacterium tuberculosis by prolonged culturing under gradually acidifying conditions. Bacteria subjected to acidification lost the capacity to form colonies on solid media, but readily resuscitated their growth in the murine host, providing a useful model to study in vivo development of infection mimicking latent and reactivation tuberculosis (TB) in humans. Here we characterize biomarkers of lung pathology and immune responses triggered by such attenuated bacteria in genetically TB-susceptible and resistant mice. In susceptible I/St mice, CFU counts in lungs and spleens were ~1.5-log higher than in resistant B6 mice, accompanied by diffuse pneumonia and excessive lung infiltration with highly activated CD44+CD62L- T-lymphocytes resulting in death between months 7-9 post challenge. B6 mice were characterized by development of local inflammatory foci, higher production of pro-inflammatory IL-6 and IL-11 cytokines and a more balanced T-cell activation in their lungs. CFU counts remained stable in B6 mice during the whole 18-mo observation period, and all mice survived. Thus, we established a mouse model of fatal reactivation TB vs. indefinite mycobacterial possession after identical challenge and characterized the features of immune responses in the lung tissue underlining these polar phenotypes.

One-Year Old Dormant, "Non-culturable" Mycobacterium tuberculosis Preserves Significantly Diverse Protein Profile.

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.

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.

Corynebacterium jeikeium Dormant Cell Formation and Photodynamic Inactivation.

Pathogenic non-spore forming bacteria enter a dormant state under stressful conditions, which likely allows them to acquire resistance to various antibiotics. This work revealed the efficient formation of dormant "non-culturable" (NC) Corynebacterium jeikeium cells in stationary phase upon gradual acidification of the growth medium. Such cells were unable to form colonies and existed in a prolonged stationary phase. At an early stage of dormancy (approximately 14 days post-inoculation), dormant cells are able for resuscitation in liquid medium. However, those stored for long time in dormant state needed addition of supernatant taking from active C. jeikeium cultures for successful resuscitation. NC cells possessed low RNA synthesis and significant tolerance to antibiotics (rifampicin and vancomycin). They also accumulated free porphyrins, and 5-aminolevulinic acid addition enhanced free porphyrin accumulation which makes them potentially sensitive to photodynamic inactivation (PDI). PDI of dormant bacteria was accomplished by exposing cells to a 565 nm wavelength of light using a SOLIS-4C light-emitting diode for 60 min. This revealed that increased porphyrin concentrations were correlated with elevated PDI sensitivity. Results shown here demonstrate the potential utility of employing PDI to minimize levels of dormant, persistent corynebacteria and the C. jeikeium dormancy model developed here may be useful for finding new drugs and techniques for combatting persistent corynebacteria.

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.

Protein Composition of Mycobacterium smegmatis Differs Significantly Between Active Cells and Dormant Cells With Ovoid Morphology.

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.

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.

Overexpression of Adenylyl Cyclase Encoded by the Mycobacterium tuberculosis Rv2212 Gene Confers Improved Fitness, Accelerated Recovery from Dormancy and Enhanced Virulence in Mice.

Earlier we demonstrated that the adenylyl cyclase (AC) encoded by the MSMEG_4279 gene plays a key role in the resuscitation and growth of dormant Mycobacterium smegmatis and that overexpression of this gene leads to an increase in intracellular cAMP concentration and prevents the transition of M. smegmatis from active growth to dormancy in an extended stationary phase accompanied by medium acidification. We surmised that the homologous Rv2212 gene of M. tuberculosis (Mtb), the main cAMP producer, plays similar physiological roles by supporting, under these conditions, the active state and reactivation of dormant bacteria. To test this hypothesis, we established Mtb strain overexpressing Rv2212 and compared its in vitro and in vivo growth characteristics with a control strain. In vitro, the AC-overexpressing pMindRv2212 strain demonstrated faster growth in a liquid medium, prolonged capacity to form CFUs and a significant delay or even prevention of transition toward dormancy. AC-overexpressing cells exhibited easier recovery from dormancy. In vivo, AC-overexpressing bacteria demonstrated significantly higher growth rates (virulence) in the lungs and spleens of infected mice compared to the control strain, and, unlike the latter, killed mice in the TB-resistant strain before month 8 of infection. Even in the absence of selecting hygromycin B, all pMindRv2212 CFUs retained the Rv2212 insert during in vivo growth, strongly suggesting that AC overexpression is beneficial for bacteria. Taken together, our results indicate that cAMP supports the maintenance of Mtb cells vitality under unfavorable conditions in vitro and their virulence in vivo.

Free Trehalose Accumulation in Dormant Mycobacterium smegmatis Cells and Its Breakdown in Early Resuscitation Phase.

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.

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.

Nonquaternary poly(diallylammonium) polymers with different amine structure and their biocidal effect on Mycobacterium tuberculosis and Mycobacterium smegmatis.

