IDENTIFICATION OF A NOVEL ANTI-APOPTOTIC GENE OF MYCOBACTERIUM TUBERCULOSIS

Over the next 20 years, more than 36 million people are expected to die of Mycobacterium tuberculosis (Mtb) related illness. We may prevent this only by learning as much as possible about the Mtb-mediated exploitation of the human immune system and successfully implementing that knowledge to combat the pathogen. One Mtb virulence mechanism involves inhibition of host apoptosis. An Mtb laden macrophage will attempt cell suicide, subsequently destroying any intracellular bacteria. In prior work, a large gain-of-function screen identified Mtb genomic regions involved in host apoptotic suppression; one such region is "K20." A loss-of-"gain-of-function" (LoGoF) screen involving in vitro transposon (Tn) mutagenesis of a K20 expressing vector identified individual gene(s) of K20 potentially responsible the virulence phenotype. This LoGoF screen found two unique K20 Tn-clones that consistently induced significant host apoptosis. These Tn's disrupted expression of K20 genes Rv2666 ( probable truncated transposase) and Rv2667 ( possible Clp ATPase). A Himar1 Tn mutant of Rv2666 was obtained through the TARGET mutant library project. Upon infection of human macrophages, TnRv2666 induced significantly more host apoptosis than wild type (WT) and complement, confirming Rv2666 as an anti-apoptotic gene of Mtb. There are no current publications suggesting a specific role for transposase-based virulence in Mtb; Rv2666 may affect an anti-apoptotic phenotype by modulating transcription of factors important for the suppression of host apoptosis. The second LoGoF identified gene, Rv2667/clpC2/clpX', was not available through TARGET; a recombinant ΔclpC2 mutant was generated. Comparing induced host apoptosis of ΔclpC2 infected macrophages to WT revealed Rv2667/clpC2 has no essential role as an anti-apoptotic gene of Mtb. ΔclpC2 was further characterized to explain the discrepancy between the initial LoGoF data and the ΔclpC2 Mtb results. Study of ΔclpC2 determined that it bears no significant differences with WT in terms of in vitro growth, host necrosis-induction, in vivo survival and induced host TNFα secretion levels. However, ΔclpC2 induces significantly more host IL-1β release than WT Mtb. The reason for this effect is unknown; ClpC2 may aid Mtb pathogenicity by limiting host inflammation, thus permitting infecting Mtb a "head start" against a host adaptive immune response

Characterizing the Role of Prophages on WHIB7 Expression and Antibiotic Resistance in Mycobacterium Chelonae

Mycobacterial pathogens are responsible for an ongoing public health crisis. Mycobacterium abscessus is the causative agent of lung infections that disproportionately affect immunocompromised individuals and is the most intrinsically antibiotic-resistant bacterial species known. These characteristics make M. abscessus infections difficult to treat, with a success rate of only 45%. While some extensively resistant isolates are caused by mutations in drug targets, others appear to be a result of increased intrinsic drug resistance. Common among these strains is the presence of integrated viral genomes (prophage) that are known to contribute to fitness and antibiotic resistance in other pathogens but whose roles are largely unknown in mycobacteria. M. chelonae is an opprtunisitc pathogen that is closely related to M. abscessus. We have demonstrated that the presence of an M. abscessus cluster R prophage, McProf, in M. chelonae, increased resistance to antibiotics, such as amikacin, relative to strains lacking the prophage. The presence of McProf also enhances amikacin resistance in response to sub-lethal concentrations of antibiotics, or other cellular stresses such as infection by a second phage, BPs. Relative to strains carrying only one of the prophages or no prophage, the strain carrying two prophages, BPs and McProf, had the highest amikacin resistance. This strain also showed increased expression of the transcriptional activator, whiB7, which promotes expression of intrinsic antibiotic resistance genes. We investigated the role of BPs lysogenic gene products in the presence of McProf and showed that individual expression of these genes does not contribute to whiB7 upregulation, indicating that McProf likely plays a larger role in mediating this intrinsic resistance. We identified a McProf-encoded polymorphic toxin system and evaluated its effect on whiB7 expression in M. chelonae carrying the BPs prophage. The polymorphic toxin system elevates whiB7 expression but does not fully account for the dramatic increase in expression observed in the M. chelonae strain carrying both prophages. This work suggests that prophages play a role in increasing intrinsic antibiotic resistance and stress adaptation in pathogenic mycobacteria. Given that most pathogenic mycobacteria carry one or more prophages, characterizing how prophages regulate antibiotic resistance genes and adaptation to stresses will provide insight for developing more effective therapies for mycobacterial diseases

