MtbEis protein: A new player in stringent response ofMycobacterium tuberculosis
Authors: Singh Nittu , Basra Vandana , Sharma Charu 
Published: October 2023

The stringent response in bacteria is an important defense mechanism in which hyperphosphorylated forms of guanosine, also known as molecular alarmones, are synthesized by RelA/SpoT Homolog (RSH) proteins. Rel protein inMycobacterium tuberculosis(RelMtb) also regulates expression of persistence or virulence associated genes. Loss of RelMtbleads to higher expression of few of the virulence and cell wall remodeling factors in addition to upregulating many secreted antigens and proteins. TheEnhanced IntracellularSurvival (MtbEis) protein is one of the upregulated virulence factors. Based on this information, the precise role of MtbEis, a GNAT family acetyltransferase, in the stringent response inM. tuberculosiswas explored. To begin with, MtbEis has been confirmed to enhance the guanosine pentaphosphate (pppGpp) synthesis activity of RelMtbby acetylating RelMtbat K513. Next, theMtbEisgene was knocked out in Mtb. The deletion of MtbEis resulted in a compromised survival accompanied by elevated levels of rRNAs under starvation conditions. Furthermore, the reduced expression of RelMtband subsequent decrease in pppGpp synthesis was also observed in MtbΔEis cells. Complementation of MtbΔEis with full length MtbEis restored the expression of RelMtb, rRNAs, pppGpp levels and survival ofM. tuberculosis. However, MtbEis lacking acetyltransferase domain (ΔAT) failed to restore this, confirming the role of MtbEis mediated acetylation in regulating stringent response. In sum, our findings not only report the unexplored role of MtbEis in the starvation survival but also the acetylation of RelMtbas a novel mechanistic aspect of stringent response inM. tuberculosis. In addition, this being the first post translational modification (PTM) report on any of the bacterial Rel proteins opens up the field for the discovery of new PTMs of Rel proteins.

Author Summary

Stringent Response is essential for bacterial survival and pathogenesis under stress conditions. The bifunctional RelMtbenzyme is the key player of stringent response inM. tuberculosiscatalyzing the synthesis of pppGpp from ATP and GTP under nutrient deprived conditions. Under stringent conditions, the tight regulation of transcriptional and translational processes aids the intracellular survival of Mtb. Amongst the translational regulations, the post-translational modification (PTM) is one of the most efficient mechanisms regulating the functions of enzymes. None of the PTMs of Rel proteins is known so far. Here, we have not only identified the acetylation of RelMtbby MtbEis but also dissected the consequences of this modification in regulation of stringent response. MtbEis is a known virulence factor belonging to GNAT family acetyltransferase that is upregulated upon stringent response activation in Mtb. The MtbEis regulates expression of rRNAs and RelMtb, synthesis of pppGpp and Mtb survival by acetylating RelMtbunder starvation conditions. The regulation of stringent response by MtbEis mediated acetylation of RelMtbhave unravelled the novel function of MtbEis in adapting Mtb to existing environmental challenges associated with nutrient deficit state.

Highlights

None of the PTMs of Rel proteins is known so far

MtbEis interacts with RelMtband acetylates RelMtbat K513

The acetyltransferase domain of MtbEis regulates rRNAs and RelMtbexpression, pppGpp synthesis and Mtb survival under starved conditions

MtbEis helps Mtb in adapting to nutrient deficit state