Abstract
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.
Highlights
The dormant state of infectious agents attracts attention from both microbiologists and physicians as their dormant forms are considered to be responsible for the development of chronic diseases characterized by phenotypic resistance to antibiotics
Comparison of the data obtained with published spectra of standard solutions of pure substances1 allowed identification of the main component of the cytoplasm of mycobacterial dormant cells as trehalose
Similar to germination of yeast or actinomycetes we found a decrease in trehalose content during resuscitation of dormant M. smegmatis cells (Figure 5) which possibly was due to activation of trehalase earlier in the resuscitation process (HeyFerguson et al, 1973)
Summary
The dormant state of infectious agents attracts attention from both microbiologists and physicians as their dormant forms are considered to be responsible for the development of chronic diseases characterized by phenotypic resistance to antibiotics. Despite recent intensive studies of the molecular mechanisms underlining the transition of viable MTB cells to dormant forms (for review see, Dutta and Karakousis, 2014) much less is known about the mechanisms and metabolic processes responsible for dormant cells surviving for long periods and their resuscitation to a viable, multiplying state. It is known that bacterial cells accumulate some storage substances (polyphosphates, glycogen, PHB, etc.) which could be used to maintain metabolic activity and survival under nutrient-limiting conditions (Preiss, 1984; Wood and Clark, 1988; Anderson and Dawes, 1990). Yeast and fungal spores accumulate another storage/protective substance – trehalose – which participates in spore stabilization in stressful conditions like desiccation and could be used in spore germination (Elbein et al, 2003)
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