Mycobacteria, especially Mycobacterium tuberculosis, are one of the most dangerous types of microorganisms to cause diseases and mortality. Due to the known distinctive structure of their cell wall, mycobacteria are resistant to majority of antibiotics and common chemical disinfectants, including quaternized low molecular weight and polymer biocides. In this work, nonquaternary protonated polydiallylamines (PDAAs) based on protonated monomers of the diallylamine (DAA) series have been synthesized, secondary s-PDAA and tertiary t-Me-PDAA and t-Et-PDAA (with Me and Et N-substituents). The antimicrobial actions of PDAAs on M. tuberculosis and Mycobacterium smegmatis have been studied, namely, dependences of the activity on the amine structure, length of alkyl N-substituents, M w of polymers, treatment time, and cell concentration. All PDAAs examined at different conditions have been found to exhibit strong bactericidal effect on M. smegmatis and M. tuberculosis, including "nonculturable" dormant M. tuberculosis cells. The quaternary counterpart poly(diallyldimethylammonium chloride) (PDADMAC) and current antibiotics rifampicin and ciprofloxacin have been also tested and shown to be significantly less efficient or inactive at all (at the maximum tested concentration of 500 µg mL(-1)). s-PDAA appeared to be the most effective or exhibited similar activity to t-Me-PDAA, while t-Et-PDAA appeared to be less active, especially against M. tuberculosis. The results obtained indicate a key role of the nonquaternary ammonium groups in the mycobactericidal action of PDAAs. Examination under an optical microscope in the epifluorescence mode has evidenced damage of the inner membrane permeability of M. smegmatis cells under the impact of PDAAs after 20 min. Studies on electrophoretic mobility (zeta-potential) of M. smegmatis cells and some model liposomes in the presence of PDAAs have revealed a small negative charge of mycobacteria outer surface and recharge in the presence of PDAAs. A conclusion was made that bactericidal activity of PDAAs is related to the disturbance of the integrity of the mycobacterial cell wall followed by damage of the inner membrane permeability.

Reactivation of dormant "non-culturable" Mycobacterium tuberculosis developed in vitro after injection in mice: both the dormancy depth and host genetics influence the outcome.

Three stocks of Mycobacterium tuberculosis H37Rv were cultured in vitro under prolonged hypoxic or acidified conditions until partial or complete loss of the capacity to form colonies on agar medium was achieved. Such dormant "non-culturable" mycobacteria were assessed for the growth resuscitation after intra-tracheal injection into mice of the two inbred strains with different genetic susceptibility to M. tuberculosis-triggered disease: hyper-susceptible I/St and relatively resistant B6. The results indicate that bacteria which are able to resuscitate spontaneously in liquid medium in vitro started to multiply in organs of infected mice, and that the outcome of such infection strongly depended upon the level of genetic TB susceptibility. However, dormant bacteria required inducers for resuscitation in vitro lost the capacity to multiply even in genetically susceptible mice. The established model of dormancy/reactivation is suitable for the studying host-pathogen interactions and testing vaccine and drug candidates specifically targeting latent TB.

Cyclic AMP-dependent resuscitation of dormant Mycobacteria by exogenous free fatty acids.

One third of the world population carries a latent tuberculosis (TB) infection, which may reactivate leading to active disease. Although TB latency has been known for many years it remains poorly understood. In particular, substances of host origin, which may induce the resuscitation of dormant mycobacteria, have not yet been described. In vitro models of dormant ("non-culturable") cells of Mycobacterium smegmatis (mc(2)155) and Mycobacterium tuberculosis H37Rv were used. We found that the resuscitation of dormant M. smegmatis and M. tuberculosis cells in liquid medium was stimulated by adding free unsaturated fatty acids (FA), including arachidonic acid, at concentrations of 1.6-10 µM. FA addition enhanced cAMP levels in reactivating M. smegmatis cells and exogenously added cAMP (3-10 mM) or dibutyryl-cAMP (0.5-1 mM) substituted for FA, causing resuscitation of M. smegmatis and M. tuberculosis dormant cells. A M. smegmatis null-mutant lacking MSMEG_4279, which encodes a FA-activated adenylyl cyclase (AC), could not be resuscitated by FA but it was resuscitated by cAMP. M. smegmatis and M. tuberculosis cells hyper-expressing AC were unable to form non-culturable cells and a specific inhibitor of AC (8-bromo-cAMP) prevented FA-dependent resuscitation. RT-PCR analysis revealed that rpfA (coding for resuscitation promoting factor A) is up-regulated in M. smegmatis in the beginning of exponential growth following the cAMP increase in lag phase caused by FA-induced cell activation. A specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate) suppressed FA-induced resuscitation. We propose a novel pathway for the resuscitation of dormant mycobacteria involving the activation of adenylyl cyclase MSMEG_4279 by FAs resulted in activation of cellular metabolism followed later by increase of RpfA activity which stimulates cell multiplication in exponential phase. The study reveals a probable role for lipids of host origin in the resuscitation of dormant mycobacteria, which may function during the reactivation of latent TB.