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.

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.

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.

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.

Dormant non-culturable Mycobacterium tuberculosis retains stable low-abundant mRNA.

BACKGROUND: Dormant Mycobacterium tuberculosis bacilli are believed to play an important role in latent tuberculosis infection. Previously, we have demonstrated that cultivation of M. tuberculosis in K(+)-deficient medium resulted in generation of dormant cells. These bacilli were non-culturable on solid media (a key feature of dormant M. tuberculosis in vivo) and characterized by low metabolism and tolerance to anti-tuberculosis drugs. The dormant bacteria demonstrated a high potential to reactivation after K(+) reintroduction even after prolonged persistence under rifampicin. In this work, we studied the transcriptome and stability of transcripts in persisting dormant bacilli under arrest of mRNA de novo synthesis. RESULTS: RNA-seq-based analysis of the dormant non-culturable population obtained under rifampicin exposure revealed a 30-50-fold decrease of the total mRNA level, indicating global transcriptional repression. However, the analysis of persisting transcripts displayed a cohort of mRNA molecules coding for biosynthetic enzymes, proteins involved in adaptation and repair processes, detoxification, and control of transcription initiation. This 'dormant transcriptome' demonstrated considerable stability during M. tuberculosis persistence and mRNA de novo synthesis arrest. On the contrary, several small non-coding RNAs showed increased abundance on dormancy. Interestingly, M. tuberculosis entry into dormancy was accompanied by the cleavage of 23S ribosomal RNA at a specific point located outside the ribosome catalytic center. CONCLUSIONS: Dormant non-culturable M. tuberculosis bacilli are characterized by a global transcriptional repression. At the same time, the dormant bacilli retain low-abundant mRNAs, which are considerably stable during in vitro persistence, reflecting their readiness for translation upon early resuscitation steps. Increased abundance of non-coding RNAs on dormancy may indicate their role in the entry into and maintenance of M. tuberculosis dormant non-culturable state.

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.

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.

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.

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.

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.

Genome sequence of the Fleming strain of Micrococcus luteus, a simple free-living actinobacterium.

Micrococcus luteus (NCTC2665, "Fleming strain") has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G+C content, 73%) predicted to encode 2,403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 insertion sequence (IS) elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and 14 response regulators, a finding indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to beta-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in the antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to most other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy, and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome.

Structural changes and cellular localization of resuscitation-promoting factor in environmental isolates of Micrococcus luteus.

Dormancy among nonsporulating actinobacteria is now a widely accepted phenomenon. In Micrococcus luteus, the resuscitation of dormant cells is caused by a small secreted protein (resuscitation-promoting factor, or Rpf) that is found in "spent culture medium." Rpf is encoded by a single essential gene in M. luteus. Homologs of Rpf are widespread among the high G + C Gram-positive bacteria, including mycobacteria and streptomycetes, and most organisms make several functionally redundant proteins. M. luteus Rpf comprises a lysozyme-like domain that is necessary and sufficient for activity connected through a short linker region to a LysM motif, which is present in a number of cell-wall-associated enzymes. Muralytic activity is responsible for resuscitation. In this report, we characterized a number of environmental isolates of M. luteus, including several recovered from amber. There was substantial variation in the predicted rpf gene product. While the lysozyme-like and LysM domains showed little variation, the linker region was elongated from ten amino acid residues in the laboratory strains to as many as 120 residues in one isolate. The genes encoding these Rpf proteins have been characterized, and a possible role for the Rpf linker in environmental adaptation is proposed. The environmental isolates show enhanced resistance to lysozyme as compared with the laboratory strains and this correlates with increased peptidoglycan acetylation. In strains that make a protein with an elongated linker, Rpf was bound to the cell wall, rather than being released to the growth medium, as occurs in reference strains. This rpf gene was introduced into a lysozyme-sensitive reference strain. Both rpf genes were expressed in transformants which showed a slight but statistically significant increase in lysozyme resistance.

Resuscitation of Dormant "Non-culturable" Mycobacterium tuberculosis Is Characterized by Immediate Transcriptional Burst.

