THE ROLE OF DE NOVO MYO-INOSITOL SYNTHESIS AND METABOLISM IN LEISHMANIA PARASITES
TIM CHUNG-TING LIU
(ORCID: 0000-0001-8500-2010)
Submitted in total fulfilment of the requirements of the
degree of Doctor of Philosophy (Ph.D.)
February 2018
Department of Biochemistry and Molecular Biology
The University of Melbourne
Abstract
Leishmania are protozoan parasites responsible for a spectrum of sandfly-borne diseases known as the leishmaniases that cause substantial morbidity and mortality in some of the most impoverished countries in the world. Conventional anti-leishmanials are limited by toxicity, high cost, and increasing resistance, highlighting an urgent need to identify novel drug targets. Leishmania parasites require the nutrient myo-inositol for a multitude of crucial cellular processes that relate to its role as an integral building block for inositol lipids, including phosphatidylinositol (PI), inositol phosphorylceramide (IPC), free and anchoring glycosylphosphatidylinositol (GPI), and organelle-specific phosphoinositides.
This work investigates the extent to which Leishmania parasites are dependent on de novo myo-inositol synthesis versus salvage from the extracellular milieu both in vitro and in vivo for growth and pathogenesis. By generating defined genetic deletion mutants in Leishmania mexicana lacking two key putative enzymes in this pathway, namely myo-inositol 3-phosphate synthase (INO1) and myo-inositol
3-phosphate monophosphatase 1 (IMP1), the importance of de novo myo-inositol synthesis was directly addressed.
As expected, the L. mexicana ∆ino1 mutant lacked the capacity for de novo myo-inositol synthesis and was an inositol auxotroph. Strikingly, this mutant could only grow in ex vivo infected macrophages which had been supplied with an exogenous sources of myo-inositol and was avirulent in the susceptible BALB/c mouse model, suggesting that myo-inositol levels in Leishmania-induced granulomas are low. Analysis of the steady state polar metabolite profile of ∆ino1
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mutant and 13C-glucose labelling experiments failed to reveal key differences
compared to wild type, demonstrating that L. mexicana does not enter
self-preserving quiescence in response to myo-inositol starvation. This, combined
with the profound changes in lipid composition revealed by lipidomic analysis,
may underlie the rapid death phenotype induced by myo-inositol starvation.
A second L. mexicana mutant lacking IMP1 was also generated.
Unexpectedly, deletion of imp1 did not lead to disruption of de novo myo-inositol
synthesis, but resulted in a mutant that displayed a modest growth defect in vitro
that was unrelated to exogenous myo-inositol levels. Despite the presence of de
novo synthesis, the Δimp1 mutant was unable to proliferate in ex vivo macrophages
or cause disease in mice. Global metabolomic and lipidomic analysis of this mutant
revealed subtle changes in central carbon metabolism and lipid profile. Further
studies showed that the loss of virulence of this mutant was linked to increased
sensitivity to elevated temperatures encountered in the mammalian host,
suggesting an unexpected role for IMP1 in the development of thermotolerance
and differentiation to the amastigote stage. Genomic analysis also revealed a
second putative IMPase isoform, IMP2, which may explain why loss of IMP1 does
not lead to pertrubation of de novo myo-inositol synthesis in the Δimp1 mutant.
In summary, this work highlights the importance of de novo myo-inositol
synthesis for Leishmania growth and survival, providing a better understanding of
a vital pathway. This improved understanding of the metabolic mechanisms
essential for Leishmania provides further insights into their pathogenesis and
validates enzymes in this pathway as new targets for the development of new
strategies to combat the rising drug resistance.
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Declaration
This is to certify that: i. This thesis comprises only my original work towards the Ph.D. ii. Due acknowledgement has been made in the text to all other material used iii. The thesis is fewer than 100,000 words in length
Tim Chung-Ting Liu
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Acknowledgements
To my supervisors, Malcolm McConville and Fiona Sansom, for bravely taking me on this project and introducing me to the wonderful world of parasitology. Your dedication to science, work ethic, and intellectual rigor set a standard that I will try and follow in all my future pursuits. I thank you for the tremendous support and guidance you have provided throughout this project.
To my PhD committee members, Paul Gleeson and Stuart Ralph, for all your helpful suggestions and encouragements during the course of my PhD.
To the National Health and Medical Research Council, for funding this very rewarding project.
To Fleur Sernee, for all your help in and outside the lab. Your immense knowledge on Leishmania and remarkable patience really helped made this “newbie” feeling less overwhelmed when first started.
To Eleanor Saunders, for being the nicest and most generous person. Thank you for keeping our old and reliable GCs running smoothly, and more importantly, I am very grateful for all the feedback you provided on the thesis drafts.
To Julie Ralton, for being the most wonderful lab-bench neighbour and always checking to make sure I have a life outside of the lab. Thank you for all your help in the lab, especially with the HPTLC work, and keeping our lab in tip-top shape.
To Joachim Kloehn and Charlie Chua, for setting the best examples as ex-PhD students. Glad to have you guys for company in the lab during all those weekends.
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To Michael Dagley, for creating DExSI and made everyone’s life just a little bit
easier.
To Stephan Klatt, for being that unique bacteria guy among all the protozoa people!
Also, thank you for all your help on ID’ing all those tricky lipids.
To Martin Blume, for occupying all that precious space in the TC room with your
Toxo stuff. Joking aside, thanks for all your advice on how to multi-task in the lab.
To Simon Cobbold, for being the other guy who can’t bake a birthday cake. Thanks
for your help on the lipid analysis.
To Katrina Binger, for seasoning this lab with some salt and kindness. Thanks for
all your helpful advice in the lab.
To Elizabeth King, for all the very useful advice you gave on preparing for my PhD
oration and on putting this thesis together.
To Jiang Nan Zhu, for valiantly taking up the baton and continuing the inositol
story.
To Metabolomics Australia (especially Konstantinos Kouremenos, David de Souza,
and Dedreia Tull), for kindly inviting me down to your Friday coffee sessions.
Thanks for all your help with the MS instruments.
To Max Walker, Shiralee Whitehead, and the staff at Bio21 animal facility, for
taking very good care of all the mice.
Finally, to my parents and sister, for all your support and encouragements, and to
whom this thesis is dedicated.
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Table of Contents
Abstract I
Declaration III
Acknowledgements V
List of Figures XII
List of Tables XV
Abbreviations XVI
Chapter 1 Literature Review 1 1.1 Leishmaniasis 1 1.2 Epidemiology of leishmaniasis 3 1.3 Current treatment options and the emergence of drug resistance 5 1.4 Leishmania life cycle 8 1.5 The role of inositol lipids in Leishmania 13 1.5.1 Phosphatidylinositol and inositol phosphorylceramide 13 1.5.2 GPI-associated glycoconjugates 15 1.5.3 Phosphoinositides 24 1.6 myo-Inositol metabolism in Leishmania 30 1.6.1 myo-inositol salvage versus biosynthesis 30 1.6.2 Compartmentalisation of inositol lipid synthesis in protozoan parasites 33 1.7 Project rationale and thesis aims 35
Chapter 2 Materials and Methods 37 2.1 Molecular biology 37 2.1.1 Bioinformatics analysis 37
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2.1.2 Primer and vector design 37 2.1.3 Genomic DNA extraction 38 2.1.4 Polymerase chain reaction (PCR) 38 2.1.5 DNA sequencing 39 2.1.6 Generation of GFP fusion constructs 39 2.1.7 Generation of HA fusion constructs 41 2.1.8 Generation of linear targeting constructs for gene deletion 42 2.1.9 Generation of episomal complementation constructs 43 2.1.10 Generation of phosphoinositide-specific GFP probes 44 2.1.11 Whole cell lysate protein extraction and quantification 45 2.1.12 SDS-PAGE and immunoblotting 46 2.2 Bacterial culture methods 47 2.2.1 Bacterial culture 47 2.2.2 Bacterial transformation 47 2.3 Leishmania culture methods 47 2.3.1 Leishmania promastigote culture 47 2.3.2 Cell density determination 48 2.3.3 Transfection of L. mexicana and clonal selection 48 2.3.4 Axenic amastigote differentiation and culture 50 2.4 In vitro macrophage infection 50 2.4.1 Extraction and proliferation of mice bone marrow-derived macrophages (BMDM) 50 2.4.2 Leishmania infection assays 51 2.4.3 Infectivity quantification 51 2.4.4 Extraction of Leishmania amastigotes from infected macrophages 52 2.5 In vivo mice infection 52 2.5.1 Intradermal infection of Leishmania parasites 52 2.5.2 Lesion assessment 53 2.5.3 Extraction of Leishmania amastigotes from cutaneous lesions 53 2.5.4 Extraction of Leishmania amastigotes from draining lymph nodes 54 2.6 Fluorescence microscopy 54 2.6.1 GFP live-cell imaging 54 2.6.2 Immunofluorescence imaging 55 2.6.3 Tracing endosomal activity by live-cell imaging 56 2.6.4 Fluorescence cell viability assay 56 2.7 Metabolomics analysis 56
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2.7.1 13C-U-glucose labelling time-course experiment 56 2.7.2 Quenching and extraction of intracellular polar metabolites 57 2.7.3 Extracellular polar metabolite samples 58 2.7.4 Gas chromatography-mass spectrometry (GC-MS) 59 2.7.5 Metabolite identification and analysis 60 2.8 Lipidomics analysis 61 2.8.1 High-performance thin-layer chromatography 61 2.8.2 Supercritical fluid chromatography-quadrupole/time-of-flight tandem mass spectrometry (SFC-QTOF-MS) 62 2.8.3 Lipid identification and analysis 64
Chapter 3 Characterisation of L. mexicana myo-Inositol 3-phophate Synthase (INO1) and its Role in Pathogenesis 65 3.1 Introduction 65 3.2 L. mexicana INO1 is localised to the cytoplasm of the parasite 68 3.3 L. mexicana ino1 is not essential in vitro in the presence of exogenous myo-inositol 70 3.4 L. mexicana Δino1 undergo rapid cell death in the absence of exogenous myo-inositol 73 3.5 Loss of INO1 does not affect growth inside macrophages in vitro 77 3.6 L. mexicana Δino1 are avirulent in the BALB/c model of cutaneous leishmaniasis 80 3.7 Discussion 82 3.7.1 INO1 localisation and the glycosomal compartmentalisation of glucose 6-phosphate 83 3.7.2 The essentiality of ino1 in Leishmania compared to other protozoan parasites 85 3.7.3 The role of ino1 in Leishmania growth and infectivity 86
Chapter 4 Defining the Molecular Basis of myo-Inositol Starvation in L. mexicana 89
4.1 Introduction 89 4.2 The de novo myo-inositol synthesis pathway is abolished in Δino1 91 4.3 Carbon metabolism of L. mexicana is unregulated in response to myo-inositol starvation 94 4.4 myo-Inositol starvation leads to depletion of specific inositol lipids 99 4.5 PI3P is disrupted in L. mexicana during myo-inositol starvation 107
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4.6 Disscussion 116 4.6.1 INO1 is essential for de novo myo-inositol synthesis 116 4.6.2 Effects of myo-inositol starvation on L. mexicana central carbon metabolism 118 4.6.3 Effects of myo-inositol starvation on L. mexicana lipid composition 120
Chapter 5 Characterisation of L. mexicana myo-Inositol Monophosphatase (IMPase) and its Role in Pathogenesis 125 5.1 Introduction 125 5.2 L. mexicana encodes two putative IMPase isoforms 128 5.3 L. mexicana IMP1 and IMP2 are co-localised to the ER 132 5.3.1 Bioinformatics prediction of localisation 132 5.3.2 Localisation by GFP-fusion proteins 132 5.3.3 Localisation by HA-tagging 133 5.4 L. mexicana imp1 is not essential in vitro 137 5.5 The L. mexicana Δimp1 mutant retains myo-inositol prototrophy but exhibits a distinct growth defect in vitro 140 5.6 L. mexicana Δimp1 is unable to survive in ex vivo infected macrophages or cause disease in the mouse model 142 5.7 Discussion 145 5.7.1 Comparison between the two L. mexicana IMPase isoforms, IMP1 and IMP2 145 5.7.2 The role of imp1 in Leishmania proliferation and virulence 148 5.7.3 The essentiality of imp2 149
Chapter 6 Functional Analysis of L. mexicana Δimp1 151 6.1 Introduction 151 6.2 The Δimp1 mutant is able to synthesise myo-inositol 152 6.3 Inositol lipid biosynthesis is unchanged in ∆imp1 mutant 156 6.4 Changes in Δimp1 promastigote central carbon metabolism are subtle 157 6.5 Changes to lipid composition in Δimp1 are minimal 162 6.6 L. mexicana Δimp1 is thermosensitive and cannot differentiate to amastigote in vitro 166 6.7 Discussion 170
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6.7.1 imp1 and de novo myo-inositol synthesis 170 6.7.2 Metabolic consequences of imp1 deletion in L. mexicana 171 6.7.3 imp1 is required for thermotolerance and amastigote differentiation 173 6.7.4 Possible links between IMP1 and IMP2 functions 175
Chapter 7 Final Remarks and Future Directions 177
References 187
Appendices 229 Appedix 1 BSA standards for Bradford colorimetric assay 229 Appedix 2 Semi-defined medium (SDM) for Leishmania 230 Appedix 3 Completely defined medium (CDM) for Leishmania 231 Appedix 4 GC-MS library for DExSI 233
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List of Figures
1.1 Disease manifestations of leishmaniasis 2 1.2 The geographical distribution and burden of leishmaniasis 4 1.3 Life cycle of Leishmania parasites 12 1.4 Biosynthesis of IPC from PI 15 1.5 Structures of GPI-anchored glycoconjugates in Leishmania 17 1.6 Phosphoinositide metabolism and subcellular distribution 25 1.7 Nutrient availability inside the Leishmania parasitophorous vacuole 33 1.8 Hypothetical model for the metabolic compartmentalisation of myo-inositol-containing lipids in T. brucei and P. falciparum 35 3.1 Reaction catalysed by the enzyme, myo-inositol 3-phosphate synthase 66 3.2 L. mexicana INO1 is localised to the cytosol 69 3.3 Targeted deletion of myo-inositol 3-phosphate synthase (ino1) in L. mexicana by homologous recombination and gene replacement 70 3.4 The Δino1 and episomal add-back mutants display the expected genotype 72 3.5 L. mexicana Δino1 have lost INO1 expression while genetic complementation with pXG-INO1 results in higher expression levels compared to wild type 73 3.6 Deletion of ino1 results in myo-inositol auxotrophic parasites 75 3.7 L. mexicana Δino1 undergo rapid loss of viability without exogenous myo-inositol 76 3.8 Infection and intracellular replication of L. mexicana Δino1 are similar to wild type in macrophages under myo-inositol-replete conditions 78 3.9 L. mexicana Δino1 still replicate in macrophages even after myo- inositol starvation pre-infection 79 3.10 L. mexicana Δino1 are unable to replicate in macrophages when the medium is myo-inositol depleted 80 3.11 L. mexicana Δino1 are avirulent in susceptible mice 81
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3.12 A subpopulation of Δino1 persist in the lymph node during mouse infection 82 4.1 The de novo myo-inositol synthesis pathway is abolished in Δino1 92 4.2 De novo myo-inositol is released by wild-type promastigotes 94 4.3 Metabolic fluxes of central carbon metabolism are unaffected by myo-inositol starvation 96 4.4 Uptake and secretion of metabolites by L. mexicana are unaffected by myo-inositol starvation 98 4.5 PI and PIP species are depleted in Δino1 in the absence of exogenous myo-inositol 100 4.6 myo-inositol starvation induces profound changes in Δino1 lipid composition 102 4.7 Identification of significant lipid species during myo-inositol starvation based on MS/MS information 104 4.8 PI species are depleted during myo-inositol starvation while other inositol lipids are less affected 105 4.9 A diverse range of lipid species are affected by myo-inositol conditions 106 4.10 Expression of phosphoinositide-specific GFP probes was unaffected during myo-inositol starvation 109 4.11 Monitoring PI3P in L. mexicana using GFP-2×FYVE reporter during myo-inositol starvation 111 4.12 Monitoring PI4P in L. mexicana using GFP-OSBP PH probe during myo-inositol starvation 113
4.13 Monitoring PI(4,5)P2 in L. mexicana using PLCD1-GFP probe during myo-inositol starvation 115 5.1 Schematics of phosphoinositide and inositol phosphate synthesis and recycling 126 5.2 Sequence alignment of L. mexicana IMP1 and IMP2 128 5.3 Sequence alignment of L. mexicana IMP1 with known and putative IMP1 homologue from other Leishmania and Trypanosoma (annotated as IMPase 2) species, as well as human, yeast and Mycobacterium IMPase 130 5.4 Sequence alignment of L. mexicana IMP2 with known and putative IMP2 homologue from other Leishmania and Trypanosoma (annotated as IMPase 1) species, as well as human, yeast and Mycobacterium IMPase 131 5.5 Episomal expression of GFP-fused IMPase is inconsistent in L.
