MtbEis protein: A new player in stringent response of Mycobacterium tuberculosis

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 in Mycobacterium tuberculosis (RelMtb) also regulates expression of persistence or virulence associated genes. Loss of RelMtb leads to higher expression of few of the virulence and cell wall remodeling factors in addition to upregulating many secreted antigens and proteins. The Enhanced Intracellular Survival (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 in M. tuberculosis was explored. To begin with, MtbEis has been confirmed to enhance the guanosine pentaphosphate (pppGpp) synthesis activity of RelMtb by acetylating RelMtb at K513. Next, the MtbEis gene 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 RelMtb and subsequent decrease in pppGpp synthesis was also observed in Mtb{Delta}Eis cells. Complementation of Mtb{Delta}Eis with full length MtbEis restored the expression of RelMtb, rRNAs, pppGpp levels and survival of M. tuberculosis. However, MtbEis lacking acetyltransferase domain ({Delta}AT) 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 RelMtb as a novel mechanistic aspect of stringent response in M. 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 SummaryStringent Response is essential for bacterial survival and pathogenesis under stress conditions. The bifunctional RelMtb enzyme is the key player of stringent response in M. tuberculosis catalyzing 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 RelMtb by 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 RelMtb under starvation conditions. The regulation of stringent response by MtbEis mediated acetylation of RelMtb have unravelled the novel function of MtbEis in adapting Mtb to existing environmental challenges associated with nutrient deficit state. HighlightsO_LINone of the PTMs of Rel proteins is known so far C_LIO_LIMtbEis interacts with RelMtb and acetylates RelMtb at K513 C_LIO_LIThe acetyltransferase domain of MtbEis regulates rRNAs and RelMtb expression, pppGpp synthesis and Mtb survival under starved conditions C_LIO_LIMtbEis helps Mtb in adapting to nutrient deficit state C_LI