Metabolism and drug resistance in Trypanosomatids

The principle aim of this project is the investigation of metabolism and mechanisms of pentamidine resistance in trypanosomatids. An understanding of these mechanisms may allow the development of novel drugs to treat Leishmaniasis and human African trypanosomiasis (HAT), caused by the protozoan parasites Leishmania spp and Trypanosoma brucei. In this study a pentamidine resistance L. mexicana promastigote cell line was generated in vitro. This cell line was 20-fold resistant to pentamidine when compared to the parental wild type cells. Furthermore, these lines were cross resistant to other diamidine compounds. A proteomic analysis of these cell lines revealed numerous changes to the proteome, with the down regulation of several flagellar proteins. A hypothesis to investigate a role of the voltage dependent anion channel (VDAC) in pentamidine resistance was also explored. The metabolomic approach involved the investigation of transketolase and the pentose phosphate pawthway. A previous study involving a transketolase knockout T. brucei cell line indicated that an increased sensitivity to pentamidine and methylene blue. A transketolase deficient L. mexicana cell line was generated to test this hypothesis in Leishmania, however the differences were minimal. A metabolomic analysis of the L. mexicana tkt null cell line (lmtkt-/-) revealed an increase in ribose 5-phosphate, a key substrate of transketolase. Erythrose 4-phosphate also increased in the lmtkt-/- cells, indicating a source of this metabolite independent of TKT. It appears that the deletion of TKT prevents any flux through the oxidative branch of the PPP returning to the glycolytic pathway. Interestingly, the lmtkt-/- cells do not acidify the medium to the same extent as the wild type cells; however a glucose assay indicated that both cell lines used similar quantities of glucose. This would suggest that there is a change in the metabolites excreted by the lmtkt-/- cell line. Finally, a global metabolomics approach was investigated using high resolution mass spectrometry. Metabolomics is a rapidly developing field in systems biology, and whilst significant improvements have been made in mass spectrometry; the ability to analyse and interpret raw metabolomic datasets on a global scale has been largely neglected. Consequently, a database program to query these complex datasets was constructed

Digital gene expression analysis of two life cycle stages of the human-infective parasite, Trypanosoma brucei gambiense reveals differentially expressed clusters of co-regulated genes

<p><b>Background</b></p> <p>The evolutionarily ancient parasite, Trypanosoma brucei, is unusual in that the majority of its genes are regulated post-transcriptionally, leading to the suggestion that transcript abundance of most genes does not vary significantly between different life cycle stages despite the fact that the parasite undergoes substantial cellular remodelling and metabolic changes throughout its complex life cycle. To investigate this in the clinically relevant sub-species, Trypanosoma brucei gambiense, which is the causative agent of the fatal human disease African sleeping sickness, we have compared the transcriptome of two different life cycle stages, the potentially human-infective bloodstream forms with the non-human-infective procyclic stage using digital gene expression (DGE) analysis.</p> <p><b>Results</b></p> <p>Over eleven million unique tags were generated, producing expression data for 7360 genes, covering 81% of the genes in the genome. Compared to microarray analysis of the related T. b. brucei parasite, approximately 10 times more genes with a 2.5-fold change in expression levels were detected. The transcriptome analysis revealed the existence of several differentially expressed gene clusters within the genome, indicating that contiguous genes, presumably from the same polycistronic unit, are co-regulated either at the level of transcription or transcript stability.</p> <p><b>Conclusions</b></p> <p>DGE analysis is extremely sensitive for detecting gene expression differences, revealing firstly that a far greater number of genes are stage-regulated than had previously been identified and secondly and more importantly, this analysis has revealed the existence of several differentially expressed clusters of genes present on what appears to be the same polycistronic units, a phenomenon which had not previously been observed in microarray studies. These differentially regulated clusters of genes are in addition to the previously identified RNA polymerase I polycistronic units of variant surface glycoproteins and procyclin expression sites, which encode the major surface proteins of the parasite. This raises a number of questions regarding the function and regulation of the gene clusters that clearly warrant further study.</p&gt

Use of reconstituted metabolic networks to assist in metabolomic data visualization and mining

Metabolomics experiments seldom achieve their aim of comprehensively covering the entire metabolome. However, important information can be gleaned even from sparse datasets, which can be facilitated by placing the results within the context of known metabolic networks. Here we present a method that allows the automatic assignment of identified metabolites to positions within known metabolic networks, and, furthermore, allows automated extraction of sub-networks of biological significance. This latter feature is possible by use of a gap-filling algorithm. The utility of the algorithm in reconstructing and mining of metabolomics data is shown on two independent datasets generated with LC–MS LTQ-Orbitrap mass spectrometry. Biologically relevant metabolic sub-networks were extracted from both datasets. Moreover, a number of metabolites, whose presence eluded automatic selection within mass spectra, could be identified retrospectively by virtue of their inferred presence through gap filling

Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana

Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not infective to mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both

Search for dark photons in Higgs boson production via vector boson fusion in proton-proton collisions at √s = 13 TeV

A search is presented for a Higgs boson that is produced via vector boson fusion and that decays to an undetected particle and an isolated photon. The search is performed by the CMS collaboration at the LHC, using a data set corresponding to an integrated luminosity of 130 fb−1, recorded at a center-of-mass energy of 13 TeV in 2016–2018. No significant excess of events above the expectation from the standard model background is found. The results are interpreted in the context of a theoretical model in which the undetected particle is a massless dark photon. An upper limit is set on the product of the cross section for production via vector boson fusion and the branching fraction for such a Higgs boson decay, as a function of the Higgs boson mass. For a Higgs boson mass of 125 GeV, assuming the standard model production rates, the observed (expected) 95% confidence level upper limit on the branching fraction is 3.5 (2.8)%. This is the first search for such decays in the vector boson fusion channel. Combination with a previous search for Higgs bosons produced in association with a Z boson results in an observed (expected) upper limit on the branching fraction of 2.9 (2.1)% at 95% confidence level

Search for strongly interacting massive particles generating trackless jets in proton-proton collisions at s = 13 TeV

A search for dark matter in the form of strongly interacting massive particles (SIMPs) using the CMS detector at the LHC is presented. The SIMPs would be produced in pairs that manifest themselves as pairs of jets without tracks. The energy fraction of jets carried by charged particles is used as a key discriminator to suppress efficiently the large multijet background, and the remaining background is estimated directly from data. The search is performed using proton-proton collision data corresponding to an integrated luminosity of 16.1 fb - 1 , collected with the CMS detector in 2016. No significant excess of events is observed above the expected background. For the simplified dark matter model under consideration, SIMPs with masses up to 100 GeV are excluded and further sensitivity is explored towards higher masses

Search for new physics in top quark production with additional leptons in proton-proton collisions at root s=13 TeV using effective field theory

Events containing one or more top quarks produced with additional prompt leptons are used to search for new physics within the framework of an effective field theory (EFT). The data correspond to an integrated luminosity of 41.5 fb(-1) of proton-proton collisions at a center-of-mass energy of 13 TeV at the LHC, collected by the CMS experiment in 2017. The selected events are required to have either two leptons with the same charge or more than two leptons; jets, including identified bottom quark jets, are also required, and the selected events are divided into categories based on the multiplicities of these objects. Sixteen dimension-six operators that can affect processes involving top quarks produced with additional charged leptons are considered in this analysis. Constructed to target EFT effects directly, the analysis applies a novel approach in which the observed yields are parameterized in terms of the Wilson coefficients (WCs) of the EFT operators. A simultaneous fit of the 16 WCs to the data is performed and two standard deviation confidence intervals for the WCs are extracted; the standard model expectations for the WC values are within these intervals for all of the WCs probed.Peer reviewe

Measurement of differential cross sections for Z bosons produced in association with charm jets in pp collisions at s \sqrt{s} = 13 TeV

Measurements are presented of differential cross sections for the production of Z bosons in association with at least one jet initiated by a charm quark in pp collisions at s √s = 13 TeV. The data recorded by the CMS experiment at the LHC correspond to an integrated luminosity of 35.9 fb−1. The final states contain a pair of electrons or muons that are the decay products of a Z boson, and a jet consistent with being initiated by a charm quark produced in the hard interaction. Differential cross sections as a function of the transverse momentum pT of the Z boson and pT of the charm jet are compared with predictions from Monte Carlo event generators. The inclusive production cross section 405.4 ± 5.6 (stat) ± 24.3 (exp) ± 3.7 (theo) pb, is measured in a fiducial region requiring both leptons to have pseudorapidity |η| 10 GeV, at least one lepton with pT > 26 GeV, and a mass of the pair in the range 71–111 GeV, while the charm jet is required to have pT > 30 GeV and |η| < 2.4. These are the first measurements of these cross sections in proton-proton collisions at 13 TeV

Search for CP violating top quark couplings in pp collisions at s \sqrt{s} = 13 TeV

Results are presented from a search for CP violation in top quark pair production, using proton-proton collisions at a center-of-mass energy of 13 TeV. The data used for this analysis consist of final states with two charged leptons collected by the CMS experiment, and correspond to an integrated luminosity of 35.9 fb−1. The search uses two observables, O1 and O3, which are Lorentz scalars. The observable O1 is constructed from the four-momenta of the charged leptons and the reconstructed top quarks, while O3 consists of the four-momenta of the charged leptons and the b quarks originating from the top quarks. Asymmetries in these observables are sensitive to CP violation, and their measurement is used to determine the chromoelectric dipole moment of the top quark. The results are consistent with the expectation from the standard model