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mexicana promastigotes 133 5.6 L. mexicana promastigotes transfected with episomal constructs expressed HA-tagged IMP1 and IMP2 recombinant proteins 134 5.7 L. mexicana IMP1 and IMP2 are associated with the promastigote ER 135 5.8 Targeted deletion of myo-inositol monophosphatase 1 (imp1) in L. mexicana 137 5.9 The Δimp1 and episomal add-back mutants display the expected genotype 139 5.10 Deletion of imp1 results in slower promastigote growth that is not rescued by addition of exogenous myo-inositol 141 5.11 L. mexicana Δimp1 are unable to replicate in macrophages in vitro 143 5.12 L. mexicana Δimp1 are unable to generate lesions in a susceptible mouse model of infection and are completely cleared by the host 144 6.1 The de novo myo-inositol synthesis pathway remains functional in Δimp1 154 6.2 Extracellular myo-inositol abundance and percentage of 13C-enrichment of L. mexicana wild type and Δimp1 155 6.3 Lipid composition of Δimp1 is unaffected by the absence of exogenous myo-inositol 157 6.4 Intracellular 13C-enrichment of key carbon metabolites show modest differences between wild type and Δimp1 159 6.5 Abundance of key extracellular metabolites from L. mexicana wild type and Δimp1 161 6.6 Volcano plot of differences in the lipid profile of wild type against Δimp1 under myo-inositol-replete condition 163 6.7 Identification of significant lipid species between wild type and Δimp1 based on MS/MS information 164 6.8 Lipid species identified as being significantly different between L. mexicana wild type and Δimp1 under myo-inositol-replete condition 165 6.9 L. mexicana Δimp1 cannot differentiate into amastigotes under axenic condition 167 6.10 L. mexicana Δimp1 retain the promastigote-associated protein marker, GP63 168 6.11 L. mexicana Δimp1 show reduced viability as a result of increased temperature during axenic amastigote differentiation 169 A.1 Calibration curve of Bradford colorimetric assay based on BSA 229
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List of Tables
2.1 PCR primer pairs for GFP fusion constructs 40 2.2 Sequencing primers for GFP, HA, and knockout constructs 41 2.3 PCR primer pairs for HA fusion constructs 42 2.4 PCR primers for linear knockout constructs 43 2.5 PCR primers for episomal complementation constructs 44 2.6 PCR primers for phosphoinositide-specific GFP fusion probes 45 2.7 List of antibodies used for immunoblotting 46 2.8 List of antibodies used for immunofluorescence imaging 55 2.9 Gas chromatography oven settings for analysing TMS-deritivised samples 59 2.10 Supercritical fluid chromatography solvent parameters 63 3.1 PCR primers for diagnosing ino1 deletion and complementation 71 5.1 PCR primers for diagnosing imp1 and imp2 deletion and complementation 138 A.1 Bovine serum albumin protein standards 229 A.2 Composition of semi-defined medium SDM-79 powder stock 230 A.3 Composition of completely defined medium base 231 A.4 Working solution of completely defined medium 232 A.5 List of metabolites and their signature ions detected and identified by GC-MS after TMS derivitisation 233
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Abbreviations
Abbreviations of the International System of Units (SI), SI-derived units and
standard notations for chemical elements and amino acids are used in this thesis.
Other abbreviations used in the text are defined below.
3PG 3-phosphoglycerate ABC ATP-binding cassette ADP adenosine diphosphate α-KG α-ketoglutarate amu atomic mass unit ATP adenosine triphosphate BH Benjamini-Hochberg BMDM bone marrow-derived macrophage bp base pair BSA bovine serum albumin BSW 1-butanol-saturated water ºC degree Celsius CDF common data format CDM completely defined medium CDP cytidine diphosphate CL cutaneous leishmaniasis DG diacylglycerol DALY disability-adjusted life years DExSI Data Extraction for Stable Isotope-labelled metabolites DHAP dihydroxyacetone phosphate
dH2O distilled water DIC differential interference contrast DNA deoxyribonucleic acid dNTPs deoxyribonucleotide triphosphate EDTA ethylenediaminetetraacetic acid
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EI electron ionisation EM electron multiplier EPB electroporation buffer ER endoplasmic reticulum EtN ethanolamine FC fold change Fru6P fructose 6-phosphate
Fru1,6P2 fructose 1,6-bisphosphate Gal galactose
Galf galactofuranose GC-MS gas chromatography-mass spectrometry gDNA genomic DNA GFP green fluorescent protein GIPL glycoinositolphospholipid Glc glucose Glc6P glucose 6-phosphate GlcN glucosamine GlcNAc N-acetyl glucosamine GPI glycosylphosphatidylinositol h hour HA haemagglutinin HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIV human immunodeficiency virus HPTLC high-performance thin-layer chromatography HRP horseradish peroxidase id inside diameter iFBS heat-inactivated foetal bovine serum IMPase myo-inositol monophosphatase Ino myo-inositol Ino3P myo-inositol 3-phosphate
IP3 inositol 1,4,5-trisphosphate IPC inositol phosphorylceramide kD kilodalton LB Lysogeny broth LPG lipophosphoglycan MCS multiple cloning site min minute Man mannose
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MCL mucocutaneous leishmaniasis MEM Eagle’s minimum essential medium MOPS 3-(N-morpholino)propanesulfonic acid MS mass spectrometry MSD mass selective detector MS/MS tandem mass spectrometry MVT multivesicular tubule MVB multivesicular body NADH nicotinamide adenine dinucleotide NADPH nicotinamide adenine dinucleotide phosphate NCBI National Center for Biotechnology Information OD optical density PBS phosphate-buffered saline PC phosphatidylcholine PCR polymerase chain reaction PE phosphatidylethanolamine PFA paraformaldehyde PG phosphoglycan
Pi inorganic phosphate PI phosphatidylinositol PI3K PI 3-kinase PI4K PI 4-kinase PI5K PI 5-kinase PI3P phosphatidylinositol 3-phosphate
PI(3,4)P2 phosphatidylinositol 3,4-bisphosphate
PI(3,4,5)P3 phosphatidylinositol 3,4,5-trisphosphate
PI(3,5)P2 phosphatidylinositol 3,5-bisphosphate PI4P phosphatidylinositol 4-phosphate
PI(4,5)P2 phosphatidylinositol 4,5-bisphosphate PIS PI synthase PKC protein kinase C PPG proteophosphoglycan PLC phospholipase C PPP pentose phosphate pathway PV parasitophorous vacuole PVDF polyvinylidene fluoride QTOF quadrupole/time-of-flight RPMI Roswell Park Memorial Institute medium
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RNA ribonucleic acid RNAi RNA interference RT room temperature SDM semi-defined medium SDS sodium dodecyl sulphate SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis SEM standard error of the mean SFC supercritical fluid chromatography Succ-CoA succinyl-Coenzyme A TCA tricarboxylic acid (cycle) TG triacylglycerol TMS trimethylsilyl Tris tris(hydroxymethyl)aminomethane U uniformly-labelled UDP uridine diphosphate UTR untranslated region VL visceral leishmaniasis v/v volume by volume WHO World Health Organisation WSB water-saturated 1-butanol w/v weight by volume w/w weight by weight
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CHAPTER 1 Literature Review
1.1 Leishmaniasis
Human leishmaniasis comprises a spectrum of diseases caused by vector-borne protozoan parasites belonging to the genus Leishmania (order Trypanosomatidae).
Over 30 species of Leishmania have been identified, 20 of which are known to cause infections in human (Pearson & Sousa, 1996). The major clinical manifestations of Leishmania infection are cutaneous (CL), mucocutaneous (MCL), and visceral (VL) leishmaniasis (Figure 1.1). The extent to which each of these different forms of disease arise depends on the species of Leishmania involved, the immune status of the host, as well as the availability of appropriate treatments
(McCall et al., 2013). The majority of cases of CL are marked by the development of a localised ulcerative skin lesion at the site of sandfly bite. These lesions are usually self-limiting, although healing can often take months to years, and often result in permanent scarring (Reithinger et al., 2007). In more severe cases, diffuse cutaneous lesions can develop, which are marked by non-ulcerative nodules that extend along the limbs of the primary infection and sometimes cover the patient’s entire body. MCL occurs when parasites metastasise from the original site of infection to mucosal tissues in the face, triggering the destruction of tissues around the nose, mouth, and throat cavities (David & Craft, 2009). Severe form of CL and
MCL are difficult to treat and the risk of secondary bacterial infections is common.
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The resulting facial damage and disfigurement associated with MCL can be
accompanied by enormous social stigmatisation and considerable impact on
psychological and economic well-being (Yanik et al., 2004; Kassi et al., 2008). VL,
also known as kala-azar, occurs when the parasites disseminate to internal organs,
such as the liver, spleen, and bone marrow, resulting in organ enlargement, as well
as clinical presentation of fever, weight-loss and weakness that progress over
weeks to months (Chappuis et al., 2007). If left untreated, VL leads to severe
wasting and multisystem disease that almost always result in death.
(A) (B)
MCL CL
VL
Figure 1.1| Disease manifestations of leishmaniasis. (A) Depending on the species of Leishmania involved and host factors (immunity and nutrition), Leishmania infection can lead to the development of localised skin ulcers at the site of sandfly bite (CL), extensive destruction of mucosal tissues (MCL), or can further disseminate to internal organs, such as liver, spleen, and bone marrow to elicit visceral form (VL). (B) Images of patients suffering from different forms of leishmaniasis. Clockwise from top left: CL, MCL, VL, and disseminated form of CL. Sources: Murray et al. (2005), Reithinger et al. (2007), WHO (2017b).
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1.2 Epidemiology of leishmaniasis
More than 350 million people are at risk of contracting human leishmaniasis which is endemic in 98 countries, with highest prevalence in East Africa, the Middle East,
Central Asia, and Latin America (Avar et al. 2012) (Figure 1.2). Disease prevalence is highest in the poorest communities due to increased risk associated with poor housing and sanitary conditions, malnutrition, lack of personal protective measures, and economically driven migration that all aid in the spread of disease
(Alvar et al., 2006; Boelaert et al., 2009). It is estimated that more than 140 million people are chronically infected with Leishmania, with approximately 1.6 million new cases (0.4 million VL and 1.2 million CL) and 12-14 million active cases reported each year (Alvar et al., 2012). However, it is likely the incidence of leishmaniasis is grossly underestimated due to the lack of organised surveillance and empirical data collection (King & Bertino, 2008; Bern et al., 2008). Overall, mortality caused by leishmaniasis is second only to malaria among all parasitic diseases, and it is the third most common cause of morbidity in terms of disability adjusted life years (DALYs) after malaria and schistosomiasis (WHO, 2017c).
The disease prevalence may be further aggravated by global events such as war and climate change. In recent years, significant leishmaniasis outbreaks have been reported in Syria and South Sudan, due to breakdown of the health care systems in these countries and mass forced migration (Al-Salem et al., 2016; Burki,
2017). A recent increase in leishmaniasis cases across Europe has been attributed to the spread of sandfly vectors due to warmer temperatures (Medlock et al., 2014) and is predicted to occur in other parts of the world (González et al., 2010; Ready
2010). Increased prevalence of leishmaniasis is also linked to co-infection with
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other serious endemic diseases such as HIV and malaria, caused by weakening of
immunity and increased vulnerability of organs such as the liver (Alvar et al, 2008;
van den Bogaart et al, 2012; 2013; van Griensven et al., 2014).
(A)
(B)
Figure 1.2| The geographical distribution and burden of leishmaniasis. Maps depict the global distribution and number of new cases reported to the World Health Organisation in 2015 for (A) cutaneous (CL) and (B) visceral leishmaniasis (VL). More than 90 % of CL were reported from Eastern Mediterranean and South American regions, with Afghanistan, Brazil, Iran, Iraq, and Syria representing 75 % of global cases. VL were distributed across East Africa (40 %), the Indian subcontinent (39 %) and Brazil (14 %), with Brazil, Ethiopia, Kenya, India, Somalia, South Sudan, and Sudan representing 90 % of global cases. Maps and statistics were sourced from WHO (2017a).
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1.3 Current treatment options and the emergence of drug resistance
There have been relatively few developments in anti-leishmanial drug therapy in the last 50 years. Currently available first line drugs are the pentavalent antimonials, different amphotericin formulations, paromomycin, and miltefosine. Each treatment option has its benefits and deficiencies, which are reviewed below.
Pentavalent antimonials, such as sodium stibogluconate and meglucamine antimoniate (Croft & Olliaro, 2011) have been in use since the 1940s and are thought to target multiple processes in the parasites, although their precise mode of action remains poorly defined (Frézard et al., 2009). Due to adequate efficacy in treating VL patients and relatively low production cost, antimonials are still the
“drug of choice” in many endemic countries. However, in epidemic regions such as the Indian state of Bihar that suffer high disease prevalence and transmission rates, treatment failure rates of up to 65 % have been reported (Sundar, 2001;
Olliaro et al., 2005), and the use of antimonials has been abandoned in many areas
(Ponte-Sucre et al. 2017). While the rise in antimonal resistance is partly attributable to overuse and inadequate monitoring, recent studies have also highlighted a possible link between elevated levels of arsenic (mimetic of antimony) in the drinking water in Bihar to the development of antimonial resistance (Perry et al., 2013). Other deficiencies of antimonials include inherent toxicity, lengthy treatment course (typically 28 days), and systemic side effects often associated with the treatment (Moore & Lockwood, 2010). Severe adverse events such as cardiac arrhythmias, can also develop in approximately 10 % of the patients (Lawn et al.,
2006).
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Amphotericin B is increasingly being used as the first-line treatment for VL
in endemic areas where antimonials resistance is widespread. These drugs exert
their anti-leishmanial activity by binding to ergosterol, the major sterol of these
parasites, leading to the formation of pore complexes that disrupt membrane
permeability (Hartsel & Bolard, 1996; Ramos et al., 1996). There is some evidence
that Amphotericin B may also actively inhibit parasite entry into macrophages by
altering cholesterol-rich membrane domains in the host cell, although this is likely
to be a minor mode of action (Chattopadhyay & Jafurulla, 2011). The original
deoxycholate form of amphotericin B was associated with severe adverse side
effects, including severe nephrotoxicity (Laniado-Laborín & Cabrales-Vargas,
2009), which are greatly reduced when delivered in a liposomal formulation,
Ambisome (liposomal-Amphotericin). Lipid-conjugated formulations allow more
effective targeting of infected tissues and host cells and can therefore be used at
lower concentrations (Sundar & Chakravarty, 2010). However, they require a cold
chain procedure and are more expensive (Balasegaram et al., 2012), which means
these treatment options are heavily dependent on subsidies from the government or
WHO for use in resource-poor settings.
The aminoglycoside, paromomycin, has been used to treat both VL and CL.
Similar to the antimonials and Amphotericin B, it has to be parenterally
administered; although topical formulations are available for treating CL (Sundar
& Chakravarty, 2008). Furthermore, clinical trials suggest that the efficacy of
paromomycin may be significantly lower than antimonials in certain endemic
regions, such as Sudan, Ethiopia, and Kenya (Hailu et al., 2010; Musa et al., 2012).
Resistance to paromomycin can arise quickly (Sundar & Chakravarty, 2013) and
its usage in combination therapies is recommended.
6
CHAPTER 1
Miltefosine, an alkyl phospholipid, was the first orally administered anti-leishmanial drug registered and approved for clinical use. Originally developed as an anticancer drug, miltefosine was discovered to have anti-leishmanial activity (Croft et al., 1987; Kuhlencord et al., 1992). The mode of action of miltefosine remains poorly defined, with possible targets including enzymes involved in lipid metabolism, cytochrome c oxidase, or other metabolic pathways in the mitochondria (Dorlo et al., 2012). As an oral agent, miltefosine has the clear advantage over other treatment options when it comes to patient acceptance. However, the drug is limited by the need to monitor gastrointestinal side effects and the occasional hepatotoxicity and nephrotoxicity (Sindermann &
Engel, 2006). Miltefosine is also foetotoxic and teratogenic and is administered with a contraceptive pill in women. Moreover, the long elimination half-life of miltefosine encourages the rapid development of drug resistance. Indeed, a decade after licencing, the efficacy of miltefosine treatment in India has declined, with relapse rates of more than 10 % in many areas (Sundar et al., 2012) and up to 20 %
(one year post-treatment) in neighbouring Nepal (Rijal et al., 2013).
A number of other treatment options have also been proposed, but each comes with its own draw-backs. Sitamaquine and azole compounds (ketoconazole, itraconazole, and fluconazole) are orally administrable drugs that have shown anti-leishmanial potential (Sundar & Chakravarty, 2013). However due to the limited clinical efficacies, their developments have since been postponed or withdrawn. Pentamidine was also once a promising alternative to treating refractory VL in India, but its usage has since been abandoned due to decreasing efficacy and life-threatening toxicities such as diabetes mellitus, and various cardiac and gastrointestinal side effects (Thakur et al., 1991; Sundar & Chakravarty,
7 37
CHAPTER 1
2013).