Under unfavorable conditions such as host immune responses and environmental stresses, human pathogen Mycobacterium tuberculosis may acquire the dormancy phenotype characterized by "non-culturability" and a substantial decrease of metabolic activity and global transcription rates. Here, we found that the transition of M. tuberculosis from the dormant "non-culturable" (NC) cells to fully replicating population in vitro occurred not earlier than 7 days after the start of the resuscitation process, with predominant resuscitation over this time interval evidenced by shortening apparent generation time up to 2.8 h at the beginning of resuscitation. The early resuscitation phase was characterized by constant, albeit low, incorporation of radioactive uracil, indicating de novo transcription immediately after the removal of the stress factor, which resulted in significant changes of the M. tuberculosis transcriptional profile already after the first 24 h of resuscitation. This early response included transcriptional upregulation of genes encoding enzymes of fatty acid synthase system type I (FASI) and type II (FASII) responsible for fatty acid/mycolic acid biosynthesis, and regulatory genes, including whiB6 encoding a redox-sensing transcription factor. The second resuscitation phase took place 4 days after the resuscitation onset, i.e., still before the start of active cell division, and included activation of central metabolism genes encoding NADH dehydrogenases, ATP-synthases, and ribosomal proteins. Our results demonstrate, for the first time, that the resuscitation of dormant NC M. tuberculosis is characterized by immediate activation of de novo transcription followed by the upregulation of genes controlling key metabolic pathways and then, cell multiplication.

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.

Adoption of the transiently non-culturable state--a bacterial survival strategy?

Microbial culturability can be ephemeral. Cells are not merely either dead or alive but can adopt physiological states in which they appear to be (transiently) non-culturable under conditions in which they are known normally to be able to grow and divide. The reacquisition of culturability from such states is referred to as resuscitation. We here develop the idea that this "transient non-culturability" is a consequence of a special survival strategy, and summarise the morphological, physiological and genetic evidence underpinning such behaviour and its adaptive significance.

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.

Toxin-antitoxin vapBC locus participates in formation of the dormant state in Mycobacterium smegmatis.

Toxin-antitoxin (TA) loci are widely spread in bacterial plasmids and chromosomes. Toxins affect important functions of bacterial cells such as translation, replication and cell-wall synthesis, whereas antitoxins are toxin inhibitors. Participation in formation of the dormant state in bacteria is suggested to be a possible function of toxins. Here we show that overexpression of VapC toxin in Mycobacterium smegmatis results in development of morphologically distinct ovoid cells. The ovoid cells were nonreplicating and revealed a low level of uracil incorporation and respiration that indicated their dormant status. To validate the role of VapBC in dormancy formation, we used a model of dormant, 'nonculturable' (NC) M. smegmatis cells obtained in potassium-limited conditions. Overexpression of VapB antitoxin prevented transition to dormancy, presumably due to a decreased level of the free VapC protein. Indeed, this effect of the VapB was neutralized by coexpression of the cognate VapC as a part of the vapBC operon. In summary, these findings reveal participation of vapBC products in formation of the dormant state in M. smegmatis.

Potassium availability triggers Mycobacterium tuberculosis transition to, and resuscitation from, non-culturable (dormant) states.

Dormancy in non-sporulating bacteria is an interesting and underexplored phenomenon with significant medical implications. In particular, latent tuberculosis may result from the maintenance of Mycobacterium tuberculosis bacilli in non-replicating states in infected individuals. Uniquely, growth of M. tuberculosis in aerobic conditions in potassium-deficient media resulted in the generation of bacilli that were non-culturable (NC) on solid media but detectable in liquid media. These bacilli were morphologically distinct and tolerant to cell-wall-targeting antimicrobials. Bacterial counts on solid media quickly recovered after washing and incubating bacilli in fresh resuscitation media containing potassium. This resuscitation of growth occurred too quickly to be attributed to M. tuberculosis replication. Transcriptomic and proteomic profiling through adaptation to, and resuscitation from, this NC state revealed a switch to anaerobic respiration and a shift to lipid and amino acid metabolism. High concordance with mRNA signatures derived from M. tuberculosis infection models suggests that analogous NC mycobacterial phenotypes may exist during disease and may represent unrecognized populations in vivo. Resuscitation of NC bacilli in potassium-sufficient media was characterized by time-dependent activation of metabolic pathways in a programmed series of processes that probably transit bacilli through challenging microenvironments during infection.