Vitamin C targets (p)ppGpp synthesis leading to stalling of long-term survival and biofilm formation inMycobacterium smegmatis

Abstract

Earlier, vitamin C was demonstrated to sterilize Mycobacterium tuberculosis culture via Fenton's reaction at high concentration. It alters the regulatory pathways associated with stress response and dormancy. Since (p)ppGpp is considered to be the master regulator of stress response and is responsible for bacterial survival under stress, we tested the effect of vitamin C on the formation of (p)ppGpp. In vivo estimation of (p)ppGpp showed a decrease in (p)ppGpp levels in vitamin C-treated M. smegmatis cells in comparison to the untreated cells. Furthermore, in vitro (p)ppGpp synthesis using Rel enzyme was conducted in order to confirm the specificity of the inhibition in the presence of variable concentrations of vitamin C. We observed that vitamin C at high concentration can inhibit the synthesis of (p)ppGpp. We illustrated binding of vitamin C to Rel by isothermal titration calorimetry. Enzyme kinetics was followed where K was found to be increased with the concomitant reduction of V value suggesting mixed inhibition. Both long-term survival and biofilm formation were inhibited by vitamin C. The experiments suggest that vitamin C has the potential to be developed as the inhibitor of (p)ppGpp synthesis and stress response, at least in the concentration range used here.

Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria

“…The V max and K 0.5 values were derived from the steady-state kinetic experiments for the full-length Rel protein. The radioactive pppGpp spots were quantified as described previously, and the standard curve was plotted (11,27). Kinetic parameters, such as K 0.5 and V max , were calculated using the Hill equation.…”

Mycobacterial DNA-binding protein 1 is critical for long term survival of Mycobacterium smegmatis and simultaneously coordinates cellular functions

“…M. smegmatis is a saprophytic fast grower that share the house keeping genes and characteristics of long-term survival with those of pathogenic mycobacteria 8 . M. smegmatis is thus used as a model for the study of mycobacterial biology and the long-term persistence 1, 2, 9–12 . However, the fundamental mechanisms of the long-term survival are mostly unknown.…”

Can vitamin C help in managing tuberculosis associated with diabetes mellitus?

“…The former activity might help in bacillary killing through the oxidative burst, 1,5 and the latter activity possibly in limiting the size of Mtb persister population, through inhibition of mycobacterial dormancy-related pathways 5-7 that likely include guanosine pentaphosphate ((p)ppGpp) signalling, as there is a structural analogy of vitamin C and guanosine diphosphate, the substrate for (p)ppGpp synthesis. 6 Vitamin C levels in subjects with type 2 diabetes have been found to be low, with fasting glucose, body mass index, smoking history and dietary vitamin intake as significant independent predictors. 11 In addition, diabetic subjects likely have increased requirement of vitamin C. 11 Several clinical studies have alluded to the improvement of glycaemic control in diabetics with vitamin C supplementation.…”

“…4 Vitamin C possibly has both pro-oxidant and antioxidant activities regarding mycobacteria (Table 1). [5][6][7][8][9][10] Biologically plausible hypotheses pertaining to the modulation of anti-TB drug efficacy by these activities are summarized as follows. The former activity might help in bacillary killing through the oxidative burst, 1,5 and the latter activity possibly in limiting the size of Mtb persister population, through inhibition of mycobacterial dormancy-related pathways 5-7 that likely include guanosine pentaphosphate ((p)ppGpp) signalling, as there is a structural analogy of vitamin C and guanosine diphosphate, the substrate for (p)ppGpp synthesis.…”

“…Furthermore, the adjunctive role of vitamin C in the targeted treatment of latent TB infection associated with diabetes mellitus might warrant exploration in macrophage and animal models. 5 Wing-Wai Yew, MB, BS, FRCP, 1 Kwok-Chiu Chang, MD, 2 Denise P. Chan, PhD, 1 and Ying Zhang, MD 3 6 Vitamin C targets (p)ppGpp synthesis leading to stalling of long-term survival and biofilm formation in Mycobacterium smegmatis Shukla et al…”