Studies on the mechanisms of action and the physiological roles of MutT1 in mycobacteria

Abstract

Mycobacterium tuberculosis (Mtb), a pathogenic bacterium, thrives and proliferates inside host macrophages, encountering a high level of reactive oxygen species and reactive nitrogen intermediates which is generated by the host’s immune system. Reactive oxygen species and reactive nitrogen intermediates can cause alternation in nucleotide bases in both the DNA/RNA and the nucleotide pool. Due to its low redox potential, guanine is highly susceptible to damage caused by oxidation. Oxidation of (d)GDP or (d)GTP leads to their conversion into 8-oxo-(d)GDP or 8-oxo-(d)GTP respectively. Misincorporation of 8-oxo-dGTP into the genome results in AT to CG transversion mutations. It also demonstrates exceptional skills in surviving within a host by employing diverse strategies to evade host defense and establish a protected environment. Continuous environmental monitoring is pivotal for pathogenic mycobacterium to ascertain their immediate location and orchestrate the expression of genes essential for adaptation. This adaptability relies mainly on two-component systems that activate intricate transcriptional networks. In the studies presented in this thesis we explored additional roles of MutT1 in maintaining genome stability and regulation of two component systems activity The thesis consists of 5 chapters. Chapter-1 summarises the relevant literature on MutT proteins and their reported functions, nucleoside diphosphate kinase (NDK), and two-component systems. Chapter-2 describes the materials and methods used throughout the experimental work of this thesis. It is followed by three chapters (3-5) containing the experimental work. The summaries of chapters 3-5 are provided as follows. Chapter-3 An exchange of single amino acid between the phosphohydrolase modules of Escherichia coli MutT and Mycobacterium smegmatis MutT1 switches their cleavage specificities. MutT proteins belong to the Nudix hydrolase superfamily that includes a diverse group of Mg2+ requiring enzymes. These proteins use a generalized substrate, nucleoside diphosphate linked to a chemical group X (NDP-X), to produce nucleoside monophosphate (NMP) and the moiety X linked with phosphate (XP). E. coli MutT (EcoMutT) and mycobacterial MutT1 (MsmMutT1) belong to the Nudix hydrolase superfamily that utilize 8-oxo-(d)GTP (referring to both 8-oxo-GTP or 8-oxo-dGTP). However, the predominant products of their activities are different. While EcoMutT produces 8-oxo-(d)GMP, MsmMutT1 gives rise to 8-oxo-(d)GDP. Here, we show that the altered cleavage specificities of the two proteins are largely a consequence of the variation at the equivalent of Gly37 (G37) in EcoMutT to Lys (K65) in the 6 MsmMutT1. Remarkably, mutations of G37K (EcoMutT) and K65G (MsmMutT1) switch their cleavage specificities to produce 8-oxo-(d)GDP, and 8-oxo-(d)GMP, respectively. Further, a time course analysis using 8-oxo-GTP suggests that MsmMutT1(K65G) hydrolyses 8-oxo-(d)GTP to 8-oxo-(d)GMP in a two-step reaction via 8-oxo-(d)GDP intermediate. Expectedly, unlike EcoMutT (G37K) and MsmMutT1, EcoMutT and MsmMutT1 (K65G) rescue an E. coli ΔmutT strain, better by decreasing A to C mutations. Chapter-4 An unusual activity of mycobacterial MutT1 Nudix hydrolase domain as a protein phosphatase regulates nucleoside diphosphate kinase (NDK) function. MutT proteins are Nudix hydrolases characterized by the presence of a Nudix box, GX5EX7REUXEEXGU, where U is a bulky hydrophobic residue and X is any residue. Major MutT proteins hydrolyse 8-oxo-(d)GTP (8-oxo-GTP or 8-oxo-dGTP) to the corresponding 8-oxo-(d)GMP, preventing their incorporation into nucleic acids. Mycobacterial MutT1 comprises an N-terminal domain (NTD) harbouring the Nudix box motif, and a C-terminal domain (CTD) harbouring the RHG histidine phosphatase motif. Interestingly, unlike other MutTs, the MutT1 hydrolyses the mutagenic 8-oxo-(d)GTP to corresponding 8-oxo-(d)GDP. Nucleoside diphosphate kinase (NDK), a conserved protein, carries out reversible conversion of (d)NDPs to (d)NTPs through phospho-NDK (NDK-Pi) intermediate. Recently, we showed that NDK-Pi converts 8-oxo-dGDP to 8-oxo-dGTP and escalates A to C mutations in a MutT deficient E.coli. We now show that both MtbMutT1, and MsmMutT1, through their NTD (Nudix hydrolase motifs) function as protein phosphatase to regulate the levels of NDK-Pi to NDK and prevent it from catalysing conversion of (d)NDPs to (d)NTPs (including conversion of 8-oxo-dGDP to 8-oxo-dGTP). To corroborate this function, we show that MsmMutT1 decreases A to C mutations in E. coli under the conditions of EcoNDK overexpression. Chapter-5 A novel mechanism of MutT1 mediated regulation of two component system in mycobacteria. Pathogenic bacteria such as Mtb, navigate the host defenses through intricate mechanisms, including two component systems (TCS) to regulate gene expression in response to the environmental cues. However, the regulation of TCS is poorly understood. We explore the potential role of MutT1, an enzyme known to hydrolyze oxidized G (8-oxo-G) nucleoside triphosphates (NTP/dNTP), in regulation of TCS. Unlike most other MutT proteins, mycobacterial MutT1 comprises two domains (N-terminal domain, NTD; and C-terminal domain, CTD). Structurally, the NTD is like MutT proteins in other organisms. However, the CTD is structurally very similar to E. coli SixA, a histidine phosphatase defined by an RHG motif. In this study, we report that the MutT1 CTD phosphatase activity dephosphorylates many of the sensor kinases and impacts their target gene expression, highlighting the role of MutT1 in TCS function and regulation. The findings are of special significance because, for the first time, they provide us with a mechanism of how to downregulate the activated TCS to return it to ground state. In addition, the study sheds light on the intricate interplay between an enzyme known to maintain clean nucleotide pool in the cell, and the bacterial signaling pathway, offering insight into bacterial adaptation mechanisms.