The Role of F420-dependent Enzymes in Mycobacteria

Abstract

Tuberculosis (TB) is the leading cause of death by an infectious disease, recently surpassing HIV/AIDS. The causative agent, Mycobacterium tuberculosis, is difficult to treat as it can survive harsh conditions and can switch between an active infection, which causes ~1.5 million deaths a year, and a latent state, which infects up to one third of the world’s population. M. tuberculosis is also becoming more resistant to frontline drugs, making it a dangerous world epidemic. It is therefore essential that new treatments are developed to help combat TB. A new enzyme superfamily has recently been discovered that utilises the rare co-factor F420, which is not produced or utilized by humans, allowing for specific drug targeting of the mostly uncharacterized enzymes that utilise it. The aim of this thesis is to better understand the roles of these enzymes in Mycobacteria and to investigate the mechanism by which M. tuberculosis might evolve resistance to a new class of prodrugs that are activated by the F420-dependent enzyme deazaflavin-dependent nitroreductase (Ddn). Prodrugs that are activated by Ddn include pretomanid and delamanid, which are effective against both an active and latent TB infection. Ddn is natively a quinone reductase, but also reacts with these drugs to reduce them, thereby releasing nitrous oxide and other breakdown products that are thought to inhibit hydroxymycolic acid dehydrogenase. Resistance against pretomanid in vitro has been documented in laboratory studies to occur via mutations to the F420 biosynthetic pathway, the enzyme F420-dependent glucose-6-phosphate dehydrogenase that reduces F420, and Ddn. However, the fitness cost of such mutations, i.e. whether they would still be virulent and transmissible, has not been studied. This important question will determine whether such genetic changes could lead to clinically relevant resistance. I explored this question with a detailed study of Ddn to establish (i) whether its activity is essential for the fitness of M. tuberculosis and (ii) whether any mutations could knock out the prodrug-activating activity without substantially affecting the native quinone reductase activity. I investigated this by better defining the physiological role of Ddn, showing that it can reduce menaquinone, and that this activity can enhance respiration of the cell by coupling with cytochrome bd. I also demonstrated that Ddn orthologues have similar quinone reductase activity as Ddn, but have no activity with pretomanid, except for the M. marinum orthologue that has activity with both. Through site directed mutagenesis I have identified a number of mutations to Ddn’s active site that eliminate activity with pretomanid while retaining some quinone reductase activity. A clinical strain that had acquired one of the tested mutations v through neutral genetic drift/variation showed resistance to pretomanid, despite never having been exposed to the compound. Interestingly, the other nitroimidazole prodrug, delamanid, was still effective Computational modelling suggets this to be due to the way that each nitroimidazole binds Ddn. The first step to developing a new drug to target an F420-dependent enzyme, we first need to identify genes/proteins that are essential for some aspect of the life cycle of M. tuberculsosis. Collaborators have shown that the F420-dependent enzyme Rv0121c (homologous to MSMEG_6526 in Mycobacterium smegmatis) from the flavin/deazaflavin oxidoreductase (FDOR) protein superfamily is essential for escape from dormancy. As a model for M. tuberculosis we made a MSMEG_6526 knockout in M. smegmatis and confirmed that it is conditionally essential. This was demonstrated by the slower growth rate of Δ6526 compared to wildtype in minimal media with several different carbon sources, and the lack of growth on acetate and pyruvate. Proteomics showed the upregulation of the methylcitrate cycle, glyoxylate cycle, and several F420-dependent enzymes, including the Ddn orthologue MSMEG_2027. Proteins that were downregulated were part of the Kerbs cycle, late stage glycolysis, and several amino acid metabolomic pathways. This was complemented with metabolomics, revealing several metabolites that were affected by the MSMEG_6526 knockout. The pathway that these metabolites were involved in included the Krebs cycle and several related amino acid metabolic pathways. This suggests that MSMEG_6526 is somehow involved in amino acid metabolism. The crystal structures of Rv0121c and MSMEG_6526 in complex with F420 were solved, revealing homodimers of the split β-barrel fold that defines the FDOR super family. The structures revealed three extended loops, two of which made a more defined active site compared to other FDORs. The third loop was not involved with the active site and is not conserved between the two enzymes, while the active site and F420 binding are highly conserved. The structure was also used to identify the type of substrates that can bind these enzymes. The other chapters presented in this thesis are collaborative projects that I had contributed towards. These include detailed characterization of the FDOR superfamily in which a number of specific sub-groups of the FDOR superfamily based on sequence similarity and structural motifs. We also identified novel F420-dependent biliverdin reductases in M. tuberculosis that reduce bilirubin, a known antioxidant. Finally, we expand the list of chemicals that FDORs has promiscuous activity with, including antimicrobials that we show are more vi potent with F420 knocked out. The final chapter is a comprehensive review of F420, it precursor Fo, and related enzymes in Mycobacteria, methanogens, and other bacteria that utilise it. Show more