Various combination regimens involving the use of at least two drugs with
different mechanisms of action are now strongly advocated in order to combat the
rising number of drug-resistant parasites (Bryceson, 2001; van Griensven et al.,
2010; Sundar et al., 2011; Musa et al., 2012; Ponte-Sucre et al., 2017). However,
improving patient compliance and enforcing policies that tighten drug usage are
equally crucial (Sundar & Olliaro, 2007). With the number of obstacles and
set-backs currently faced in the development of vaccines against Leishmania
parasites (Kumar & Engwerda, 2014; Gillespie et al., 2016; Srivastava et al., 2016),
there is an urgent need to identify novel drug targets and expand current treatment
options.
1.4 Leishmania life cycle
Leishmania have a complex digenetic life cycle, involving sandfly vectors and
vertebrate hosts. During this life cycle, parasites alternate between two major
developmental stages and multiple physiological states, reflecting the need to adapt
to markedly different niches within each host (Figure 1.3).
Life within the Sandfly
Leishmania are transmitted by 30 species of sandflies belonging to the genus
Phlebotomus and Lutzomyia (Bates, 2007). Female sandflies become infected with
Leishmania when they take a blood meal from an infected vertebrate host. The
blood meal is initially encased in a peritrophic membrane in the abdominal midgut
of the sandfly, and parasites released from lysed cells differentiate to flagellated
8
CHAPTER 1
procyclic promastigotes, which replicate rapidly in the nutrient-rich milieu of the blood meal (Bates, 1994). Following the gradual breakdown of the peritrophic membrane, the massively expanded population of promastigotes differentiate to nectomonad promastigotes that are less replicative but highly motile (Rogers et al.,
2003). Nectomonads bind to epithelial cells that line the midgut, preventing their expulsion with the remains of the blood meal (Kamhawi, 2006). These stages subsequently migrate anteriorly, and upon reaching the stomodeal valve transform into leptomonad promastigotes, where they undergo a second stage of replication
(Gossage et al., 2003). Leptomonads also produce an extracellular gel-like substance rich in high molecular weight glycoproteins (Rogers et al., 2002) and subsequently differentiate into the highly-infective metacyclic promastigotes, which are pre-adapted for life in the vertebrate host. The accumulation of large parasite aggregates in the sandfly feeding apparatus, and the secretion of parasite chitinases, leads to damage of the stomodeal valve and alterations in sandfly feeding behaviour (Schlein et al., 1992; Volf et al., 2004; Bates, 2007; Rogers &
Bates, 2007). In particular, infected sandflies tend to probe the skin of their vertebrate host multiple times (Killick-Kendrick et al., 1977; Beach et al., 1984) and regurgitate large parasite aggregates (Rogers et al., 2004), resulting in increased parasite transmission.
Life within the Mammalian Host
The sandfly bite elicits a strong localised innate immune response, as a result of the introduction of metacyclic promastigotes, sandfly saliva and associated tissue damage in the upper dermal layer (Kamhawi, 2000; Rogers et al., 2004; Rohousová
& Volf, 2006). A wave of neutrophils are initially recruited to the site and actively
9 37
CHAPTER 1
phagocytose metacyclic promastigotes (Laskay et al., 2003; Peters et al., 2008).
Internalised promastigotes survive within the phagolysosomes of these host cells
but do not differentiate or divide. Neutrophils are short-lived cells and
subsequently undergo spontaneous apoptosis. The released parasites or infected
pre-apoptotic neutrophils are phagocytosed by the second wave of macrophages
that are recruited to the site of the bite, and are the definitive host cells of
Leishmania parasites (van Zandbergen et al., 2004). The abundance of apoptotic
debris in these tissues suppresses the pro-inflammatory responses of macrophages
that are required for effective clearing of intracellular Leishmania stages,
promoting the establishment of infection (van Zandbergen et al., 2007;
Ribeiro-Gomes & Sacks, 2012).
Following macrophage phagocytosis, the parasite-containing phagosomes
undergo maturation through fusion with lysosomes, resulting in the formation of
the mature phagolysosome or parasitophorous vacuole (PV) (Forestier et al., 2011).
The elevated temperature in the mammalian host and low pH of the macrophage
phagolysosome are sufficient to trigger promastigote differentiation into the non-
flagellated amastigotes that appear to be uniquely adapted for life in this
intracellular niche (Zilberstein & Shapira, 1994). The PV has all the hallmarks of
a mature phagolysosme, with a luminal pH of 5.5 and a full complement of
hydrolytic enzymes (proteases, lipases and glycosidases). While there is evidence
that amastigote molecules can modulate host cell signalling pathways or
microbicidal processes, such as the NADH-dependent phagosome oxidase,
amastigotes also appear to be intrinsically resistant to both hydrolases and reactive
oxygen species and minimally activate macrophages via classic antigen
presentation pathways (Kaye & Scott, 2011; Arango Duque & Descoteaux, 2015;
10
CHAPTER 1
Podinovskaia & Descoteaux, 2015). Recent studies have shown that amastigotes switch to a metabolically quiescent, slow growth state, but nonetheless continue to divide slowly, eventually resulting in tens to hundreds of amastigotes per host cell
(Kloehn et al., 2015). How amastigotes disseminate to new macrophages within the large tissue granuloma remains poorly defined. Uninfected macrophages recruited to these tissues may phagocytose parasites that have been released from apoptotic macrophages (Rai et al., 2017). Alternatively, there is evidence for direct transfer of phagosome-encased parasites from one macrophage to another (Real et al., 2014). Amastigotes can also be partitioned into daughter macrophages upon cell division (Kima, 2007). Regardless, parasite burdens in susceptible murine models of infection can reach >1010 parasites over several weeks. Finally, parasites in skin, either in the primary lesion or at disseminated sites of infection, are taken up by another sandfly thereby completing the life cycle.
11 37
CHAPTER 1
(5)
(6)
(4)
SANDFLY STAGE (7)
(3)
(2)
(8)
(1)
MAMMALIAN STAGE
(9)
(11) (10) Figure 1.3| Life cycle of Leishmania parasites. (1) Amastigotes are taken up when the sandfly takes a blood meal and (2) are delivered to the midgut, encased in peritrophic matrix (3) where they transform into procyclics and undergo first round of replication. (4) Procyclics transform into nectomonads and begin migration towards the foregut. (5) Upon reaching the stomodeal valve, the parasites transform into leptomonads, and undergo another round of replication. (6) The leptomonads then transform into metacyclic promastigotes and (7) are transmitted into human host by the infected sandfly. (8) The parasites are first engulfed by neutrophils, (9) and subsequently by macrophages recruited to the bite site. (10) Parasites taken up by the macrophage enter the phagolysosome and (11) differentiate into amastigotes to continue to proliferate. The cycle is completed when the infected macrophages are taken up by the sandfly during the next blood meal.
12
CHAPTER 1
1.5 The role of inositol lipids in Leishmania
All stages of Leishmania express a range of cell surface molecules that play an important role in protecting these parasite from host microbicidal processes in both sandfly and vertebrate hosts, and in mediating specific host-parasite interactions.
Work over several decades has highlighted several unusual properties of the
Leishmania cell surface, including the predominance of inositol lipids and glycosylated inositol lipids. These lipids function as bulk components of the plasma membrane and serve as membrane anchors for a range of surface glycoproteins and phosphoglycans that contribute to the formation of a complex surface glycocalyx. Given the importance of these lipids in facilitating Leishmania infection and their sometimes unique structures compared to mammalian cells, there has been considerable interest in targeting aspects of myo-inositol metabolism.
In the following sections, the structure and function of the major inositol lipids of
Leishmania are reviewed.
1.5.1 Phosphatidylinositol and inositol phosphorylceramide As in other eukaryotes, the major inositol lipid in Leishmania is phosphatidylinositol (PI). PI is synthesised by the transfer of myo-inositol to
CDP-diacylglycerol (CDP-DAG) by endoplasmic reticulum (ER) and/or
Golgi-localised PI synthase, and constitutes approximately 15 % of the total phospholipid pool of promastigotes (Wassef et al 1985). PI is thought to be predominantly located on the inner (cytoplasmic) leaflet of the plasma membrane and intracellular organelles, although a significant fraction may be transported into the lumen of the Golgi and converted to the sphinogolipid, inositol phosphorylceramide (IPC) (Denny et al., 2006). IPC is synthesised by IPC synthase
13 37
CHAPTER 1
that transfers the myo-inositol headgroup of PI to a ceramide lipid acceptor (Figure
1.4), and accounts for about 10 % of the total phospholipids in Leishmania, making
it an important bulk lipid (Kaneshiro et al., 1986). While, IPC is synthesised by
many protists, fungi and plants, it is not produced by animal cells, and thus
constitutes an important difference between Leishmania and their hosts (Zhang &
Beverley, 2010). IPC is thought to be primarily located in the outer leaflet of the
plasma membrane and may play a role in regulating the physical properties of the
Leishmania membrane and the formation of lipid rafts (Ralton et al., 2002). In other
eukaryotic cells, sphingolipids have also been shown to have important roles in
membrane trafficking, signal transduction, and regulation of cell growth and
survival (Hannun & Obeid, 2008; Gault et al., 2010; Merrill, 2011). IPC is
expressed in high levels by both promastigotes and amastigotes, and gene knockout
studies suggest that this lipid is important for amastigote differentiation.
Specifically, Leishmania Δspt2 mutants, which are unable to synthesise the
sphingoid bases (precursors for ceramide) and are therefore IPC-deficient, display
normal promastigote growth but showed defective vesicular trafficking in
stationary phase and subsequently failed to differentiate into the infective
metacyclic form (Zhang et al., 2003; 2005; Denny et al., 2004). Interestingly,
intracellular Δspt2 amastigotes are fully virulent in mice and still possess high
levels of IPC, suggesting they are able to acquire the IPC precursors from
macrophages (Zhang et al., 2005). Indeed, studies of Leishmania amastigotes
indicate they contain host-derived sphingolipids (McConville & Blackwell, 1991;
Schneider et al., 1993; Winter et al., 1994). The importance for Leishmania to
salvage sphingolipids from the mammalian host (in the form of sphingomyelin) is
further emphasised by the severely attenuated virulence observed in parasite
14
CHAPTER 1
mutants that lack neutral sphingomyelinase, which is responsible for the degradation of both host-derived sphingomyelin and parasite-derived IPC (Zhang et al., 2009; Pillai et al., 2012).
IPC synthase
PI Ceramide IPC Diacylglycerol
Figure 1.4|Biosynthesis of IPC from PI. The inositol phosphate headgroup (with myo-inositol highlighted in red) is transferred from PI to the ceramide moiety by the IPC synthase. This results in the production of IPC and a free diacylglycerol molecule.
1.5.2 GPI-associated glycoconjugates A second major class of inositol-lipid produced by Leishmania are the glycoinositol phospholipids (GIPLs) and related glycosylphosphatidylinositol
(GPI) glycolipid anchors (Figure 1.5). GIPLs are abundant surface components in both development stages and, based on estimates of headgroup size, are thought to effectively cover >75% of the plasma membrane to form a loosely linked glycocalyx (McConville & Bacic, 1989). Leishmania also express a number of
GPI-anchored proteins (gp63, PSA2), as well as the GPI-anchored lipophosphoglycan (LPG), which together form a conspicuous glycocalyx during
15 37
CHAPTER 1
promastigote stages that can be readily detected by electron microscopy (Orlandi
& Turco, 1987; McConville & Blackwell, 1991; McConville & Ferguson, 1993).
This repertoire of surface glycans is further extended by non-anchored
phosphoglycans (PG) and proteophosphoglycans (PPG) that form the extracellular
mucilage gels that allow promastigotes to form large aggregates (Ilg et al, 1994;
1996). Strikingly, promastigote-to-amastigote differentiation is associated with
decreased expression of all GPI-anchored glycoproteins, as well as massively
decreased expression of LPG. As a result, amastigotes have a much thinner surface
glycocalyx than promastigotes, comprised mainly of GIPLs, although intracellular
amastigotes can actively scavenge complex host-derived glycosphinogolipids
within the PV, which become incorporated into their surface membrane
(McConville & Blackwell, 1991; Winter et al., 1994; Naderer et al., 2004).
A number of studies have dissected the pathways for LPG, GPI, and GIPL
biosynthesis in Leishmania (McConville & Ferguson, 1993) and many of the steps
involved in the assembly of these glycolipids are similar to those in other
eukaryotes (McConville & Menon, 2000). These involve the sequential addition of
different sugars (N-acetylglucosamine and mannose) and ethanolamine phosphate
to a PI base (Figure 1.5). The pathway for LPG anchor biosynthesis deviate from
GPI protein anchor biosynthesis with the addition of the second mannose residue
in α1,3 instead of α1,6 linkage, followed by three galactose residues (Ralton &
McConville, 1998). Interestingly, GIPLs can have glycan backbones that contain
either α1,6-linked and/or α1,3 mannose residues to form three distinct lineages that
are structurally related to the protein anchors, the LPG anchors, or both anchor
classes (Ralton & McConville, 1998). The current understandings of the functions
that each of these GPI-associated glycoconjugates play during different stages of
16
CHAPTER 1
Leishmania life cycle are summarised below.
Type I Type II Hybrid GPI- type GIPL LPG anchored protein
Figure 1.5| Structures of GPI-anchored glycoconjugates in Leishmania. GPI-associated molecules form a major part of the surface glycocalyx in Leishmania parasites. The crucial myo-inositol linker is highlighted in red. In LPG, the phosphoglycan polymer is comprised of a series of Gal-Man-PO4 repeating units (n) that can be modified with branching units, and a variable terminating oligosaccharide cap (m).
Abbreviations used: EtN, ethanolamine; Gal, galactose (galactopyranose); Galf, galactofuranose; Glc, glucose; GlcN, N-acetylglucosamine; GIPL, glycoinositol phospholipid; GPI, glycosylphosphatidylinositol; LPG, lipophosphoglycan; Man, mannose; P, phosphate.
17 37
CHAPTER 1
Lipophosphoglycan (LPG)
LPG is the principal surface glycoconjugate of Leishmania promastigotes and is
composed of four distinct domains: a 1-O-alkyl-2-lyso-phosphatidyl(myo)inositol
lipid anchor, a glycan core, the disaccharide-phosphate polymer consisting of
Gal(β1,4)Man(α1)-PO4 repeating units, and a terminal oligosaccharide cap
(McConville et al., 1990; Ilg et al., 1992). While the GPI anchor (comprised of the
lipid anchor and glycan core) and the Gal-Man-PO4 backbone of the
phosphoglycan repeating units are conserved between all Leishmania species,
species- and stage-specific variations can occur in the number of these repeating
units, the extent to which they are modified with glycan side-chain extensions, as
well as alterations in the glycan cap structure (McConville et al., 1992; 1995;
Mahoney et al., 1999).
LPG is thought to perform a multitude of functions in the sandfly vector
(Sacks et al., 2000). Specifically, although mutant parasites lacking LPG are able
to initiate infection in their natural sandfly vector, they are rapidly cleared from the
midgut when the digested blood meal is excreted. LPG has been shown to serve as
the major parasite ligand for lectins on the surface of the sandfly midgut epithelium,
which allow promastigotes to bind to the epithelial wall and avoid expulsion.
Furthermore, structural polymorphisms in LPG structure appear to have evolved to
allow attachment to lectin receptors in different sandfly species, and underlie the
marked vector tropism or promiscuity shown by different Leishmania species
(Sacks et al., 1994). As infection progresses and the parasites transform into
metacyclic promastigotes, the number of disaccharide-phosphate repeat units in
LPG approximately doubles, while terminal side chain glycan epitopes are capped
with other sugars (McConville et al., 1992; Sacks et al., 1995; Mahoney et al.,
18
CHAPTER 1
1999). These modifications are thought to directly or indirectly (through conformation changes in LPG structure) mask epitopes recognised by the epithelial receptors, allowing the parasites to detach and migrate towards the mouth parts of the sandfly.
LPG also has a number of functions during early stages of mammalian infection. The long LPG chains on the surface of metacyclic promastigotes form the glycocalyx that inhibits the lytic attack of complement system by preventing the attachment and activation of complement components (Puentes et al., 1989;
1990; Späth et al., 2003). LPG is also recognised by multiple macrophage receptors which promote parasite internalisation by phagocytosis. This process involves the recognition of C3 complement bound to LPG by the complement receptors, CR1 and CR3, expressed on the surface of macrophage host (Mosser & Rosenthal, 1993).