Mycobacterium tuberculosis inhibitors: action and resistance

Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, has been a global health problem for years. The emergence of drug resistance in this organism generates the necessity of exploring novel targets and developing new drugs. Topoisomerases are enzymes found in all kingdoms of life responsible for overcoming the topological barriers encountered during essential cellular processes. The genomes of mycobacteria encode only one type IA topoisomerase (MtopI), which has been validated as a novel TB drug target. The goal of this study is to obtain new information on the mechanism and resistance of endogenous and synthetic inhibitors of MtopI. Rv1495 is a M. tuberculosis toxin that belongs to the MazEF family (MazE is the antitoxin and MazF is the toxin), with endoribonuclease activity. Rv1495 (MazF homolog in M. tuberculosis) toxin has been shown to interact directly with the C-terminal domain of MtopI for mutual inhibition. In this study the interaction of Rv1495 with the positively charged C-terminal tail in Mtop I is reported. This new information is useful for rational design and discovery of antibiotics for mycobacteria. Ethacridine, an FDA approved drug has shown activity against MtopI. In this project we studied the mechanisms of resistance associated with this drug as well the use of Ethacridine in combination with Moxifloxacin, to potentiate the bactericidal effect of this current second line drug for TB treatment. Results from sequencing of the genomic DNA isolated from the resistant mutants suggested the involvement of the Holliday-junction Ruv resolvase. Further studies showed that co-treatment with Ethacridine can enhance the moxifloxacin-mediated killing of M. smegmatis. FP-11g, a novel fluoroquinophenoxazine inhibitor of bacterial topoisomerase I, has shown promising activity against M, tuberculosis. We explored the bactericidal activity and resistance mechanisms associated to FP-11g using M. smegmatis as model organism. Additionally, the inhibitory effect of FP-11g was also evaluated on M. abscessus. FP-11g at concentration 4X MIC showed complete bactericidal activity against M. smegmatis after 24 hours. Inhibitory activity against M. abscessus was also observed. Results from sequencing of the genomic DNA isolated from the M. smegmatis resistant mutants revealed mutations in genes associated with general drug resistance

Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 2.0

This book, "Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity", presents five papers on human cytochrome P450 (CYP) and P450 reductase, three reviews on the role of CYPs in humans and their use as biomarkers, six papers on CYPs in microorganisms, and one study on CYP in insects. The first paper reports the in silico modeling of human CYP3A4 access channels. The second uses structural methods to explain the mechanism-based inactivation of CYP3A4 by mibefradil, 6,7-dihydroxy-bergamottin, and azamulin. The third article compares electron transfer in CYP2C9 and CYP2C19 using structural and biochemical methods, and the fourth uses kinetic methods to study electron transfer to CYP2C8 allelic mutants. The fifth article characterizes electron transfer between the reductase and CYP using in silico and in vitro methods, focusing on the conformations of the reductase. Then, two reviews describe clinical implications in cardiology and oncology and the role of fatty acid metabolism in cardiology and skin diseases. The second review is on the potential use of circulating extracellular vesicles as biomarkers. Five papers analyze the CYPomes of diverse microorganisms: the Bacillus genus, Mycobacteria, the fungi Tremellomycetes, Cyanobacteria, and Streptomyces. The sixth focuses on a specific Mycobacterium CYP, CYP128, and its importance in M. tuberculosis. The subject of the last paper is CYP in Sogatella furcifera, a plant pest, and its resistance to the insecticide sulfoxaflor

