Arabidopsis thaliana DNA gyrase: expression, characterisation and in vivo insight

DNA gyrase is a type II topoisomerase distinguished by its ability to introduce negative supercoils into double-stranded DNA in a reaction linked to ATP hydrolysis. The essentiality of gyrase in bacteria has permitted its exploitation as an antibacterial target. The unanticipated discovery of gyrase within the nuclear genomes of eukaryotes including Arabidopsis and Plasmodia, was made near to two decades ago. Despite this, our understanding of gyrase within these species remains limited. The work here aimed to heterologously generate eukaryotic gyrases in order to biochemically characterise and better understand their mechanism of actions, gain an insight into their in vivo functions and explore their potential for inhibition. The specific inhibition of gyrase within these species would facilitate the generation of novel herbicidal and antimalarial drugs. In vivo knockdown experiments of A. thaliana gyrase have confirmed the embryo-lethality of GyrA. Arabidopsis plants able to propagate with a knockdown of GyrB1 are dwarfed, chlorotic, have reduced numbers and lengths of lateral roots and altered thylakoid ultrastructure. An increase of GyrB1 transcript mediates a stress response within Arabidopsis. The functional cooperation to achieve supercoiling of a reconstituted gyrase comprising A. thaliana GyrA and E. coli GyrB has been shown. The catalysis of A. thaliana enzyme (GyrA and GyrB2) is differentially mediated by potassium glutamate levels. The A. thaliana DNA gyrase has been determined to be 45-fold more efficient for ATP-independent DNA relaxation than E. coli gyrase. A novel sensitive DNA decatenation substrate, ‘bis-cat’, comprising two singly-linked supercoiled plasmids of disparate sizes has been generated and compared to the current marketed decatenation substrate. The novel substrate determined A. thaliana gyrase to be 35-fold more effective for DNA decatenation than the E. coli enzyme. The herbicidal and bactericidal specificities of novel fluoroquinolone compounds have also been compared

Functional analyses of sphingolipid biosynthesis in an apicomplexan parasite

The phylum Apicomplexa includes many protozoan parasites that cause serious human and animal disease, for example Plasmodium, Eimeria and Toxoplasma. Treatments against these parasites are limited and novel solutions are urgently required. Recently, research has focused on parasite specific features of lipid biosynthesis as drug targets. In particular the biosynthesis of sphingolipids, which have essential roles in many processes, has been highlighted as a potential target. Using the model apicomplexan Toxoplasma gondii we are studying the role of parasite and host sphingolipid biosynthesis in invasion and proliferation. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis, and our results demonstrated that the expression of host cell SPT is unaffected by Toxoplasma infection. In mammals the primary complex sphingolipid is sphingomyelin (SM), again our data demonstrated that the SM synthases (1 and 2) are not influenced by infection. Together these data indicated that parasite manipulation of host sphingolipid biosynthesis does not occur, supporting the hypothesis that Toxoplasma is dependant on de novo sphingolipid biosynthesis. To characterise this pathway, we showed that the Toxoplasma TgSPT1 and 2 are, like other eukaryotes, localised and active in the endoplasmic reticulum. However, uniquely, they have a prokaryotic origin. Metabolic labelling showed that several distinct complex sphingolipids are synthesized independently by the parasite. The fungal inositol phosphorylceramide (IPC) synthase inhibitor aureobasidin A (AbA) has been reported to target Toxoplasma IPC synthesis. However, our results demonstrated that whilst AbA, and an orthologue, are active against the parasite, their effect on Toxoplasma de novo sphingolipid biosynthesis is negligible. In addition, by using Leishmania major as a model we have analysed the global effect of compounds recognised as IPC synthase inhibitors in this kinetoplastid protozoan parasite. The results showed that ceramide levels increased in treated parasites, perhaps leading to parasite death via secondary signalling dysfunction. These data confirmed that the sphingolipid biosynthetic pathway is targeted by these anti-leishmanial compounds. Finally, the anti-leishmanial drug miltefosine showed reduced activity against a transgenic strain of L. major lacking sphingolipid biosynthesis ΔLCB2 compared to wild type. This suggested the sphingolipid synthesis has a role in sensitivity to the drug, metabolomic analyses supported this. Taken together, the present findings further characterised the T. gondii sphingolipid biosynthetic pathway and indicated the potential to target this in drug discovery efforts. In addition, metabolomic and lipidomic approaches confirmed that clemastine targets L. major IPCS

Structural studies of enoyl acyl carrier protein reductase from Plasmodium falciparum and Toxoplasma gondii.