More importantly, uptake via these receptors may reduce the activation of oxidative burst and inhibit IL-12 production, a key facilitator of cell-mediated immunity
(Descoteaux & Turco, 1999). Once the promastigotes are internalised by the macrophage, studies in L. donovani and L. major have shown that LPG delays the process of PV maturation during early stages of infection by preventing phagosome-endosome fusion (Desjardins & Descoteaux, 1997; Dermine et al.,
2000). This delay may provide promastigotes with enough time to differentiate into amastigotes and avoid being destroyed by elevated levels of oxidative and hydrolytic stress in the mature PV. LPG may also protect the parasites against oxidative damage by targeting nitric oxide production. An in vitro study of murine macrophages has demonstrated that LPG isolated from Leishmania promastigotes can act in a synergistic manner with interferon-γ to significantly reduce the expression of inducible nitric oxide synthase and consequently, nitric oxide
19 37
CHAPTER 1
generation (Proudfoot et al., 1996). Consistent with the view that LPG is important
in resistance to oxidative stress, LPG-deficient L. major promastigotes have been
shown to display increased sensitivity to killing by oxidative stress during
phagocytosis (Späth et al., 2003).
Interestingly, LPG does not appear to be essential for initial mammalian
infection in all species. For example, an L. mexicana LPG-deficient mutant, Δlpg1,
establishes an infection in macrophages and susceptible mice at the same rate as
wild type parasites (Ilg, 2000). Another L. mexicana mutant strain, Δlpg2, which
lacks the Golgi GDP-mannose transporter and is therefore unable to synthesise the
phosphoglycan repeating units, also remains infectious in both macrophages and
mice (Ig et al., 2001; Turco et al., 2001). In all species, LPG expression is greatly
down-regulated following amastigote differentiation suggesting that it is not
required once infection has been established (McConville & Blackwell, 1991;
Turco & Sacks, 1991; Bahr et al., 1993). Indeed, surviving L. major LPG-deficient
parasites, such as Δlpg1, generate normal lesions in mice after an initial delay,
while infection of mice with Δlpg1 amastigotes results in normal lesion
development without a delay (Späth et al., 2000).
GPI-anchored proteins
The most abundant glycoprotein found on the surface of Leishmania promastigotes
is GP63, a zinc metalloprotease tethered to the cell membrane by the GPI anchor
(Bouvier et al., 1985; 1989; Etges et al., 1986). The protein is thought to perform
a series of functions for promastigotes during early stages of mammalian infection.
GP63 is capable of cleaving C3 of the complement system (Chaudhuri & Chang,
1988), enhancing complement fixation and opsonisation but avoiding its lytic
20
CHAPTER 1
effects, thereby assisting the parasites in entering the macrophage host
(Brittingham et al., 1995). Evidence suggests that GP63 may also target other antimicrobial peptides produced by host macrophages (Kulkarni et al., 2006; Wang et al., 2011) and/or degrade components of the extracellular matrix, and help promote intermacrophage dissemination (McGwire et al., 2003).
Although GP63 is also expressed by Leishmania amastigotes, levels of expression are much lower, and the protein is either located in the lysosome or secreted (Medina-Acosta et al., 1989; Frommel et al., 1990; Schneider et al., 1992).
While intracellular pools of GP63 may have roles in protein degradation and nutrition, there is some evidence that secreted GP63 may be transported to the cytoplasm of the host cells, where it may target host proteins involved in regulating macrophage microbicidal responses (Olivier et al., 2005; Gomez et al., 2009;
Jaramillo et al., 2011). Recently, GP63 has been shown to stall phagosome maturation by hydrolysing the membrane fusion mediator, VAMP8, and inhibiting
LC3-associated phagocytosis (Matte et al., 2016). However L. major
GP63-deficient amastigotes remain virulent (Joshi et al. 1998), while a L. mexicana mutant completely devoid of all GPI-anchored proteins (due to the deletion of gene encoding the GPI transamidase) also retain infectivity in both macrophages and mice (Hilley et al., 2000). Therefore, the significance of GP63 in Leishmania amastigotes remains to be clearly defined.
The other major class of GPI-anchored proteins presented on the surface of
Leishmania parasites is the promastigote surface antigen-2 complex (PSA-2).
PSA-2 is a protein family comprised of antigenically similar polypeptides (Murray et al., 1989; Jiménez-Ruiz et al., 1998). These proteins are expressed by both
21 37
CHAPTER 1
promastigotes and amastigotes, but are particularly enriched in metacyclic
promastigotes (Handman et al., 1995; Beetham et al., 2003). PSA-2 may contribute
to promastigote resistance to complement lysis (Lincoln et al., 2004), and the
multiple leucine-rich repeats found in PSA-2 may interact with the CR3
complement receptor, aiding the parasites in attaching and subsequently entering
the macrophage host (Kedzierski et al., 2004).
Glycoinositol phospholipids (GIPL)
As outlined above, both major developmental stages of Leishmania express high
levels of GIPLs (McConville & Blackwell et al., 1991; Bahr et al., 1993). These
glycolipids are classified as either type-1, type-2 or hybrid GIPLs depending on
whether their glycan headgroups contain the same trimannose backbone as the
protein GPI anchor, the LPG GPI anchors, or branched glycans with both motifs
(Figure 1.5) (McConville & Blackwell et al., 1991; Schneider et al., 1994). GIPLs
form a protective glycocalyx over the surface of promastigotes and amastigotes
and confer resistance to lipases in the sandfly midgut and the phagolysosome of
macrophages, by virtue of containing ether-linked fatty chains (Naderer &
McConville, 2008). Type 1 and hybrid GIPLs with terminal mannose residues
could also act as ligands for mannose-binding lectin in the sandfly and on the
surface of macrophages (Wilson & Pearson, 1986; Schneider et al., 1994). In
addition, GIPLs have also been shown to modulate host signalling to suppress nitric
oxide generation and oxidative burst (Proudfoot et al., 1995; Tachado et al., 1997).
However, a number of genetic studies have suggested that GIPLs may not be
essential for virulence in animals. In particular, Garami et al. (2001) generated an
L. mexicana Δdpms mutant deficient in dolichol-phosphate mannose synthase,
22
CHAPTER 1
which exhibited selective defects in LPG, protein GPI anchor and GIPL biosynthesis. Unexpectedly, this mutant was found to remain infectious in both macrophages and mice, although with slightly lowered infectivity. A second L. mexicana mutant, termed DIG1, was generated by chemical mutagenesis of the phosphoglycan-deficient L. mexicana Δlpg2 strain, and was defective in both GPI and phosphoglycan biosynthesis (Naderer & McConville, 2002). The DIG1 mutant was still able to proliferate in macrophages, although at a slightly reduced rate
(Ralton et al., 2003). Finally, the L. major Δads1 mutant lacking the enzyme, alkyldihydroxyacetonephosphate synthase required for synthesising all ether lipids, including LPG and GIPLs, also remain infectious in mice, albeit with a delayed onset of cutaneous lesion formation (Zufferey et al., 2003). A closer examination by the authors found that the virulence of Δads1 closely resembled that of the
LPG-deficient Δlpg1 (Späth et al., 2000; 2003), indicating the loss of LPG, instead of GIPLs, may be responsible for the attenuated infectivity observed in Δads1.
Altogether, these findings suggest that the GIPLs are not essential for amastigote survival in the mammalian host, possibly because of compensatory uptake of host-derived glycosphingolipids. Indeed, endogenously synthesised and exogenously acquired sphinogolipids may be present at 10-fold higher levels than
GIPLs during amastigote stages, and appear to be essential for the intracellular survival of Leishmania parasites (Zhang et al., 2005; Zhang & Beverley, 2010).
23 37
CHAPTER 1
1.5.3 Phosphoinositides As in other eukaryotes, PI can also be phosphorylated in Leishmania to generate a
complex array of phosphoinositides and lipid-free inositol phosphates.
Phosphoinositides are generated by phosphorylation at three of the five free
hydroxyl groups on the myo-inositol headgroup (positions 3, 4, and 5), giving rise
to a total of seven possible phosphoinositide species (Figure 1.6A) (Falkenburger
et al., 2010). Despite comprising only about 1 % of the total phospholipids found
in the eukaryotic cell membrane, they participate in a multitude of biological
processes (Di Paolo & De Camilli, 2006). In recent years, various
phosphoinositide-specific probes have been introduced to assist in delineating the
spatiotemporally regulated production and turnover of phosphoinositides (Várnai
& Balla, 2006; Balla, 2007; Lemmon, 2008). These include fluorescent proteins
with phosphoinositide-binding domains that can be expressed in cells and used to
localise specific phosphoinositide species (Figure 1.6B). While the Leishmania
genome encodes all of the enzymes needed to make the full complement of
different phosphoinositides, very few of these lipid kinases, phosphatase and
lipases have been characterised in any detail and comparatively little is known
about the function of different phosphoinositides in these parasites. The following
sections highlights possible functions for these lipids, based on current
understanding of model systems (yeast and mammals).
24
CHAPTER 1
(A)
(B)
Figure 1.6| Phosphoinositide metabolism and subcellular distribution. (A). Phosphoinositides are generated from PI through phosphorylation of the myo- inositol headgroup at 3, 4, and/or 5-OH positions. All possible reactions based on eukaryote models for the synthesis and degradation of phosphoinositide species are shown. Reactions currently known to be possible in Leishmania are indicated by solid lines. (B) Subcellular distribution of phosphoinositide species based on yeast and mammalian models. The predominant species in each organelle are indicated in bold. Schematics adapted from Di Paolo & De Camilli (2006).
Abbreviations used: DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI4P, phosphatidylinositol 4-phosphate; PI5P, phosphatidylinositol 5-phosphate;
PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,5)P2, phosphatidylinositol
3,5-bisphosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate.
25 37
CHAPTER 1
PI(4,5)P2
Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is perhaps the most-studied
species. It is one of the most abundant phosphoinositide species found in
eukaryotes and is localised to the inner leaflet of the plasma membrane. PI(4,5)P2
interacts with a number of effector molecules to regulate a multitude of cellular
processes that occur in proximity of the plasma membrane, such as actin
polymerisation for anchorage of plasma membrane and vesicular structures,
assembly/disassembly of vesicular coatings, formation of endocytic vesicles,
regulation of exocytosis and secretion, and operation and turnover of membrane
ion channels and transporters (Toker, 1998; Takenawa & Itoh, 2001; Roth, 2004;
Schink et al., 2016). PI(4,5)P2 is synthesised by the enzyme PI 5-kinase (PI5K)
from phosphatidylinositol 4-phosphate (PI4P), and is itself the substrate for two
receptor-stimulated enzymes. The first enzyme, Class I PI 3-kinase (PI3K), is
responsible for converting PI(4,5)P2 to phosphatidylinositol 3,4,5-trisphsophate
[PI(3,4,5)P3] (discussed in more details in the section below). The second is
phospholipase C (PLC), which, upon stimulation by a wide range of membrane
receptors, cleaves the phospholipid to produce two second messengers,
diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) (Rhee, 2001; Fukami
2+ et al., 2010). The soluble IP3 triggers the release of Ca from intracellular storage
2+ by binding to IP3 receptors and the increase in cytosolic Ca levels can
subsequently regulate a number of Ca2+-sensitive proteins, including PLC itself.
Conversely, the lipid DAG is recognised by proteins such as protein kinase C (PKC)
and results in their activation (Newton, 1995).
In Leishmania, the downstream actions of both IP3 and DAG have been
implicated in parasite growth and infectivity (Alvarez-Rueda et al., 2009;
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Docampo & Huang, 2015). The PI5K responsible for generating PI(4,5)P2 is also found to be essential in the related trypanosomatid, Trypanosoma brucei (Cestari
& Stuart, 2015). However, unlike yeast and mammalian cells, the IP3 receptors in
Leishmania and other trypanosomatids are mainly found on the acidocalciosomes instead of the ER, suggesting differences in the mechanism by which IP3 may modulate Ca2+ fluxes in Leishmania compared to animal cells (Huang et al, 2013;
Docampo & Huang, 2015).
PI(3,4,5)P3 and PI(3,4)P2
As mentioned above, PI(3,4,5)P3 is synthesised by Class I PI3K through the phosphorylation of PI(4,5)P2. In mammalian cells, this can occur upon the activation of a number of growth factor receptors (Cantley, 2002). PI(3,4,5)P3 itself is a substrate for 5-phosphatase, which hydrolyses the phosphate group at position
5 to generate phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]. Similar to
PI(4,5)P2, the majority of PI(3,4,5)P3 is localised to the plasma membrane; whereas
PI(3,4)P2 is formed at the plasma membrane, as well as the early endocytic pathway
(Watt et al., 2003; Ivetac, et al., 2005). PI(3,4,5)P3 is an important messenger molecule that recruits protein kinases to the plasma membrane and is part of a complex signalling network that regulates cell growth and proliferation, membrane dynamics, and intracellular vesicular transport (Cantley, 2002, Vanhaesebroeck et al., 2010; Riehle et al., 2013). On the other hand, PI(3,4)P2 was once thought to be a catabolic product generated during PI(3,4,5)P3 degradation. However, recent evidence suggests that it is also likely to play a prominent role in a number of membrane and cytoskeletal responses associated with the PI 3-kinase pathway, either in a co-ordinated fashion with PI(3,4,5)P3, or by exerting its own distinct
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signalling effects (Hawkins & Stephens, 2016).
Very little information is available regarding the roles of PI(3,4,5)P3 and
PI(3,4)P2 in Leishmania and other protozoa. However, a putative Class I PI3K and
5-phosphatase have both been annotated on the Leishmania genome (Figure 1.6A).
The latter is essential for T. brucei (Cestari & Stuart, 2015), which suggest these
phosphoinositides are likely to have equally important functions in these parasites.
PI4P
Phosphatidylinositol 4-phosphate (PI4P) is another prominent phosphoinositide
species in eukaryotic cells, usually present at a level comparable to that of PI(4,5)P2.
It was originally thought to be present only on the trans-Golgi network (TGN) and
the associated secretory vesicles, but significant pools of PI4P can also occur in the
plasma membrane (Balla et al., 2005). PI4P can be generated either from the
phosphorylation of PI by PI 4-kinase (PI4K), or from the degradation of PI(4,5)P2
by 5-phosphatase. One of the main functions of PI4P in the TGN is to co-ordinate
the formation of clathrin-coated pits during vesicular budding by interacting with
cytoplasmic proteins of specific-lipid binding domains (Dowler et al., 2000; Wang
et al., 2003; De Matteis et al., 2005). In contrast, PI4P in the plasma membrane is
viewed as an intermediate for PI(4,5)P2 production or as an cycling intermediate
between PI5K and 5-phosphatase activities. This has recently been challenged by
studies which indicate that PI4P may be important in supporting functions
previously attributed to PI(4,5)P2, including stabilising ion channels and anchoring
of lipid-specific proteins (Hammond et al., 2012).
Leishmania and Trypanosoma contain two putative PI4K genes which might
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be involved in synthesising PI4P (Figure 1.6A). Knock-down of one of these kinases in T. brucei by RNA interference (RNAi) results in Golgi distortion, mislocalisation of lysosomal and flagellar pocket proteins, and irregular cell morphology (Rodgers et al., 2007), suggesting that PI4P is required for normal
Golgi and vesicular functions in these parasites, as in other eukaryotes.
PI3P and PI(3,5)P2
Phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol
3,5-bisphosphate [PI(3,5)P2] are two phosphoinositide species that are predominantly associated with the endo-lysosome system; with PI3P forming at the early endosomes, and PI(3,5)P2 being converted from PI3P at regions of the sorting endosomes that are destined to become the luminal inside the multivesicular bodies (Gillooly et al., 2000; Roth, 2004). PI3P is synthesised from PI by the Class
III PI3K (and by Class II PI3K in higher eukaryotes) and is subsequently converted to PI(3,5)P2 by Class III PI 5-kinase (PI5K). PI3P controls several aspects of endosomal biology by recruiting a whole set of regulatory proteins, many of which contain the FYVE domain (Stenmark et al., 1996). These cellular processes include endosomal membrane fusion, sorting of endocytic cargoes for recycling or degradation, targeting newly synthesised proteins to the lysosomes as well as protein retrieval, and regulating autophagosome formation (Simonsen et al., 1998;
Gillooly et al., 2001; Obara et al., 2008; Schink et al., 2013).
On the other hand, while PI(3,5)P2 is considerably less abundant, studies in yeast, plant, and mammalian cells have highlighted the importance of this phosphoinositide species in maintaining cellular homeostasis in response to extracellular stimuli or environmental shock (Dove et al., 2009; Jin et al., 2016).
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This lipid is also required for normal endosomal/lysosomal functions, as well as
regulating autophagy in response to nutrient starvation (Rutherford; 2006; Jin et al.,
2014), however, the underlying mechanism remains to be elucidated.
In Leishmania, a putative Class III PI3K and PI5K have both been annotated
(Figure 1.6A). Moreoever, RNAi knock-down of PI3K in T brucei indicate that
PI3K is essential in these parasites (Hall et al, 2006).
