MtbEis Mediated Acetylation of RelMtb Regulates Stringent Response in Mycobacterium tuberculosis
Nittu Singha, Vandana Basraa,b and Charu Sharmaa
aCSIR-Institute of Microbial Technology, Sector 39-A, Chandigarh-160036, India; bAcademy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, India
Stringent response is an important defense mechanism employed by bacteria for their enhanced survival under hostile conditions by synthesizing hyperphosphorylated Guanosine, a molecular alarmone, using Rel protein. Rel protein in Mycobacterium tuberculosis (RelMtb) also regulates expression of persistence or virulence associated genes. Loss of RelMtb causes higher expression of some of the virulence and cell wall remodeling factors besides upregulating many secreted antigens and proteins. Out of these, enhanced intracellular survival (eis Rv2416c) gene is one of the upregulated virulence factors. Based on this information, we have explored the precise role of MtbEis in modulating the stringent response in M. tuberculosis. To begin with, MtbEis has been confirmed as a novel interacting partner of the RelMtb protein that enhances the guanosine pentaphosphate (pppGpp) synthesis activity of both the recombinant and endogenous RelMtb forms. The MtbEis protein, a GNAT family acetyltransferase, acetylated RelMtb at K513. The acetylated RelMtb was found to have enhanced pppGpp synthesis activity whereas the acetylation defective mutant RelMtbK513A failed to do so. To ascertain the explicit role of MtbEis in stringent response, MtbEis gene was knocked out in MtbH37Rv. The deletion of MtbEis resulted in a compromised survival accompanied by elevated levels of rRNAs under starvation. Furthermore, the reduced expression of RelMtb and subsequent decrease in pppGpp synthesis was also observed in MtbΔEis cells. Complementation of MtbΔEis with full length wild type MtbEis restored the expression of RelMtb, rRNAs, pppGpp levels and survival of M. tuberculosis. However, these could not be restored with MtbEis lacking acetyltransferase domain (ΔN3), 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 post translational modification of RelMtb as a novel mechanistic aspect of stringent response in M. tuberculosis.