Enoyl acyl carrier protein reductase enzyme (ENR) catalyses one of the two reductive steps in fatty acid elongation within the fatty acid synthesis type II cycle that is common to plants and prokaryotes. Since enzymes of this pathway are absent in humans they have become the target for several potent antibacterial compounds including triclosan which inhibits ENR in the picomolar range. As part of this thesis the gene for a type II ENR was located in the genomes of the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii. Analysis of the derived protein sequences suggested that these enzyme reside in the apicoplast. X-ray crystallographic techniques have been used to solve the structure for Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg) ENR in complex with the NAD+ cofactor and triclosan by molecular replacement to 2.2A and 2.6A, respectively. Both enzymes. are tetrameric with the approximate dimensions of 90A x 90A x 50A. Each subunit is formed by a 7 stranded parallel β-sheet flanked by 9α helices, reminiscent of a Rossmann nucleotide binding fold common to several NAD+ binding enzymes. Analysis of the ENR family reveals that a characteristic of apicomplexan ENRs is an insert which varies in size from 42 residues in the P jalciparum enzyme to 6 residues in T.gondii ENR and which flanks the inhibitor/substrate binding site. In PfENR this loop is disordered but in the structure of TgENR the loop can be clearly seen and the structure shows that the loop lies close to the bound inhibitor but makes no direct contacts. Comparisons of the binding sites of a range of different ENR inhibitor complexes has led to a better understanding of the plasticity of the enzyme in response to inhibitor (and possibly substrate) binding. Moreover analysis of the substrate/inhibitor binding pocket in P jalciparum and T.gondii ENR shows that whilst they are similar to the bacterial enzymes there are distinct differences which could be exploited for the development of novel antiparasitic agents. A major hurdle in the delivery of inhibitors targeted towards the apicoplast organelle is the need to cross several barriers including the parasite membranes and host cell walls. However the addition of a releasable eight arginine linker to the phenolic OH group of triclosan significantly improved the speed of delivery and enabled triclosan to enter both the extracellular and intracellular T.gondii tachyzoites and bradyzoites. The identification of both a novel inhibitor for the apicomplexan family and a possible general delivery mechanism may provide a foundation for the development of ENR inhibitors that will efficiently treat several key parasitic diseases

The role of DNA gyrase in illegitimate recombination

DNA, due to its double-helical structure, is subject to changes in topology due to the nature of transcription and replication. To overcome this, cells have processes and enzymes that ameliorate these changes. One such group of enzymes are the DNA topoisomerases, which are responsible for the maintenance of DNA topology. Despite this important role, these enzymes participate in illegitimate recombination (IR), which is genetic recombination between regions of DNA that share little or no homology. This can result in chromosomal rearrangements and is often a consequence of DNA-damaging agents. A consequence of topoisomerase-induced IR is thought to be therapy-related acute myeloid leukaemia (tAML). Analogously, there is evidence that exposure to sublethal concentrations of ciprofloxacin, a topoisomerase inhibitor, can cause resistance to non-quinolone antibiotics. This may work by a similar mechanism as that proposed for t-AML. This project centres around the examination of DNA gyrase-mediated IR focussing on the proposed subunit-exchange model. Using Blue-Native PAGE, I set up an assay to examine subunit exchange in topoisomerases. I have also characterised previously identified gyrase hyper-recombination mutations, known to increase the frequency of IR. Furthermore, I have investigated quinolone-induced antibiotic resistance and what the mechanism is. Here, I show that DNA gyrase can undergo subunit exchange, and that this seems to occur within higherorder oligomers of the enzyme, which have not been investigated before. Biochemical characterisation of the hyper-recombination mutations shows that they impair DNA gyrase activity which, in vivo, may have downstream consequences that may lead to IR. Using an in vivo assay where E. coli is treated with subinhibitory levels of quinolones, I have seen resistance to other non-quinolone antibiotics. This is not seen when other antibiotics, including other topoisomerase inhibitors, are tested. Whole genome sequencing has revealed point mutations that explain the resistances seen, however other larger chromosomal modifications have been observed as well