1.6 myo-Inositol metabolism in Leishmania
Leishmania and other eukaryotes are entirely dependent on de novo synthesis or
extracellular salvage of myo-inositol for the synthesis of inositol lipids (Reynolds,
2009). A number of studies have shown that Leishmania and other trypanosomatids
constitutively express the enzymes needed for de novo myo-inositol synthesis, as
well as dedicated myo-inositol transporters. These are discussed in more detail
below.
1.6.1 myo-inositol salvage versus biosynthesis myo-Inositol levels in the sandfly midgut and mammalian macrophage are
expected to differ substantially, and it is likely that Leishmania obtains this
essential substrate from both endogenous and exogenous sources during its life
cycle. Within the sandfly midgut, the blood meal would initially contain
approximately 35 µM myo-inositol (Leung et al., 2013), which provides a sufficient
source during differentiation of amastigotes to promastigotes and early rapid
growth within the peritrophic membrane. While these pools of exogenous
myo-inositol may be rapidly depleted, subsequent sugar meals by the sandfly
(comprising plant sap and aphid honeydew) are likely to provide sustained levels
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CHAPTER 1
of myo-inositol and other sugars (Auclair, 1963; Sabri et al., 2013). On the other hand, the nutrient composition of the macrophage phagolysosome remains poorly defined. The highly dynamic nature of the compartment means various nutrients may be delivered via multiple vesicular trafficking pathways, including endosomes, phagosomes, autophagosomes, as well as the ER-Golgi network (Figure 1.7)
(Veras et al., 1994; Schaible et al., 1999; Burchmore & Barrett, 2001; Ndjamen et al., 2010). However, phenotypic analysis of Leishmania mutants with defects in gluconeogenesis, suggest that this niche may be relatively sugar poor (Naderer et al 2006). Nonetheless, intracellular amastigotes are dependent on sugars as major carbon sources, suggesting that levels of at least some sugars are sufficient to sustain the slow growth rates of these stages (Burchmore et al., 2003; Saunders et al., 2018).
Parasite uptake of myo-inositol in each of these host niches is mediated by a myo-inositol/H+ symporter, which transports myo-inositol via a proton gradient
(Drew et al., 1995). The transporter system has a high substrate specificity and kinetics analyses suggest it is likely to be active at neutral and acidic pH in both sandfly and mammalian environments (Mongan et al., 2004). Significantly, the surface expression of this transporter is regulated in response to intracellular myo-inositol levels, as well as in a stage-dependent matter (Seyfang & Landfear,
1999; Vince et al., 2011).
Alternatively, Leishmania constitutively express the enzymes needed to catalyse the 2-step pathway of de novo myo-inositol synthesis. This pathway involves the conversion of glucose 6-phosphate to myo-inositol 3-phosphate, and subsequent dephosphorylation of the latter to myo-inositol, catalysed by
31 37
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myo-inositol 3-phosphate synthase (INO1) and myo-inositol monophosphatase
(IMPase), respectively. In an early study, Ilg (2002) generated an L. mexicana
∆ino1 mutant that exhibited expected myo-inositol auxotrophy in vitro. The ∆ino1
mutant exhibited attenuated infectivity in susceptible mice, although
complementation of the loss-of-virulence phenotype could not be demonstrated
following ectopic expression of wild type ino1 gene.
While nothing is known about the function or role of the Leishmania IMPase,
recent studies in T. brucei showed that these parasites contained two isoforms of
IMPase, TbIMPase1 and TbIMPase2 (Cestari & Stuart, 2015; Cestari et al., 2016).
Surprisingly, neither of these genes were shown to be essential when deleted
individually, although the TbIMPase2-null mutant displayed an attenuated growth
during in vivo mouse infection. These studies suggest that the two IMPase proteins
may have redundant function in T. brucei and/or that T. brucei can salvage most of
its myo-inositol needs from the host.
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(A)
(C)
(D) Carbohydrates Amino acids (B) Lipids Vitamins Nucleotides Fe2+
Figure 1.7| Nutrient availability inside the Leishmania parasitophorous vacuole. Macromolecules and small metabolites can be delivered to the phagolysosomal compartment following fusion with phagosomes, late endosomes (A), and autophagosomes (B). Host material may also be delivered directly from the ER- Golgi complex (C), while small metabolites and essential ions can be convey to the intracellular niche via transporters (D). Schematics adapted from Naderer & McConville (2008).
1.6.2 Compartmentalisation of inositol lipid synthesis in protozoan parasites
Intriguingly, gene deletion and conditional knock-down studies suggest that the T. brucei INO1 is essential for parasite survival even in the presence of exogenous myo-inositol (Martin & Smith, 2005; 2006a). This unexpected finding suggested that the INO1 pathway may have functions other than the synthesis of myo-inositol and/or that the products of de novo synthesis are compartmentalised in different ways from intracellular myo-inositol derived from exogenous pools. Using a
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conditional double knockout mutant and radiolabelled substrates, Martin & Smith
showed that the knock-down of T. brucei ino1 resulted in a significant reduction in
the synthesis of GPI glycolipids, while having no effect on the cellular levels of
bulk PI. These studies suggested that de novo synthesised myo-inositol is
preferentially channelled into a subpool of PI that is subsequently used for GPI
biosynthesis. Consistent with this model, early studies had shown that metabolic
labelling of T. brucei bloodstream forms with [3H]-myo-inositol results in efficient
labelling of bulk PI but not of GPI glycolipids. Conversely, knock-down of the
myo-inositol transporter in T. brucei bloodstream forms in the presence of
exogenous glucose, resulted in a defect in bulk PI synthesis, but no defect in GPI
biosynthesis (Gonzalez-Salgado et al., 2012). Based on these findings, it was
proposed that de novo synthesised myo-inositol is targeted to the ER, where it is
used to make a pool of ER-located PI that is used by UDP-GlcNAc-dependent
N-acetyl-glucosamine:PI transferase, the first committed enzyme in GPI
biosynthesis (Figure 1.8). On the other hand, the cytoplasmic pools of myo-inositol
(sourced primarily from exogenous myo-inositol under normal growth conditions)
is used to generate bulk PI by a Golgi-located PI synthase (Figure 1.8). This model
of compartmentalised myo-inositol synthesis and pathway channelling is supported
by reports that in T. brucei bloodstream forms, PI synthase is localised to both the
ER and Golgi complex (Martin & Smith, 2006b), and that the myo-inositol
transporter is also targeted to the Golgi apparatus, in addition to the plasma
membrane (Gonzalez-Salgado et al., 2012). Interestingly, evidence for the
channelling of endogenous myo-inositol into GPI anchor biosynthesis has since
been obtained in Plasmodium falciparum (Macrae et al., 2014).
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CHAPTER 1
Figure 1.8| Hypothetical model for the metabolic compartmentalisation of myo- inositol-containing lipids in T. brucei and P. falciparum. De novo myo-inositol is directed into the ER to be utilised by PI synthase for the synthesis of PI that is subsequently used for GPI production. On the other hand, exogenous myo- inositol is directed into the Golgi and only used to synthesise bulk PI. Schematics adapted from Macrae et al. (2014). Abbreviation used: Glc, glucose; Glc6P, glucose 6-phosphate; GlcN, glucosamine; GlcNAc, N-acetyl glucosamine; IMPase, myo-inositol monophosphatase; Ino, myo-inositol; INO1, myo-inositol 3- phosphate synthase; Ino3P, myo-inositol 3-phosphate; PI, phosphatidylinositol; PIS, PI synthase; PPP, pentose phosphate pathway.
1.7 Project rationale and thesis aims
Throughout its digenetic life cycle, Leishmania resides in vastly different environments and simultaneously undergoes dramatic morphological transformations. Consequently, the parasite must be proficient in adapting and regulating its metabolism in order to survive and exert its virulence. Uncovering these metabolic mechanisms not only will contribute towards our understanding of
Leishmania and the unique environments it inhabits, it is also paramount for our quest towards discovering potential novel drug targets.
Previous studies have highlighted the crucial roles that various
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CHAPTER 1
myo-inositol-containing molecules play during the course of the Leishmania life
cycle. The early study by Ilg (2002) suggested that de novo myo-inositol
biosynthesis may be essential for parasite virulence, although it was acknowledged
that further work was required to validate these findings as the phenotype of the
mutant could not be genetically complemented. The major aim of the current study
is to further investigate the consequences of disrupting de novo myo-inositol
synthesis in Leishmania, in order to validate this pathway as a potential drug target.
Chapters three and four investigate the role of the enzyme INO1, its role in
mammalian infection, and the metabolic consequences of myo-inositol starvation.
Chapter five describes the two L. mexicana IMPase isoforms, IMP1 and IMP2, and
investigates the role of IMP1 in parasite virulence. Finally, the function of Δimp1
is further explored in Chapter six.
36
CHAPTER 2 Materials and Methods
2.1 Molecular biology
2.1.1 Bioinformatic analysis
Nucleotide and protein sequences were sourced from the National Center for
Biotechnology Information (NCBI) online database (www.ncbi.nlm.nih.gov) or
TriTrypDB (tritrypdb.org). Multiple protein sequence alignments were performed using UniProt (www.uniprot.org) in conjunction with the Clustral Omega programme. Additional sequence alignments were performed by BLAST
(blast.ncbi.nlm.nih.gov/BlastAlign.cgi).
Protein localisation predictions were performed using the web-based servers,
PSORTII (psort.hgc.jp) and SignalP 4.0 (www.cbs.dtu.dk/services/SignalP-4.0).
Predictions of transmembrane regions were carried out using the servers, Phobius
(phobius.sbc.su.se), CCTOP (cctop.enzim.ttk.mta.hu), DAS-TMfilter
(mendel.imp.ac.at/DAS), and TMHMM (www.cbs.dtu.dk/services/TMHMM).
2.1.2 Primer and vector design
OLIGO 7 primer analysis software (Molecular Biology Insights) and the online Tm
Calculator (tmcalculator.neb.com, New England Biolabs) were used to assist primer design. All vector constructs engineered in this thesis were designed with the help of Clone Manager (Sci-Ed), SnapGene Viewer (GSL Biotech), and
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NEBcutter V2.0 (nc2.neb.com/NEBcutter2, New England Biolabs).
2.1.3 Genomic DNA extraction
Leishmania genomic DNA was isolated using DNAzol (Invitrogen), following a
protocol adapted from the manufacturer’s guidelines. Briefly, 1 mL of late-log
phase cells were pelleted by centrifugation (10,000×g, RT, 1 min) and supernatant
removed. The pellet was resuspended in 400 μL DNAzol and incubated at RT for
3 min before the addition of 200 μL absolute ethanol, followed by mixing and
further incubation at RT for 5 min to precipitate DNA. The DNA was then pelleted
by centrifugation (15,000×g, 0 °C, 10 min) and washed with 500 μL 75 % ethanol
(v/v in Milli-Q dH2O). Following centrifugation (15,000×g, 0 °C, 5 min), the
supernatant was aspirated and the pellet was dried in a rotational vacuum
concentrator (Martin Christ) at 38 °C for10 min. Finally, the DNA pellet was
resuspended in Milli-Q dH2O and the concentration determined by a NanoDrop
spectrophotometer (Thermo Fisher Scientific).
2.1.4 Polymerase chain reaction (PCR)
All PCR reactions for cloning were performed with Phusion High-Fidelity DNA
polymerase (New England Biolabs), while general PCR for confirmation and
verification were performed with Mango Taq DNA polymerase (Bioline).
A standard 25 μL Phusion reaction mix consisted of the following: 1× reaction
buffer, 200 μM dNTPs, 0.5 μM each of forward and reverse primer, 10 ng DNA
template, 0.3 units of Phusion DNA polymerase, and made up to volume with
Milli-Q dH2O.
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A standard 25 μL Mango Taq reaction mix consisted of the following: 5 μL
1× reaction buffer, 2 mM MgCl2, 200 μM dNTPs, 0.2 μM each of forward and reverse primer, 10 ng DNA template, 0.75 units of Mango Taq DNA polymerase, and made up to volume with Milli-Q dH2O.
PCR reactions were performed using a Mastercycler Pro S thermal cycler
(Eppendorf). For Phusion reactions, the cycle consisted of initial denaturation (98
°C, 2 min), followed by 35 cycles of denaturation (98 °C, 1 min), annealing (primer specific temperature for 45 s), and extension (72 °C, with time adjusted for product size), and finished with a final extension (72 °C, 5 min).
For Mango Taq reactions, the cycle consisted of initial denaturation (96 °C, 2 min), followed by 35 cycles of denaturation (96 °C, 1 min), annealing (57 °C , 45 s), and extension (72 °C, 1 min), and finished with a final extension (72 °C, 5 min).
2.1.5 DNA sequencing
100 ng of DNA per kb of template and 0.083 μM sequencing primer were made up in a 12 μL sample with Milli-Q dH2O. Sanger sequencing of the DNA samples was provided by either the Centre for Translational Pathology at University of
Melbourne, or the Australian Genome Research Facility.
2.1.6 Generation of GFP fusion constructs
To generate GFP fusion products for expression in L. mexicana, the Leishmania
GFP fusion vector, pXG-GFP+2’ (N-terminal GFP) or pXG-’GFP+ (C-terminal
GFP) were used (Ha et al., 1996).
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Table 2.1|PCR primer pairs for GFP fusion constructs. The engineered restriction sites are underlined.
Product Primer sequence Size (bp) INO1 N-terminal ACGAGATCTCCACCATGCCGGCAGTGCACGTG 1598 GFP TTACAATTGTCACTTGCCGTTGACGTGCT INO1 C-terminal CGAGGATCCATGCCGGCAGTGCACGTG 1590 GFP GCTCAGATATCCTTGCCGTTGACGTGCTCCT IMP1 N-terminal ACGAGATCTCCACCATGACGCAGCCCTCCCT 884 GFP TTACAATTGTCAAGATGACGTGTTCAACACC IMP1 C-terminal CGAGGATCCATGACGCAGCCCTCCCT 876 GFP GCTCAGATATCAGATGACGTGTTCAACACCGA IMP2 N-terminal CGAGGATCCACCATGAGCATCAGCGTCCTGT 1433 GFP TTACAATTGTCACTCGCTCTCGACCAA IMP2 C-terminal CGAGGATCCACCATGAGCATCAGCGTCCTGT 1425 GFP GCTCAGATATCCTCGCTCTCGACCAACCA
Each PCR product (Table 2.1) was purified using the ISOLATE II PCR and
gel kit (Bioline), digested with the corresponding restriction enzymes, then ligated
into the digested vector using T4 DNA ligase (New England Biolabs) as per
manufacturer’s instructions. The ligated product was transformed into Escherichia
coli XL1-Blue competent cells (see Section 2.2.2) and individual clones were
screened for the presence of the ORF by colony PCR. The selected clone was
propagated in LB broth (see Section 2.2.1) and the plasmid DNA was extracted
using the ISOLATE II plasmid mini kit (Bioline) as per manufacturer’s instructions.
Correct insertion of the gene of interest to fusion vector construct was confirmed
by sequencing using primers 516 and 690 for N-terminal GFP, or 450 and 71 for
C-terminal GFP (Table 2.2).
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CHAPTER 2
Table 2.2|Sequencing primers for GFP, HA, and knockout constructs.
Primer Sequence Target 450 GCGTGCACATCATCAACTGT 5’ of MCS of pX and pxG1A 690 GACGGTATCCTGCTGCACC 3’ of MCS of pXG1A T7 GTAATACGACTCACTATAGGGC 5’ of MCS of pBlueScript M13 GGAAACAGCTATGACCATG 3’ of MCS of pBlueScript and pX 516 GCTCGCCGACCACTAC N-terminal GFP forward 71 TGCGGTTCACCAGGGTGTCG C-terminal GFP reverse 667 CGTGGGCTTGTACTCGGTC Puromycin reverse 1332 GCACTGGTCAACTTGGCC Bleocin reverse
2.1.7 Generation of HA fusion constructs
For C-terminal triple haemagglutinin (3×HA) tagging, a pX-’3×HA vector was used (Mullin et al., 2001). For N-terminal 3×HA tagging, a pXG-3×HA’ vector construct was generated by PCR amplifying the 3×HA segment from pX-’3×HA using primers in Table 2.3, purified and digested with XmaI and BamHI, then cloned into the XmaI/BamHI sites of pXG1A vector (Ha et al., 1996). Following transformation into XL1-Blue (see Section 2.2.2), clones were isolated and screened for HA insert by colony PCR. Correct insertion of 3×HA was confirmed by sequencing using primer 450 (Table 2.2).
Similar to Section 2.1.6, the desired ORF was PCR-amplified (Table 2.3), digested with restriction enzymes, cloned into the digested pX-’3×HA or pXG-3×HA’ vector, before selection of the desired plasmid construct. Correct insertion of the ORF in frame with the 3×HA epitope tag was verified by sequencing using primers 450 and 690 for N-terminal 3×HA, or 450 and M13 for
C-terminal 3×HA (Table 2.2).
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CHAPTER 2
Table 2.3| PCR primer pairs for HA fusion constructs. The engineered restriction sites are underlined.
