Mycobacterium tuberculosis Transcriptional Adaptation, Growth Arrest and Dormancy Phenotype Development Is Triggered by Vitamin C

Abstract

BackgroundTubercle bacilli are thought to persist in a dormant state during latent tuberculosis (TB) infection. Although little is known about the host factors that induce and maintain Mycobacterium tuberculosis (M. tb) within latent lesions, O2 depletion, nutrient limitation and acidification are some of the stresses implicated in bacterial dormancy development/growth arrest. Adaptation to hypoxia and exposure to NO/CO is implemented through the DevRS/DosT two-component system which induces the dormancy regulon.Methodology/Principal FindingsHere we show that vitamin C (ascorbic acid/AA) can serve as an additional signal to induce the DevR regulon. Physiological levels of AA scavenge O2 and rapidly induce the DevR regulon at an estimated O2 saturation of <30%. The kinetics and magnitude of the response suggests an initial involvement of DosT and a sustained DevS-mediated response during bacterial adaptation to increasing hypoxia. In addition to inducing DevR regulon mechanisms, vitamin C induces the expression of selected genes previously shown to be responsive to low pH and oxidative stress, triggers bacterial growth arrest and promotes dormancy phenotype development in M. tb grown in axenic culture and intracellularly in THP-1 cells.Conclusions/SignificanceVitamin C mimics multiple intracellular stresses and has wide-ranging regulatory effects on gene expression and physiology of M. tb which leads to growth arrest and a ‘dormant’ drug-tolerant phenotype, but in a manner independent of the DevRS/DosT sytem. The ‘AA-dormancy infection model’ offers a potential alternative to other models of non-replicating persistence of M. tb and may be useful for investigating host-‘dormant’ M. tb interactions. Our findings offer a new perspective on the role of nutritional factors in TB and suggest a possible role for vitamin C in TB.

The pleiotropic transcriptional response of Mycobacterium tuberculosis to vitamin C is robust and overlaps with the bacterial response to multiple intracellular stresses

“…A similar trend was noted for the Wayne hypoxia model between days 4 and 20 (nonreplicating persistence stages) (Voskuil et al, 2004). Vit C causes an immediate drop in the pH of the growth medium tõ 5.5 (Taneja et al, 2010), and lowers it further to pH~4.5 over a period of 4 days (data not shown). The occurrence of pH stress is indicated by an overlap of the vit C response with the acid stress response (Fisher et al, 2002;Rohde et al, 2007).…”

“…A serendipitous discovery was made in our laboratory that vit C treatment of Mtb resulted in a rapid induction of the DevR (DosR) dormancy regulon and led to the development of bacterial 'dormancy'. Vit C also elicited acidic and oxidative stress responses and induced an isoniazid (INH) tolerant phenotype in the treated bacteria both in axenic cultures and in the THP-1 cell infection model (Taneja et al, 2010). These responses suggested that vit C potentiates an early induction of the 'dormancy phenotype' under aerobic conditions, and bacteria may exhibit physiological adaptations similar to those observed under conditions of gradual O 2 depletion (hypoxia) and NO exposure.…”

“…Forty million THP-1 cells in 175 cm 2 tissue culture flasks were infected with Mtb at an m.o.i. of 10 : 1 (bacteria : cell) as described previously (Taneja et al, 2010). At 8 h post-infection, THP-1 cells were lysed and bacteria isolated as described previously (Monahan et al, 2001), with minor variations.…”

“…Reaction conditions were 94 uC (10 min) followed by 40 cycles of 94 uC (20 s), 55-65 uC (20 s) and 72 uC (20 s). Transcript levels in various RNA samples were normalized using 16S rRNA, and the fold change in expression in vit C-treated axenic cultures or intracellular Mtb was calculated with respect to untreated aerobic axenic cultures as described previously (Taneja et al, 2010 Comparison of dormancy models. Mtb H37Rv genes differentially regulated in response to vit C treatment were compared to the data from other dormancy models.…”

“…4c). The extent of hypoxia generation by vit C corresponds to 30 % dissolved oxygen concentration (Taneja et al, 2010), which is adequate for GFP to mature to its fluorescent form in the plate assay (Chauhan & Tyagi, 2008). The generation of hypoxia by vit C was further confirmed by the physiological response of treated Mtb cultures; a rapid and preferential induction of the DevR regulon was noted after vit C treatment in Mtb Comp dosT and not Comp devS cultures in RT-qPCR study (~83-fold versus 13-fold induction of hspX and~13-fold versus~4-fold induction of tgs1 in Comp dosT versus Comp devS strains).…”