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.

Dormant states of bacteria with drastically decreased metabolic activity, enhanced resistance to harmful factors, and absence of cell division is a form for surviving unfavorable environmental conditions. This state does not necessarily imply formation of highly differentiated spores and cysts; it has been demonstrated for non-spore-forming bacteria, including pathogenic ones. The latency of a number of infectious diseases is generally believed to be related to the capacity of bacteria (including Mycobacterium tuberculosis, an infective agent of tuberculosis) to produce dormant forms. Indeed, some results of histological investigation and modeling of latent infections in animals, as well as results obtained with in vitro models, support the hypothesis of production of dormant forms by tuberculosis bacteria. In the present review, existing experimental models of dormant form production in mycobacteria are considered, as well as modern data concerning the mechanisms of their formation and their relation to the “nonculturable” state. The mechanisms of reversion to culturability and the role of extracellular factors in reactivation of dormant forms are discussed in detail.

The spread of multidrug-resistant mycobacterium strains requires the development of new approaches to combat diseases caused by these pathogens. For that, photodynamic inactivation (PDI) is a promising approach. In this study, a tricarbocyanine (TCC) is used for the first time as a near-infrared (740 nm) activatable PDI photosensitizer to kill mycobacteria with deep light penetration. For better targeting, a novel tricarbocyanine dye functionalized with two trehalose units (TCC2Tre) is developed. The photodynamic effect of the conjugates against mycobacteria, including Mycobacterium tuberculosis, is evaluated. Under irradiation, TCC2Tre causes more effective killing of mycobacteria compared to the photosensitizer without trehalose conjugation, with 99.99% dead vegetative cells of M. tuberculosis and M. smegmatis. In addition, effective photoinactivation of dormant forms of M. smegmatis is observed after incubation with TCC2Tre. Mycobacteria treated with TCC2Tre are more sensitive to 740 nm light than the Gram-positive Micrococcus luteus and the Gram-negative Escherichia coli. For the first time, this study demonstrates the proof of principle of in vitro PDI of mycobacteria including the fast-growing M. smegmatis and the slow-growing M. tuberculosis using near-infrared activatable photosensitizers conjugated with trehalose. These findings are useful for the development of new efficient alternatives to antibiotic therapy.

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.

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.