Product Primer sequence Size (bp) ATCCCGGGATGTACCCGTACGACGTCCC N-terminal 3×HA 107 ATTGGATCCTCTAGAGGCGTAGTCCGGGACGT IMP1 N-terminal GCTCTAGAATGACGCAGCCCTCCCT 884 3×HA CGAGGATCCTCAAGATGACGTGTTCAACACC IMP1 C-terminal CTACCCGGGATGACGCAGCCCTCCCT 882 3×HA CGAGGATCCAGATGACGTGTTCAACACCGA IMP2 N-terminal GCTCTAGAACCATGAGCATCAGCGTCCTGT 1433 3×HA TTACAATTGTCACTCGCTCTCGACCAA IMP2 C-terminal CTACCCGGGATGAGCATCAGCGTCCTGT 1431 3×HA CGAGGATCCCTCGCTCTCGACCAACCA
2.1.8 Generation of linear targeting constructs for gene deletion
Targeted gene deletion was carried out via sequential homologous gene
replacement using linear DNA constructs containing drug resistance cassettes as
described previously (Cruz et al., 1991; Sansom et al., 2013). A 5’ untranslated
region (UTR) fragment containing a 5’ HindIII site and a 3’ BamHI/EcoRI/linker
region and a 3’ UTR fragment containing a 5’ BamHI/EcoRI/linker region and a 3’
XbaI site were PCR-amplified using primers listed in Table 2.4. Overlap extension
PCR was then performed using these PCR products as template, and the resulting
product was gel purified and cloned into the HindIII/XbaI sites of pBlueScript II
SK(+) (Stratagene). Correct insertion was confirmed by sequencing with primers
T7 and M13 (Table 2.2). pXG-PAC or PXG-PHLEO vectors (Freedman &
Beverley, 1993) were then digested with BamHI and EcoRI, and the 2339 and 2046
bp fragments, corresponding to puromycin (PURO) and bleomycin (BLEO) drug
resistance cassettes, respectively, were cloned into the BamHI/EcoRI sites of the
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CHAPTER 2
deletion targeting construct. Correct insertion of the resistance cassette was verified by sequencing with primer 667 for PURO and 1332 for BLEO (Table 2.2).
For gene deletion experiments, the targeting construct was digested with
HindIII and XbaI, and the gel-purified linear DNA construct was transfected into
L. mexicana promastigotes by electroporation (Section 2.3.3).
Table 2.4| PCR primers for linear knockout constructs. Each UTR construct contains a BamHI/EcoRI/linker region for subsequent overlap extension PCR. The engineered restriction sites are underlined.
Product Primer sequence Size (bp) INO1 CCCAAGCTTCATTGTCCATCGCTAGAACCTC 1021 5’UTR CCGCTGGGATCCGAATTCTAGAAACTGGTGTGCGTGCTT INO1 GAATTCGGATCCCAGCGGTACTCCCTTGACAACCCCTT 1057 3’UTR ATGCGGCCGCTGTGTATGTCGTCGATTTGC IMP1 CCCAAGCTTCGATGCCGCTGTTACGAT 1043 5’UTR CCGCTGGGATCCGAATTCCAGAGCAACCACTGTATTGGGAG IMP1 GAATTCGGATCCCAGCGGCTGCTCTTCCTCCGTCTCTG 991 3’UTR GCTCTAGAGAAGTTCGCAACAAACGGAT
2.1.9 Generation of episomal complementation constructs
To reintroduce the gene of interest into L. mexicana deletion mutants, the pXG1A
Leishmania expression vector was used (Ha et al., 1996). Similar to Section 2.1.6, the ORF of the gene of interest was PCR-amplified using primers listed in Table
2.5, purified, digested with restriction enzymes, and cloned into pXG1A.
Constructs were then verified by sequencing using primers 450 and 690 (Table
2.2).
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CHAPTER 2
Table 2.5| PCR primers for episomal complementation constructs. The engineered restriction sites are underlined.
Product Primer sequence Size (bp) CTACCCGGGATGCCGGCAGTGCACGTG INO1 ORF 1599 CGAGGATCCTCACTTGCCGTTGACGTGCT CTACCCGGGATGACGCAGCCCTCCCT IMP1 ORF 885 CGAGGATCCTCAAGATGACGTGTTCAACACC
2.1.10 Generation of phosphoinositide-specific GFP probes
Construction of episomally-expressed GFP affinity probes that target specific
phosphoinositide species was based on the concept of fusing GFP with protein
domains that interact with these molecules (Balla, 2007; Lemmon, 2008). The
vector construct pX-GFP-2×FYVE was previously generated in our lab by Dr. Judy
Callaghan for targeting phosphatidylinositol 3-phosphate (PI3P). In brief,
GFP-2×FYVE fragment was PCR amplified from pGEM-GFP-2×FYVE (Gillooly
et al., 2000) using primers listed in Table 2.6, purified, digested with
corresponding restriction enzymes, and cloned into Leishmania pX expression
plasmid (LeBowitz et al, 1990).
In this work, OSBP PH domain for targeting phosphatidylinositol 4-phosphate
(PI4P) was PCR-amplified from vector pRS406-PHO5-GFP-OSBP PH (Levine &
Munro, 1998), and PLCD1 PH domain for targeting phosphatidylinositol 4,5-
bisphosphate [PI(4,5)P2] was amplified from vector PH-PLCD1-GFP (Varnai &
Balla, 1998), using primers listed in Table 2.6. The fragments were purified,
digested, and cloned into pXG-GFP+2’ and pXG-’GFP+, respectively, as per
Section 2.1.6.
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CHAPTER 2
Table 2.6| PCR primers for phosphoinositide-specific GFP fusion probes. The engineered restriction sites are underlined.
Product Primer sequence Size (bp) CCGTGATCACATGGTGAGCAAGGGCGA GFP-2×FYVE 1264 CGGTCTAGAGTCGACTTATGCCTTCTTGTT TTAGGATCCACCTCGGGCTCGGCTCGAGAC OSBP PH 330 TTACAATTGTCACGAATTCTTCTTCACAGCTTTGG CGAGGATCCATGGACTCGGGCCGGGAC PLCD1 PH 535 TGTCAGATATCCTTCAGGAAGTTCTGCAGCTCCTT
2.1.11 Whole cell lysate protein extraction and quantification
Approximately 2×107 L. mexicana cells were harvested from culture and washed once with sterile phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4). Cells were resuspended in 60 µL RIPA buffer supplemented with cOmplete EDTA-free protease inhibitor (Roche) and incubated on ice for 30 min. Cell lysates were centrifuged (16,000×g, 4 ˚C, 15 min) and the clarified supernatant transferred to new microfuge tubes. Protein concentration of the cell lysates were then determined by Bradford colorimetric assay against BSA standards (see Appendix 1). In brief, the clarified cell lysates were diluted by 1 in 10 and 10 µL of the samples in duplicates were transferred to a clear flat-bottom 96-well plate where BSA standards had been set up in duplicates.
200 µL Bradford reagent (Bio-Rad) was then aliquoted to each sample, thoroughly mixed and incubated at RT for 5 min, before absorbance at 595 nm was measured by the plate reader (Biotek). The protein concentrations of the undiluted cell lysates were determined based on the BSA standard curve generated.
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2.1.12 SDS-PAGE and immunoblotting
Reduced sample buffer [ at 1×: 62.5 mM Tris, 10 % (v/v) glycerol, 2% (w/v) SDS,
0.01 % (w/v) bromophenol blue, 50 mM dithiothreitol, pH 6.8] was added to the
cell protein lysates of equivalent protein amount (see Section 2.1.11), and the
samples were boiled for 5 min in a water bath, followed rapid cooling on ice. 20
µg protein-equivalent samples of cell lysates were analysed by sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using “Any kD”
polyacrylamide gel (Bio-Rad) in SDS running buffer [25 mM Tris, 192 mM
glycine, 0.1 % (w/v) SDS, pH8.3] and electrophoresed at 100 V for approximately
1 h. The gel was briefly washed with Milli-Q dH2O and the proteins were
transferred onto 0.45 µm PVDF in Towbin buffer [25 mM Tris, 192 mM glycine,
20 % (v/v) methanol, pH 8.3] at 100 V for 1 h. Even protein loading was confirmed
by Ponceau stain (Sigma-Aldrich) and the membrane was blocked with 5 % skim
milk (w/v in Tris-buffered saline with Tween 20) at 4 ˚C for overnight. The blot
wasprobed with the desired primary antibody (Table 2.7), followed by horseradish
peroxidase-conjugated secondary antibody, and was visualised by luminol-based
enhanced chemiluminescence on ChemiDoc imaging system (Bio-Rad).
Table 2.7| List of antibodies used for immunoblotting Antibody Source and clone Conjugate Provider anti-α-tubulin Mouse monoclonal (DM1A) - Sigma anti-BiP Rabbit polyclonal - JD Bangs anti-GFP Mouse monoclonal (c7.1 & 13.1) - Roche/Sigma anti-HA Rat monoclonal (3F10) - Roche/Sigma anti-GP63 Mouse monoclonal - T Ilg anti-Rabbit Goat polyclonal HRP Merck anti-Mouse Rabbit polyclonal HRP Merck
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2.2 Bacterial culture methods
2.2.1 Bacterial culture
E. coli XL1-Blue (Agilent Technologies) were routinely cultured in Lysogeny broth [LB; 1 % (w/v) tryptone, 0.5 % (w/v) yeast extract, 1 % (w/v) NaCl] containing 100 ug/ml ampicillin or 50 ug/ml kanamycin for plasmid selection, and incubated in an orbital shaking incubator at 37 °C for overnight. Strains were stored at -80 °C in LB broth containing 25 % glycerol as a cryoprotectant.
2.2.2 Bacterial transformation
To introduce foreign DNA into XL1-Blue, 5 µL ligation mixture was mixed with
100 μL XL1-Blue competent cells and incubated on ice for 30 min. Cells were then heat shocked (42 °C, 1 min) and transferred back to ice for 2 min before 700 μL
LB broth was added. Cells were allowed to recover on the shaking incubator at 37
°C for 1 h, then pelleted (10,000×g, RT, 1 min) and resuspended in 100 μL fresh
LB broth, before being plated and incubated on LB agar plate containing the appropriate antibiotic at 37 °C for overnight.
2.3 Leishmania culture methods
2.3.1 Leishmania promastigote culture
Leishmania mexicana (MNYC/BZ/62/M379) promastigote cultures were revived from low-passage number frozen stabilates previous isolated from cutaneous lesion of infected BALB/c mice. Cells were maintained in 10 mL RPMI 1640 medium
(pH 7.4) (Gibco, Thermo Fisher Scientific), supplemented with 10 % (v/v) heat-inactivated foetal bovine serum (iFBS) (Bovogen Biologicals), in sealed 25
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cm2 tissue culture flask (Falcon, Corning) at 27 °C. Cell cultures were routinely
passaged every three to four days to maintain log-phase growth. All experiments
were conducted with cells that have undergone less than 15 passages following
initial isolation from mice lesion.
Frozen stabiliates were routinely prepared by centrifuging 10 mL late-log
phase culture in screw cap conical tube (800×g, 4 °C, 10 min) and the supernatant
aspirated. The cell pellet was then resuspended in 1 mL storage medium [9:1 (v/v)
iFBS/dimethyl sulfoxide], and aliquoted into 5 cryotubes (Nunc, Thermo Fisher
Scientific). Samples were then slowly chilled to -80 °C in a Mr. Frosty freezing
container (Thermo Fisher Scientific).
2.3.2 Cell density determination
L. mexicana culture was first homogenised by gentle agitation before sampling.
The cell sample was then diluted in PBS, mixed thoroughly, and 10 μL of the
diluted sample was counted using a haemocytometer visualised under 20×
objective of a compound light microscope to allow determination of the cell density
of the original culture.
2.3.3 Transfection of L. mexicana and clonal selection
Low-passage number (< 5) L. mexicana promastigote culture was allow to undergo
at least two passage cycles in SDM-79 (pH7.4) (see Appendix 2 for components)
supplemented with 10 % (v/v) iFBS. Cells were cultured to mid-log phase (0.8 to
1×107 cells/mL), and 4×107 cells were harvested (800×g, 4 °C, 10 min), washed
with 10 mL of ice-cold electroporation buffer (EPB; 21 mM HEPES, 137 mM NaCl,
5 mM KCl, 0.7 mM Na2PO4, 5 mM glucose, pH 7.5) (800×g, 4 °C, 10 min), and
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resuspended in 500 μL of ice-cold EPB. For gene deletion experiment, the suspended cells were then transferred into a chilled 4 mm electroporation cuvette
(BTX, Harvard Apparatus), and 5 µg linear DNA knockout construct was added and electroporated using Gene Pulser Xcell electroporator (Bio-Rad) at 1.7 kV and
25 μF. An initial pulse was followed by a second pulse after 10 s. The cells were then allowed to stand briefly at RT before being transferred into a flask containing
10 mL SDM + 10 % iFBS medium and recovered at 27 °C for 24 h, before the addition of appropriate selection drug (10 μg/mL puromycin or 5 μg/mL bleocin) and further incubation at 27 °C. The recovered heterozygous mutants generated from the first round of transfection were maintained as a mixed population.
To create null mutants, the transfection was repeated with the mixed population. However, 24 h post-electroporation, clonal selection was also carried out. Cells were diluted by 1/10 or 1/20 in culture medium containing selection drug to a volume of 20 mL, then aliquoted into a 96-well flat-bottom tissue culture plate
(Falcon, Corning). After sealing with parafilm (Bemis Company), the plate was incubated at 27 °C for up to two weeks. Wells containing live parasites were transferred to 2 mL cultures in 24-well tissue culture plates (Falcon, Corning), with the selection considered “clonal” if less than one-third of the wells of the 96-well plate contained live parasites at the end of selection assay. After recovery, gDNA was extracted (see Section 2.1.3) for genotype verification by PCR.
To introduce plasmid constructs for episomal gene expression, transfection was carried out with 20 μg of purified plasmid. Cells were allowed to recover for
24 h before the addition of selection drug (100 μg/mL G418). Clonal selections were not performed on cells with episomal expression.
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2.3.4 Axenic amastigote differentiation and culture
To induce axenic differentiation into amastigotes, 4×107 stationary-phase
(non-dividing) L. mexicana promastigotes were centrifuged (800×g, RT, 10 min)
then the cell pellet was resuspended in 10 mL acidified RPMI + 20 % iFBS (pH
5.5), and incubated in a sealed flask at 34 °C for three days.
2.4 In vitro macrophage infection
2.4.1 Extraction and proliferation of mice bone marrow- derived macrophages (BMDM)
Bone marrow progenitor cells were obtained by harvesting the femurs and fibulas
of female BALB/c mice derived from pathogen-free facility [Bio21 Institute,
University of Melbourne; approved by the Institutional Animal Care and Use
Committee (ethics number 1212647.1)]. The bone cavities were flushed with
RPMI + 10 % iFBS and gently homogenised. The cell suspension was diluted in
RPMI + 15 % iFBS + 20 % (v/v) L-929 cell-conditioned (L cell) medium + 100
U/mL penicillin/streptomycin (Gibco, Thermo Fisher Scientific), plated into tissue
culture petri dishes (Falcon, Corning), followed by incubation (37 °C, 10 % CO2,
24 h). Next day, non-adherent cells were resuspended by gentle swirling, diluted
two-fold with the same culture medium, then aliquoted onto non-tissue culture petri
dishes and returned back into the incubator. Cells were allowed to differentiate into
macrophages over the next five to six days.
When required, adherent macrophages were lifted off the plate by removing
the supernatant and incubating the cells with 10 mL pre-warmed cell dissociation
buffer (Gibco, Thermo Fisher Scientific) at 37 °C for 10 min.
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2.4.2 Leishmania infection assays
L. mexicana promastigotes were cultured in SDM + 10 % iFBS to stationary phase, as determined under light microscope by the elongated flagellum and increased cell motility. BALB/c BMDMs were washed with warm sterile PBS and resuspended at 2×105 cells/mL in RPMI + 15 % iFBS + 10 % L-cell medium and plated onto 10 mm round microscope cover slips in 24-well tissue culture plates at 1×105 cells per well. Cells were incubated (37 °C, 10 % CO2, overnight) to allow adherence onto the glass coverslips, then 3×105 stationary-phase promastigotes were added directly into each well. The plate was then incubated (34 °C, 5 % CO2, 4 h) to allow parasite internalisation. The supernatant containing non-internalised parasites was subsequently aspirated, and the wells were washed three times with warm PBS before the addition of 1 mL RPMI + 10 % iFBS and the plate was returned back to incubation at 34 °C with 5 % CO2.
