Studies on NMPylase and Valine Catabolism Enzymes from Mycobacterium

Abstract

Post-translational modifications constitute an important arm of the regulatory mechanism in mammalian cells. Therefore, bacterial pathogens, which thrive by mimicking the host cell signaling, are proving to be difficult to be tamed. Mycobacterium tuberculosis, for example, the morbid pathogen manipulates the host cellular pathways such as to block the host phagosomal maturation to avoid its killing as well as creates a favorable environment for its replication. NMPylation (transfer of nucleotide monophosphate; NMP from nucleotide triphosphate; NTP) mediated by Fic domain containing proteins has been an area of immense interest in the recent years and has been established as a stratagem of bacterial effectors to manipulate host cell signaling. This thesis entitled “Studies on NMPylase and valine catabolism enzymes from Mycobacterium” presents a study of Fic domain proteins from Mycobacterium sp. in pathogenesis as well as in bacterial dormancy. The work covers de novo motif identification based on sequence analysis of Fic protein from 22 Mycobacterium sp. and in vitro/ in vivo characterization of Fic domain proteins from Mycobacterium tuberculosis, Mycobacterium smegmatis and Mycobacterium marinum. The thesis begins with the literature survey on Fic mediated AMPylation. The “Chapter 1” commences with a brief introduction on the newly discovered PTM, “AMPylation” and its resemblance with “Adenylylation” which was discovered in 1968 by Stadtman in adenylylation mediate regulation of the reaction catalyzed by Glutamine synthase in E. coli. As the modification is mediated by Fic protein, belonging to Fic/Doc superfamily, the chapter further describes the characterization of Fic protein from E. coli where Fic (Filamentation induced by cAMP) name was coined for the first time. The chapter also describes the effect of Fic domain containing bacterial effectors from Vibrio parahaemolyticus, Histophilus somni, Legionella pneumophila and Bartonella sp. It also discusses in detail the proposed catalytic mechanism of AMPylation and other known PTMs mediated by Fic domain proteins. Further, it summarizes the structural features for substrate recognition and nucleotide binding. This is followed by a description of the regulation of Fic domain containing proteins. As another SxxxEG motif containing domain regulates the activity of Fic proteins, these together represent a new type II toxin-antitoxin module. Therefore, a classification based on the antitoxin motif has been detailed. Apart from having a defined role in perturbing host cell signaling, these proteins also modify targets in the bacteria itself, the biological relevance of the same has also been discussed in this chapter. Fic domain proteins are found not only in bacteria but are distributed ubiquitously in archaea and metazoans as well. Chapter 1 also covers the recent findings about the role of Fic proteins from archaea and metazoans. Chapter 1 further details the characteristic features of Mtb, briefly describing the infection cycle in the host, the pathogen-host interplay in the lung macrophages and the strategies elicited by Mtb to evade the host response with a specific emphasis on the Mtb mediated PTMs in infected macrophages.

As set in the objectives, the principal aim was to understand the role of AMPylation in the pathogenesis of Mycobacterium. To this end, Chapter 2 covers a detailed analysis of Fic domain proteins across 22 Mycobacterium sp. where 71% of Fic domain proteins shared non-canonical Fic motif and 14% with canonical Fic motif and 15% with Doc motif. Further, the variability in the protein sequences was utilized for de novo identification of linear motifs. The putative functions these Fic domain proteins disrupt in their mammalian host were deciphered from the motifs they display to localize in a particular cellular compartment. De novo identification was supported by the functional characterization of the localization signal in two Fic domain proteins from M. smegmatis. This chapter identified a new class of TA module in Fic domain proteins from Mycobacterium, i.e, mobile mystery protein B and identified putative small XRE family transcriptional regulator protein, which may be the antitoxin counterpart as mobile mystery protein A.