Drug targets in Mycobacterium tuberculosis α-glucan synthesis

α-Glucans are important energy storage polysaccharides in bacteria, plants and animals. In Mycobacterium tuberculosis, α-glucan also functions as a virulence factor that is exported to the mycobacterial capsule and interacts with human immune receptors. In M. tuberculosis and other actinomycetes, α-glucan is synthesised from maltose-1-phosphate by the maltosyl transferase GlgE and the α-1,6-branching enzyme GlgB. These enzymes have been genetically validated as tuberculosis drug targets. The loss of the α-glucan virulence factor is exacerbated by the toxic accumulation of maltose-1-phosphate, which results in a pleiotropic stress response and cell death. α-Glucan produced by this pathway has shorter branches than classical bacterial α-glucan, but the mechanistic basis of this was not fully understood. To address this, I produced α-glucan in vitro, demonstrating GlgE and GlgB alone were sufficient to generate the distinct architecture. To investigate the determinants of α-glucan branch lengths, I solved high resolution crystal structures of Mycobacterium smegmatis GlgB bound to different oligosaccharides. These enabled the identification of several substrate binding sites, including the donor and acceptor sites of a newly forming branchpoint, giving novel insights into the action of glucan branching enzymes. To address M. tuberculosis GlgB as a drug target, I developed a high-throughput screening assay and used this to identify a potent, small-molecule inhibitor. I synthesised a selection of analogues to investigate structure-activity relationships. I also assessed the action of the compounds in vivo, demonstrating growth inhibition at high concentrations, but not via the GlgB target. Finally, I explored trehalose analogues as potential precursors for GlgE inhibitors, focussing on the synthesis, uptake and processing of 4-deoxy trehalose analogues. This multidisciplinary work gives a deeper understanding of mycobacterial α-glucan synthesis and how this can be targeted to develop new tuberculosis therapeutics

Molecular, biochemical and pharmacological characterisation of Mycobacterium tuberculosis cytochrome bd-I oxidase: a putative therapeutic target

Tuberculosis (TB) remains one of the most devastating diseases in humans. Nowadays, tuberculosis therapy is not sufficient to control the TB epidemic and only lasts for 6 months to cure patients and prevent relapse; therefore, the treatment of Mycobacterium tuberculosis (Mtb) is particularly challenging (1). New antibiotics, mainly those that are derived from new chemical classes, are more likely to be more effective against resistant strains. Moreover, expanding the knowledge of the mode of action of drugs has important implications in tackling TB. Only empirical approaches can be adopted in the journey of discovering new anti-tubercular drugs until a clear picture of latency and persister cells’ physiology is achieved. Mtb has the extraordinary ability to survive under hypoxia, suggesting a high degree of metabolic plasticity. The flexibility conferred by a modular respiratory system is critical to the survival of Mtb, thereby also making it a promising area of research for new drug targets. This thesis aimed towards the characterisation of cytochrome bd-I quinol oxidase (bd-I), a respiratory component that is believed to operate during both the replicative and “dormant” Mtb phenotypes. The essential nature of Mtb bd-I, which has no human homologue, has been confirmed in a recent deep sequencing study of genes required for Mtb growth by Griffin et al. (2), further confirming its potential as a novel target. Recombinant Mtb bd-I was successfully expressed under the control of the pUC19 lac promoter in the Escherichia coli ML16 bo3/bd-I and MB44 bo3/bd-I/bd-II knockout strains, allowing “noise-free” measurement of the enzyme. Initial steady-state kinetics of the enzyme was presented using a range of quinol substrates, revealing a substrate preference for dQH2 over Q1H2 and Q2H2. A number of bd-I inhibitors were identified and their pharmacodynamic profiles against Mtb H37Rv were determined. In addition, a pharmaco-metabolomics platform was initiated to explore the cellular response of Mtb to current first-line TB drugs as well as in house bd-I and type II NADH inhibitors. The initial findings are discussed in the context of the known mode of action of the drugs and future research needs in drug discovery of this devastating disease

Bactericidal disruption of magnesium metallostasis in Mycobacterium tuberculosis is counteracted by mutations in the metal ion transporter CorA

A defining characteristic of treating tuberculosis is the need for prolonged administration of multiple drugs. This may be due in part to subpopulations of slowly replicating or nonreplicating Mycobacterium tuberculosis bacilli exhibiting phenotypic tolerance to most antibiotics in the standard treatment regimen. Confounding this problem is the increasing incidence of heritable multidrug-resistant M. tuberculosis. A search for new antimycobacterial chemical scaffolds that can kill phenotypically drug-tolerant mycobacteria uncovered tricyclic 4-hydroxyquinolines and a barbituric acid derivative with mycobactericidal activity against both replicating and nonreplicating M. tuberculosis. Both families of compounds depleted M. tuberculosis of intrabacterial magnesium. Complete or partial resistance to both chemotypes arose from mutations in the putative mycobacterial Mg2+/Co2+ ion channel, CorA. Excess extracellular Mg2+, but not other divalent cations, diminished the compounds’ cidality against replicating M. tuberculosis. These findings establish depletion of intrabacterial magnesium as an antimicrobial mechanism of action and show that M. tuberculosis magnesium homeostasis is vulnerable to disruption by structurally diverse, nonchelating, drug-like compounds