2.4.3 Infectivity quantification
At defined time points, the culture medium was aspirated and the cells were washed three times with warm PBS. The coverslips were then fixed with ice-cold methanol for 10 min and washed for a further three times with PBS. The fixed cells were stained with 1 μg/mL propidium iodide in PBS (Molecular Probes, Thermo Fisher
Scientific) for 5 min, washed once with PBS, and mounted onto the microscope slide with Fluoromount-G mounting medium (Southern Biotech) containing 1
μg/mL Hoechst 33342 dye (Molecular Probes, Thermo Fisher Scientific). The parasite load per macrophage and the number of infected macrophages were determined by counting the fluorescently-stained cells (100 macrophages per coverslip) using the Zeiss Axioplan 2 Microscope System (Carl Zeiss).
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2.4.4 Extraction of Leishmania amastigotes from infected macrophages
Macrophage infection assays were performed in 6-well tissue culture plates as
outlined in Section 2.4.2, by infecting 4×105 BMDMs with 1.2×106 L. mexicana
promastigotes per well. After 6-7 days, the infected macrophages from four wells
were harvested by scraping, and the cell suspension was pooled in a screw cap
conical tube. After centrifuging (800×g, RT, 10 min), the cells were resuspended
in 5 mL of sterile PBS. The macrophages were then lysed by passing the cell
suspension through a 25-gauge needle syringe 10 times. A two-stage differential
centrifugation was then performed with the first spin (60×g, RT, 5 min) that
pelleted large debris. The supernatant, enriched with amastigotes, was kept and
pelleted by a second spin (1300×g, RT, 10 min).
2.5 In vivo mice infection
2.5.1 Intradermal infection of Leishmania parasites
BALB/c cutaneous mouse model was used to determine the virulence of specific
L. mexicana strains as previously described (Naderer et al., 2008; Sansom et al.,
2013). In brief, female BALB/c mice (5-7 week old) were infected with L mexicana
stationary-phase promastigotes cultured in SDM + 10 % iFBS. Prior to infection,
cells were washed, resuspended in sterile PBS at 4×104 cells/μL, and kept on ice.
In groups of five mice per experimental condition, each mouse was injected at the
left rump under the skin with 50 μL cell suspension (2×106 total parasites) using a
25-gauge needle syringe.
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2.5.2 Lesion assessment
The mice were maintained at 21-23 °C on a 14 h light/10 h dark cycle, with free access to drinking water and standard rodent chow diet (Barostoc, Ridley
Corporation). A weekly assessment of the injection site was conducted to monitor for the development of cutaneous lesion. The results were scored based on the previously described numbering system (Mitchell and Handman, 1983). Briefly, a score of 0 = asymptomatic, 1 = small swelling, 2 = large swelling or lesion < 5 mm in diameter, 3 = lesion 5 to 10 mm in diameter, 4 = lesion > 10 mm in diameter.
2.5.3 Extraction of Leishmania amastigotes from cutaneous lesions
When the cutaneous lesion reached a score of 4, the mouse was euthanised by CO2.
The lesion was then excised with a sterile scalpel and transferred to 5 mL RPMI +
10 % iFBS kept on ice. To extract the cells from the lesion granuloma, the tissue was passed through a 45 μm cell strainer (Falcon, Corning) together with the storage medium. The resulting homogenate was then passed through a 25-gauge needle syringe 10 times in order to lyse the macrophages and release the intracellular amastigotes. A differential centrifugation (as described in Section
2.4.4) was then performed to isolate the amastigotes. The total number of cells were determined by diluting a small sample in PBS and counted using the haemocytometer. The cells were then immediately frozen as stabilates (see Section
2.3.1), or differentiated back to promastigotes by resuspending in fresh RPMI + 10
% iFBS, with appropriate selection antibiotics and/or 100 units/mL penicillin/streptomycin and incubating at 27 °C.
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2.5.4 Extraction of Leishmania amastigotes from draining lymph nodes
The euthanised mouse was secured onto the surgical board in dorsal recumbent
position. The abdominal skin was incised, elevated, and the draining inguinal
lymph node on the same side as the infection site was located. Using two pairs of
sterilised fine forceps, the lymph node was carefully excised and the surrounding
fat tissue cleaned off. The lymph node was placed in 2 mL RPMI + 10 % iFBS and
kept on ice. Cells were extracted as for lesion amastigotes (see Section 2.4.4)
In order to determine the total parasite number within the tissue, the
homogenate was divided into four 200 μL aliquots, and a serial dilution with RPMI
+ 10 % iFBS at five-fold increments was carried out across a 96-well plate. The
plate was sealed with parafilm and incubated at 27 °C for seven days before
determination of cell number.
2.6 Fluorescence microscopy
2.6.1 GFP live-cell imaging
To immobilise L. mexicana for live-cell imaging, 22 mm square glass coverslips
were pre-treated with 0.01 % poly-L-lysine (w/v in Milli-Q dH2O) overnight then
air-dried. Coverslips were stored for up to one month protected from light.
Log-phase GFP-expressing L. mexicana promastigotes were harvested (10,000×g,
RT, 1 min), resuspended in PBS, and mounted onto the microscope slide with a
poly-L-lysine coated coverslip. The immobilised cells were then visualised under
488 nm excitation and 509 nm emission using the DeltaVision Elite Microscopy
System (GE Healthcare Life Sciences).
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2.6.2 Immunofluorescence imaging
To prepare fixed cell sample for immunofluorescence staining, 500 μL log-phase
L. mexicana promastigotes were centrifuged (10,000×g, RT, 1 min), washed once, then resuspended in 100 μL sterile PBS. In a cold room, the cell suspension was transferred onto a poly-L-lysine coated coverslip (see Section 2.6.1), incubated for
10 min, excess buffer aspirated, then the cells were covered with ice-chilled 4 %
(w/v in PBS) paraformaldehyde (PFA) for 15 min. Back to RT, excess PFA was removed and the fixed sample was covered in 0.1 M glycine in PBS for 5 min.
Cells were then permeabilised with 0.2 % Triton (v/v in PBS) for 20 min then blocked with 2 % BSA (w/v in 0.2 % Triton/PBS) at 4 °C for overnight. Sample was incubated with primary antibodies (Table 2.8) (diluted with BSA/Trition/PBS) at RT for 1 h, washed three times with 0.2 % Triton, incubated in the dark with secondary antibodies (Table 2.8) (diluted with BSA/Trition/PBS) at RT for 45 min, followed by another three washes with 0.2 % Triton, and incubated with 1 μg/mL
Hoechst in PBS at RT for 5 min. After one final wash in PBS, the coverslip was mounted onto the microscope slide with Fluoromount-G mounting medium
(Thermo Fisher Scientific) and visualised using the DeltaVision Elite Microscopy
System.
Table 2.8| List of antibodies used for immunofluorescence imaging Antibody Source and clone Conjugate Provider anti-Bip Rabbit polyclonal - J.D. Bangs anti-HA Rat monoclonal (3F10) - Roche/Sigma anti-Rabbit Goat polyclonal Alexa 594 Molecular Probes anti-Rat Goat polyclonal Alexa 488 Molecular Probes
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2.6.3 Tracing endosomal activity by live-cell imaging
To stain the endo-lysosomal compartments of live cells, 500 μL L. mexicana cells
were incubated with 8 μM FM 4-64 (Molecular Probes, Thermo Fisher Scientific)
at 27 °C either for 15 min (to stain endosomal structures) or 40 min (to visualise
endo-lysosomal structures). Cells were then centrifuged (10,000×g, RT, 1 min),
washed once with sterile PBS, and mounted onto the microscope slide with
poly-L-lysine-treated coverslip (see Section 2.6.1). The sample was then examined
using the DeltaVision Elite Microscopy under 515 nm excitation and 640 nm
emission.
2.6.4 Fluorescence cell viability assay
To test cell viability, 500 μL L. mexicana cells were incubated with propidium
iodide (final concentration 1μg/mL) at RT for 5 min, pelleted (10,000×g, RT, 1 min)
then washing once with sterile PBS to remove excess dye. After centrifugation
(10,000×g, RT, 1 min), cells were resuspended with PBS and visualised by Zeiss
Axioplan 2 Microscope System under 535 nm excitation and 617 nm emission.
2.7 Metabolomics analysis
2.7.1 13C-U-glucose labelling time-course experiment
For each experimental condition tested, L. mexicana mid-log phase promastigotes
(2.5×108) were cultured in RPMI + 10 % iFBS were washed with warm PBS and
resuspended in 30 mL completely defined medium (CDM) without glucose and
with either 200 µM myo-inositol or no myo-inositol (see Appendix 3 for
components). Just before the start of labelling, 4×107 parasites were transferred to
a new flask as zero time-point sample, followed by immediate quenching of
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intracellular metabolites (see next section). Labelling experiment was initiated by the addition of 6 mM 13C-U-glucose to the cell cultures, and 4×107 cell-equivalent samples were collected at pre-defined time points: 0.5, 1, 2, 10, 24 h, and quenched immediately.
2.7.2 Quenching and extraction of intracellular polar metabolites
Preparation of intracellular samples were conducted according to protocol described by Saunders et al. (2015). In brief, 4x107 cell-equivalent L. mexicana samples were rapidly chilled to 2 °C in an ethanol dry-ice slurry then kept cold in an ice water bath. Quenched samples were transferred to pre-chilled 15 mL conical centrifuge tubes (Falcon, Corning) and the cells were harvested by centrifugation
(3200×g, 0 °C, 10 min). The supernatant was transferred to fresh pre-chilled tubes and stored at -20 °C for subsequent extracellular analysis (see Section 2.7.3). The cells were resuspended in 1 mL ice-cold PBS and transferred to pre-chilled 1.5 mL microfuge tubes (Eppendorf), followed by centrifugation (15,000×g, 0 °C, 1 min) and three washes with ice-cold PBS. After the supernatant was carefully aspirated, cells were resuspended in 50 μL ultra-pure chloroform (Ajax Finechem) and immediately stored at -80 °C for subsequent processing.
Samples were briefly thawed at RT and 200 μL 3:1 (v/v) ultra-pure methanol
(Fisher Scientific)/Milli-Q dH2O containing 5 µM scyllo-inositol as an internal standard was used to thoroughly resuspend the cell pellets with the help of sonication. The homogenised samples were then incubated in a 60 °C water bath for 15 min, briefly mixed, and centrifuged (16,000×g, 4 °C, 5 min). The supernatant was transferred to a fresh 1.5 mL microfuge tube containing 100 μL Milli-Q dH2O,
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thoroughly mixed, then the two phases were separated by centrifugation (16,000×g,
4 °C, 5 min). The upper aqueous phase (containing the polar metabolites) was
transferred into a new microfuge tube. To concentrate each sample, the aqueous
phase was sequentially transferred in 75 µL aliquots to a 250 µL glass vial insert
(Agilent Technologies) and dried in vacuo at 55 °C using a rotational vacuum
concentrator. The samples were then washed twice with 50 µL ultra-pure methanol
followed by drying to ensure they were completely dried.
The autosampler vial inserts with the concentrated samples were then
transferred into the autosampler vials (Agilent Technologies) and 20 µL
methoxyamine (20 mg/mL in pyridine) (Sigma-Aldrich) was added. The vials were
capped, thoroughly mixed, then the samples were incubated at RT for at least 15 h
under continuous mixing. Finally, the samples were TMS-derivitised by adding 20
µL N,O-bis(trimethylsilyl)trifluoroacetamide with 1 % trimethylchlorosilane
(Thermo Fisher Scientific), and incubated at RT for 1 h before analysing by gas
chromatography-mass spectrometry (GC-MS) (see Section 2.7.4).
2.7.3 Extracellular polar metabolite samples
To prepare the extracellular samples for analysis, 10 µL of the stored culture
supernatant after collection of cell pellets (see Section 2.7.2) was added (either
neat or diluted 1/20 in Milli-Q dH2O) to 250 µL glass vial inserts containing 10 µL
of 0.1 mM scyllo-inositol (in Milli-Q dH2O) as an internal standard. The samples
were mixed, dried in vacuo at 55 °C, and washed twice with 50 µL ultra-pure
methanol followed by drying to ensure they were completely dried. The remaining
steps of methoxymation and TMS derivitisation of the metabolites were as
described in Section 2.7.2.
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2.7.4 Gas chromatography-mass spectrometry (GC-MS)
Analysis of TMS-derivitised samples was conducted by injecting 1 µL of sample into the GC-MS system comprised of gas chromatograph HP 6890 (Agilent
Technologies) coupled to the quadrupole mass spectrometer HP 5973 (Agilent
Technologies). Detailed parameters of the system are listed below.
MSD Inlet EM voltage: <1800 Carrier gas: Helium Ion source: EI mode, 70eV, 230 ˚C Flow: 53.9 mL/min Interface temperature: 250 ˚C Injection mode: Splitless Quadrupole temperature: 150 ˚C Temperature: 270 ˚C GC-capillary column Operation: Pressure at 13.23 psi Type: J&W DB-5ms (Agilent) for l min after injection Dimension: 30 m × 250 µm (id) × followed by split flow 0.25 µm (film thickness) at 50 mL/min Carrier gas: Helium Flow: 1.1 mL/min
The temperature settings of the GC oven during a sample run are denoted in
Table 2.9. In addition, the GC column was allowed to equilibrate for 5 min in between samples. The mass spectrometer was operated in scan mode, with scanning starting after 5 min of solvent delay and covered mass range 50 to 550 amu at 2.91 scans/second.
Table 2.9| Gas chromatography oven settings for analysing TMS-deritivised samples. Oven stage ˚C/min Next ˚C Hold (min) Initial 70 2 1 12.50 295 18 2 25.00 320 1 3 320 3
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2.7.5 Metabolite identification and analysis
Metabolite identification and data mining were conducted using in-house
Python-based software, Data Extraction for Stable Isotope-labelled metabolites
(DExSI) (Dagley & McConville, 2018). In brief, GC-MS raw data were first
converted to Common Data Format (CDF) using ChemStation. The files were then
imported to DExSI where background correction and noise reduction smoothing
were applied as previously described (Savitzky & Golay, 1964; van der Walt et al.,
2014). Metabolite identification was based on an in-house metabolite library
containing information on the metabolite-specific signature ions (monoisotopic
and related mass isotopologues) and retention time of 51 metabolites (see
Appendix 4). The best matching peak for each metabolite was then recognised
using a scoring algorithm based on retention time, the isotopic series, and peak
height. After automated peak identification and integration, manual curation was
conducted to correct for any inaccuracies or missing values. The final output for
absolute metabolite abundance was determined through calculating the response
factor for each metabolite against a reference mix of metabolites-of-interest of
known quantities included in the same GC-MS run, relative to the integrated area
of the internal standard, scyllo-inositol.
The software implementation also enabled the analysis of stable isotope
labelling experiment with substrate such as 13C-U-glucose, by quantifying all the
expected mass isotopologues for each metabolite, then applying natural isotope
abundance correction based on the extent of labelling and isotopologue distribution
(van Winden et al., 2002; Nanchen et al., 2007). The final output of fractional
labelling was then generated.
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2.8 Lipidomics analysis
2.8.1 High-performance thin-layer chromatography
To analyse total cellular lipids, 4x107 cell-equivalent samples of L. mexicana promastigotes were harvested (10,000×g, RT, 1 min) and washed twice with cold
PBS (10,000×g, RT, 1 min). After aspirating the supernatant, cell pellets were resuspended in 380 µL 1:2:0.8 (v/v) chloroform/methanol/water, sonicated and incubated at 4 ˚C for overnight. Cell debris was removed by centrifugation
(16,000×g, RT, 5 min) and the supernatant was transferred to new microfuge tubes and dried under nitrogen gas. The dried samples were then resuspended in a biphasic mixture of 150 µL 1-butanol-saturated water (BSW) and 200 µL water-saturated 1-butanol (WSB) to achieve lipid-mannogen separation. After thoroughly mixed and centrifuged (16,000×g, RT, 2 min), the upper butanol phase was transferred to new tubes and the remaining aqueous phase was extracted again with an additional 200 µL of fresh WSB. Following another mixing and centrifugation cycle, the second butanol phase was pooled with the first butanol phase. These new samples were then back-extracted with 150 µL of fresh BSW.
The final butanol phase was dried in vacuo at 50 ˚C and resuspended in 9 µL 50 % propanol (v/v in Milli-Q dH2O).
Samples (2×107 cell-equivalent or 4.5 µL) were loaded onto a sheet of high-performance thin-layer chromatography (HPTLC) silica gel 60 (Merck) by sequenctial application of 1.5 µL volume at the origin, allowing for drying between loading. In a closed solvent tank, the samples were then run on a 180:140:9:9:23
(v/v) chloroform/methanol/13 M ammonia/1 M ammonium acetate/Milli-Q water solvent system across a 90 mm vertical distance. Orcinol spray (0.2 % orcinol in
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11 % sulphuric acid and 50 % ethanol) was applied to the gel to stain for glycolipids
and developed in an oven at 100 ˚C for approximately 5 min. Subsequently, the
total lipid profile was revealed by charring at 160 ˚C for about 3 min.