With the identification of a new set of putative regulators; TA system; multifunctional enzymes from mycobacterium, all equipped with a Fic domain, either canonical or non-canonical, we characterized Fic protein from Mtb, which is part of its core genome. Chapter 3 describes the role of AMPylation activity and identifies its cognate partner both in Mycobacterium and mammalian cells. The identified substrate from Mtb includes DNA gyrase subunit B. DNA gyrase is the only type II topoisomerase present in Mtb, which catalyzes negative supercoiling of DNA. AMPylation of GyrB by MtFic decreases its ATPase activity required for introduction of negative supercoiling in closed circular DNA. Further, this chapter identifies that apart from Gyrase B, there are multiple interacting partners of MtFic in the bacterial cell lysate. Further, the Chapter describes transient transfection of MtFic in HeLa and HEK293 cells result in cell rounding which eventually culminates in detachment and cell death. Subsequent investigation of sub-cellular localization of MtFic protein shows nuclear and ER lumen as the major sites of localization, which is corroborated by the identification of its interacting partners in nuclear and ER/membrane enriched fractions. A detailed characterization revealed Rac1, Cdc42, and vimentin as targets for MtFic in mammalian host cells. Altogether identification of multiple interacting partners in bacterial and mammalian host including GyraseB, Rac1, Cdc42 and vimentin, opens new avenues for further exploration of the role of AMPylation activity in Mycobacterium survival, dormancy, and pathogenicity. Fic motif residues bind with phosphates of NTPs, however, the residues from flap region and core helices support interactions with the base of NTPs and place NTP in an orientation suitable for NMPylation. As Fic proteins sequences curated from the Mycobacterium sp. showed variabilities in these regions, nucleotide selectivity exhibited by Fic proteins were explored next, Chapter 4 thus covers identification of a unique motif supporting selectivity for CTP over ATP. MtFic preferred AMPylation whereas Fic from M. smegmatis and M. marinum preferred CMPylation. The auto-modifications viz. auto-AMPylation and auto-CMPylation activity were also shown by the antitoxin mutants, however the M. marinum Fic protein, even with antitoxin domain showed auto-CMPylation. The chapter further explores regulation of Fic activity by antitoxin motif SxxxEG. Presence of arginine/asparagine at glycine position interferes with the inhibitory effect of glutamate, antagonizing the activity of antitoxin motif, thus resulting in enhanced toxin activity even in the presence of antitoxin. Moreover, this chapter also covers preliminary studies on residual pyrophosphatase activity exhibited by some CMPylating Fic enzymes. Chapter 5 covers characterization a Fic protein from M. marinum, with winged helix turn helix domain that showed DNA binding properties and a novel PTM, CMPylation, where CMP molecule gets transferred to the cognate substrates Gyrase A and Gyrase B subunits in bacteria and PCNA and Histone1 in the mammalian host. Additionally, this chapter identifies CMPylation, as a reversible PTM, which can be reversed with the same enzyme. CMPylation and de-CMPylation mediated by MmFic is highly regulated by glutamate present in the anti-toxin domain, thus MmFic protein is well-coordinated toxin-antitoxin system also. This chapter, hence, identifies and characterizes a novel TA system from Mycobacterium and identifies its targets as well. Moreover, this chapter also describes functional significance of auto-AMPylation in class I Fic proteins where auto-modification opens the N-terminus region thus enhancing the accessibility of substrate binding residues from the flap region. While these studies were in progress, functional characterization of some of the enzymes involved in valine catabolism, were also explored. In this context, it has been shown that addition of valine in the culture media inhibits the growth of Mycobacterium. Since the components of valine catabolism are critical for the survival and infection cycle of Mtb, it constitutes a potential drug target. In Chapter 6, detailed structural and functional characterizations of HIBADH enzyme catalyzing the sixth step in valine catabolism are described. MtHIBADH utilizes NAD+ as a cofactor and S-HIBA as the substrate. The locations of substrates in the active site region of HIBA dehydrogenases have been defined for the first time. Using a plethora of structural and biochemical techniques, the entry and active sites of the substrate have been unambiguously characterized. The chapter also describes a plausible reaction mechanism, inferred based on the structures and modeling. Further, studies highlight Cys210 as the critical cysteine residue for maintenance of the conformation of the active site of the enzyme. Further, the chapter identifies a novel FRET pair in NAD+ bound MtHIBADH enzyme, where tryptophan (Trp211) acts as a donor and NAD+ bound ({NAD}* acts as the acceptor. Chapter 7 briefly summarizes all the findings of the research carried out and presents an overview of our present understanding of the mycobacterial Fic proteins and HIBADH enzyme. Appendix A summarizes the preliminary results of MMSA and fadE9 enzymes involved in Mtb valine catabolism.