Identification of a Chaperone for the SecA2 Protein Export Pathway of Mycobacterium tuberculosis

The bacterial pathogen Mycobacterium tuberculosis is responsible for the disease tuberculosis. To promote disease, M. tuberculosis exports proteins from the cytoplasm to the bacterial cell surface or out into the host environment. Exported proteins are in an ideal location to manipulate the host. All bacteria, including mycobacteria, utilize the Sec export system for the bulk of protein export. The Sec system is composed of an ATPase protein, SecA, and a membrane channel complex, SecYEG. Mycobacteria, along with some Gram-positive bacteria, contain a second, functionally distinct paralog of the SecA protein. In mycobacteria, the SecA responsible for housekeeping export is called SecA1 and is essential for bacterial survival, while the second SecA is called SecA2 and exports a smaller subset of proteins and is important for M. tuberculosis virulence. The mechanism of SecA2-dependent export is not well understood. Past data support a model where the mycobacterial SecA2 export pathway is integrated into the housekeeping Sec pathway, and SecA2 shares use of the same SecYEG channel as SecA1 to export its substrates. Like SecA1, SecA2 requires ATPase activity to function. In this dissertation, we take the approach of characterizing suppressors of a secA2 mutant allele to better understand the mechanism of SecA2-dependent export. Intragenic suppressor mutations map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG, SecA2 substrates or other partners of SecA2. Extragenic suppressor mutations map to a new component of the SecA2 pathway that we named SatS. In M. tuberculosis, SatS is required for the export of a subset of SecA2 substrates and for pathogenesis. SatS functions as a protein export chaperone that protects and promotes export of its specific substrates. Structural studies of SatS reveal a new fold combined with hydrophobic grooves representing potential sites of substrate binding. Taken together, the findings presented in this dissertation advance our understanding of the mechanism of the SecA2 export pathway and expand our appreciation of the diversity among chaperones by identifying SatS as a new type of protein export chaperone.Doctor of Philosoph

Investigating the response of Mycobacterium tuberculosis to anti-tuberculosis drugs

Following the introduction of streptomycin in 1947, the main issue for new drug treatments has been to avoid drug resistance. This has been achieved, in part, by identifying the most effective drug combinations. Conventional methods used for testing combinations of current and new drug regimens and the response of M. tuberculosis to treatment are complicated and time consuming. Other issues include the need for accurate and early detection, drug resistance screening and follow-up measures to ensure treatment completion as intended. Thus, there is an urgent need to develop robust and rapid tools to assess drug efficacy and measure the response to treatment. The main scope of this thesis is to understand current available methods to predict the action of novel drugs in combination, and to determine drug responses to inform/improve patient management. Therefore, I evaluated the Molecular Bacterial Load (MBL) assay as a tool for diagnosis of M. tuberculosis and monitoring treatment response. This has confirmed that the MBL assay is a faster and more sensitive approach compared to culture-based methods. I then investigated the interaction between anti-tuberculosis drugs in vitro, and determined the transcriptomic profile of 16 key genes in response to drugs such as rifampicin (the backbone of treatment for TB), para-amino salicylic acid (PAS) (used in the treatment of MDR TB) and bedaquiline (a new drug increasingly used worldwide, which acts on persister type of bacilli). I also performed phenotypic synergism studies using these drugs. Both approaches demonstrated an antagonistic interaction between rifampicin, PAS and bedaquiline in different physiological states. Finally, the utility of whole-genome sequencing (WGS) in tracking the evolution of drug resistance was evaluated in comparison to conventional phenotypic DST profiles in clinical isolates. This confirmed a good correlation between these methods. My WGS work identified uncommon mutations associated with resistance to isoniazid and moxifloxacin which had been only rarely reported prior to this in the literature