2.8.2 Supercritical fluid chromatography- quadrupole/time-of-flight tandem mass spectrometry (SFC-QTOF-MS)
L .mexicana promastigotes were harvested in 4×107-cell equivalent aliquots
(10,000×g, RT, 1 min) and washed three times in cold PBS (10,000×g, RT, 1 min).
To extract total lipids, cell pellets were thoroughly resuspended in 300 µL 1:9 (v/v)
chloroform/methanol by sonication and incubated at RT for 30 min. After
centrifugation (12,000×g, RT, 5 min), the supernatant was collected and dried in
vacuo in glass vial inserts (Agilent Technologies). At the same time, the pellets
were re-extracted with 150 µL 2:1 (v/v) chloroform/methanol for 30 min and
centrifuged (12,000×g, RT, 5 min). This new supernatant was then pooled with the
previous samples in the glass inserts and dried under nitrogen gas. The dried
samples were then resuspended with 40 µL WSB.
Untargeted analysis of the lipids samples was carried out using
SFC-QTOF-MS. In brief, 10 µL samples were injected into a SFC-MS system
comprised of the supercritical fluid module 1260 Infinity (Agilent Technologies)
coupled to the quadrupole-time of flight tandem mass spectrometer (QTOF-MS)
6550 (Agilent Technologies). The mass spectrometer was operated on both positive
and negative ionisation modes. The mobile phase of SFC was comprised of carbon
dioxide (Solvent A) and 95 % methanol/5 % Milli-Q dH2O + 5 mM ammonium
acetate (Solvent B). The solvent conditions for a single sample run are listed in
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Table 2.10. Detailed system parameters are listed below.
SFC Acquisition Column type: Zorbax 300SB-C18 Mode: Auto MS/MS (Agilent) Dimension: 150 mm × 4.6 mm × 3.5 Ion mode: ESI AJS positive µm (film thickness) ESI AJS negative Column temperature 40 ˚C Storage mode: Centroid Flow rate: 0.75 mL/min MS range: 500-1700 m/z QTOF MS scan rate: 2 spectra/s Source parameters MS scan time 500 ms/spectrum Gas temperature: 200 ˚C MS/MS range: 500-1700 m/z Gas flow: 14 L/min MS/MS scan rate: 500 4 spectra/s Nebuliser 15 psig MS/MS scan time: 250 ms/spectrum Sheath gas temperature 300 ˚C Isolation width: Narrow (~1.3 m/z) Sheath gas flow 11 L/min Scan source parameters
Vcap: 3500 V Nozzle voltage: 1000 V Fragmentor: 175 V Octopole RF Vpp 750 V
Table 2.10| Supercritical fluid chromatography solvent parameters. Solvent A was carbon dioxide and solvent B was comprise of 95 % methanol/5 % Milli-Q dH2O + 5 mM ammonium acetate. Solvent A Solvent B Flow Max pressure Time (min) (%) (%) (mL/min) limit (bar) 0 1 99 0.75 400 2 1 99 - - 7 11 89 - - 20.50 70 30 - - 21.50 95 5 - - 23.50 95 5 - - 24.50 1 99 - -
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2.8.3 Lipid identification and analysis
Raw MS/MS data obtained from SFC-QTOF-MS were processed and analysed
using MS-DIAL (Tsugawa et al., 2015). The programme performs peak detection
and deconvolution of fragment ions in order to extract the original spectra and to
re-establish the precursor-fragment links. The programme also implements peak
alignment and filtering to allow sample comparison. The final MS-DIAL output
containing the identified features was fed into a Metabolomics Australia in-house
R package, which implemented missing value correction, median normalisation,
and Student t-test using the Benjamini-Hochberg adjustment to correct for false
discovery. The statistically significant features were then manually identified based
on MS/MS information, using internal in silico MS/MS spectra libraries based on
LipidBlast, and the online lipid database Lipid Maps (www.lipidmaps.org).
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CHAPTER 3 Characterisation of L. mexicana myo-Inositol 3-phophate Synthase (INO1) and its Role in Pathogenesis
3.1 Introduction myo-Inositol 3-phosphate synthase (INO1) is responsible for catalysing the conversion of D-glucose 6-phophate (Glc6P) to 1-D-myo-inositol 3-phosphate
(Ino3P), the first committed step in the de novo biosynthesis of myo-inositol. This enzyme thus plays a key role in regulating the availability of myo-inositol for synthesis of inositol lipids and associated inositol polyphosphates. The catalytic reaction performed by INO1 is generally thought to involve three steps: the oxidation of Glc6P, intramolecular aldol cyclisation, and reduction (Figure 3.1)
(Majumder et al., 1997). In the process, the enzyme generates two transient intermediates, 5-keto-D-glucose 6-phosphate and myo-inosose-2 1-phosphate, which both remain tightly bound to the active site (Barnett et al., 1973). NAD+ is both consumed and produced during the reaction, resulting in no net change in
NADH and NAD+ cofactors. In addition, INO1 requires a monovalent or divalent cation cofactor in order to achieve activity (Chen et al., 2000; Ju et al., 2004).
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(1) (2) (3) Figure 3.1| Reaction catalysed by the enzyme, myo-inositol 3-phosphate synthase. The reaction is proposed to involve three steps: (1) oxidation of D- glucose 6-phosphate to 5-keto-D-glucose 6-phosphate, (2) internal cyclisation to myo-inosose-2 1-phosphate, and (3) reduction to 1-D-myo-inositol 3-phosphate.
Considered an ancient protein, INO1 is found across all three domains of life,
from eubacteria (Pittner et al., 1979; Bachhawat & Mande, 1999), archaea (Chen
et al, 1998), and protozoa (Lohia et al., 1999; Ilg, 2002; Martin & Smith, 2005), to
higher plants and animals (Maeda & Eisenberg Jr, 1980; Adhikari & Majumder,
1988; RayChaudhuri, et al., 1997). Phylogenetic analysis has revealed that the
origin of INO1 likely precedes the evolutionary divergence of eukaryotes and
prokaryotes (Majumder et al., 2003), with subsequent evolution resulting in unique
protein features further separating taxonomic families into clusters that conform to
existing evolutionary models (Bachhawat & Mande, 2000; Majumder et al., 2003).
Despite these variances, the primary structures surrounding the core catalytic
region of INO1 has been found to be remarkably well-conserved across all domains
(Majumder et al., 2003; Basak et al, 2017).
Genetic deletion of ino1 in a number of bacteria, fungi, and protists leads to
myo-inositol auxotrophy, demonstrating that the de novo myo-inositol synthesis
initiated by INO1 is a non-redundant pathway in these organisms (Dean-Johnson
& Henry 1989; Movahedzadeh et al., 2004; Fischbach et al., 2006). In the
protozoan parasites Trypanosoma brucei and Plasmodium falciparum, ino1
appears to be essential for cell survival under myo-inositol-replete growth
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conditions in vitro even though these protists express myo-inositol transporters, suggesting that de novo myo-inositol may play a unique metabolic role in these organisms (Martin & Smith, 2005; 2006; Macrae et al, 2014).
As described in Section 1.6.1, it has previously been reported that deletion of ino1 gene in L. mexicana results in myo-inositol auxotrophy (Ilg, 2002). However, the author found that the mutant displayed reduced growth rate in comparison to wild-type parasites even in the presence of excess exogenous myo-inositol.
Furthermore, episomal add-back of the Δino1 mutant resulted in only partial restoration of myo-inositol prototrophy and did not complement the growth defect under myo-inositol-replete condition. Complementation of this ∆ino1 mutant also failed to restore virulence in the mouse infection model. It is not clear if the failure of complementation in this study was due to problems with expression from the complementation plasmid, or if the growth defect and loss of virulence seen in the ino1 null mutant were due to secondary genetic changes unrelated to the deletion of ino1. Therefore, further investigation into the role of ino1 in the pathogenesis of
Leishmania is warranted.
In this chapter, genetic techniques were used to localise INO1 and investigate its role in the pathogenesis of L. mexicana. Epitope-tagging of INO1 using GFP expression vectors was employed to localise the protein, and gene deletion by homologous recombination was used to create an ino1-null mutant to assist in characterising the significance of INO1 for parasite survival and infectivity.
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3.2 L. mexicana INO1 is localised to the cytoplasm of the parasite
In order to determine the subcellular localisation of L. mexicana INO1, the open
reading frame of LmxM.14.1360 (corresponding to ino1) was cloned into either
pXG-GFP+2’ or pXG-’GFP+ vector (see Section 2.1.6). The engineered constructs
pXG-GFP+2-INO1 and pXG-INO1-GFP were subsequently transfected into L.
mexicana promastigotes (see Section 2.3.3) to generate transfectants that expressed
an N-terminal or C-terminal GFP fusion protein, respectively. Parasites containing
the plasmid were selected using the antibiotic G418, and immunoblotting of the
cell lysates using anti-GFP antibody confirmed the expression of GFP-fusion
proteins of the expected molecular weight in both cell lines (Figure 3.2A).
Live-cell fluorescence imaging of these GFP-expressing cells (Figure 3.2B)
revealed that INO1 has a primarily cytosolic localisation, and was excluded from
the nucleus and acidocalciosomes regardless of the position of the GFP fusion. This
cytoplasmic location concurs with the lack of specific localisation motifs predicted
by the bioinformatics tools, PSORT II (Nakai & Horton, 1999) and SignalP 4.0
(Petersen et al., 2011).
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(A) N C
260 160 110 GFP-fused INO1 80 60 (85 kD) 50 40
30 20
80 60 BiP
(B)
DIC GFP Hoechst Merge
N nal GFP nal
K
termi
-
N
terminal GFP
- C
Figure 3.2| L. mexicana INO1 is localised to the cytosol. (A) Immunoblotting of whole cell lysate with anti-GFP mouse antibody confirmed that L. mexicana promastigotes transfected with pXG-GFP+2-INO1 (N) or pXG-INO1-GFP (C) expressed intact fusion protein (expected molecular weight of 85 kD). BiP was used as a loading control. (B) Live cell imaging of L. mexicana promastigotes transfected with pXG-GFP+2-INO1 or pXG- INO1-GFP, expressing INO1 fused with N-terminal or C-terminal GFP, respectively. DIC and fluorescence microscopy were conducted on DeltaVision Elite microscopy system (see Section 2.6.1). Green: GFP, blue: Hoechst [nucleus (N) and kinetoplast (K)]; scale bars = 10 μm.
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3.3 L. mexicana ino1 is not essential in vitro in the presence of exogenous myo-inositol
To investigate the role of INO1 in L. mexicana growth and infection, an ino1-null
mutant was generated by replacing both ino1 chromosomal alleles with drug
resistance cassettes through homologous recombination (Cruz et al., 1991) (Figure
3.3). The process involved an initial step of engineering linear DNA fragments
consisting of a puromycin- or bleocin-resistance cassette flanked by homologous
regions targeting the 5’ and 3’ untranslated regions (UTRs) upstream and
downstream of LmxM.14.1360 [ino1 open reading frame (ORF)] (see Section
2.1.8).
5’ UTR PURO 3’ UTR
LMXM.14.1355 LMXM.14.1360 LMXM.14.1370
LMXM.14.1355 LMXM.14.1360 LMXM.14.1370
5’ UTR BLEO 3’ UTR Figure 3.3| Targeted deletion of myo-inositol 3-phosphate synthase (ino1) in L. mexicana by homologous recombination and gene replacement. Linear knockout constructs, consisting of puromycin and bleocin resistance cassettes flanked by approximately 1 kb of ino1 (LMXM.14.1360) 5’ and 3’ UTRs were sequentially introduced into wild type L. mexicana by electroporation. Successful replacement of ino1 loci with the puromycin (PURO) and bleomycin (BLEO) cassettes in drug-resistant parasites was confirmed by triplicate PCR.
After the first round of transfection with the puromycin resistance cassette and
subsequent drug selection (see Section 2.3.3), the recovered parasites were
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subjected to a second round of transfection with the bleocin resistance cassette. The double drug-resistant clones were isolated by limiting dilution and screened by
PCR using primer sets denoted in Table 3.1, which were designed to demonstrate the correct integration of the drug-resistance cassettes onto the chromosome, as well as the complete loss of the ino1 gene. A clone was selected for further characterisation based on its genotype showing the loss of ino1 ORF and the correct integration of both puromycin and bleocin resistance gene cassettes (Figure 3.4).
The PCR reactions were repeated three times in order to verify the results. In addition, immunoblotting of whole cell lysate from the mutant cell line with anti-
INO1 antibody (kindly provided by Thomas Ilg) further confirmed that the clone no longer expressed INO1 (Figure 3.5). This clone was designated Δino1 and used for all subsequent analyses.
Table 3.1| PCR primers for diagnosing ino1 deletion and complementation. Primer ID Sequence ino1 ORF Fwd A CTACCCGGGATGCCGGCAGTGCACGTG ino1 ORF Rev B CGAGGATCCTCACTTGCCGTTGACGTGCT ino1 5’ Flank Fwd C ACATCTTCTCCAAGCACAGCTAC PURO Rev D CGTGGGCTTGTACTCGGTC BLEO Rev E GCACTGGTCAACTTGGCC pXG 5’ Flank Fwd F GCGTGCACATCATCAACTGT
In order to definitively attribute any phenotypes observed in the mutant cell line to the loss of ino1 instead of any secondary unknown genetic changes, an add-back strain was created by introducing pXG-INO1 (see Section 2.3.7), a vector expressing native L. mexicana ino1 ORF, into the Δino1 mutant (see Section 2.4.4).
Restoration of the ino1 ORF was verified by PCR, which showed the new
Δino1+pXG-INO1 cell line retained the drug resistance cassettes and the
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re-introduced copy of ino1 was strictly on the pXG episome (Figure 3.4). Likewise,
restoration of INO1 expression in Δino1+pXG-INO1 cell line was demonstrated
by immunoblotting with anti-INO1 antibody (Figure 3.5). As expected with the
over-expressing pXG vector, the complementation resulted in INO1 being
expressed at higher levels in the add-back strain than in the original wild-type strain.
C D/E
LMXM.14.1355 PURO or BLEO 3’ UTR LMXM.14.1370
LMXM.14.1355 ino1 LMXM.14.1370
A B F Δino1 WT Δino1 +pXG-INO1 B O P B E O P B E O P B E pXG-INO1
5000 4000 3000 2500 2000 Expected sizes: 1500 1000 O = 1599 bp 800 600 P = 1393 bp B = 1327 bp E = 1735 bp
Figure 3.4| Δino1 and episomal add-back mutants display the expected genotype. PCR were performed using gDNA as template extracted from L. mexicana wild-type, Δino1, and Δino1+pXG-INO1 cell lines to confirm the presence or absence of ino1 ORF (O) using primers A and B (Table 3.1), integration of puromycin resistance cassette (P) using primers C and D, integration of bleocin resistance cassette (B) using primers C and E and episomal pXG-INO1 (E) using primers B and F.
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KD 260 160 110 80 60 INO1 50 (58.2 kD) 40
30
80 60 BiP
Figure 3.5| Δino1 mutant has lost INO1 expression while genetic complementation with pXG-INO1 results in higher expression levels compared to wild type. Whole cell protein extracts from L. mexicana wild-type, Δino1, and Δino1+pXG-INO1 add-back cell lines were probed with anti-INO1 antibody to confirm the expression or absence of the INO1 protein. BiP was used as a loading control, demonstrating that INO1 levels are higher in the add-back strain compared to wild type, as expected with genetic complementation using the multi-copy pXG vector.
3.4 L. mexicana Δino1 undergo rapid cell death
in the absence of exogenous myo-inositol
To investigate whether the loss of ino1 results in myo-inositol auxotrophy, the growth of the ∆ino1 mutant and add-back line was measured in media containing varying concentrations of myo-inositol. Growth of wild-type, Δino1, and
Δino1+pXG-INO1 strains were virtually indistinguishable in standard RPMI medium supplemented with 10 % iFBS (Figure 3.6A). This medium contains
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approximately 200 μM myo-inositol, as determined by GC-MS. In order to assess
growth in medium lacking exogenous myo-inositol, wild-type and Δino1
promastigotes were cultivated in completely defined medium (CDM)
supplemented with 1 % BSA and 200 μM myo-inositol. Growth of wild-type and
mutant promastigotes in this minimal medium was initially slow, but increased over
three passages to support similar growth rates to that observed in RPMI containing
10 % iFBS. Following adaptation to full CDM, the promastigotes were cultivated
in CDM supplemented with 1 % BSA, and varying amounts of myo-inositol. As
expected, the growth of wild-type promastigotes was unaffected by changes in the
concentration of myo-inositol in the culture medium (Figure 3.6B), suggesting that
de novo synthesis can supply the myo-inositol needs of rapidly dividing
promastigotes. In contrast, L. mexicana Δino1 promastigotes failed to proliferate
in the absence of myo-inositol. Addition of 20 μM myo-inositol partly restored
growth of the mutant, while addition of 200 µM myo-inositol (or higher) restored
growth rates to wild-type levels.
Closer examination of Δino1 under myo-inositol-starved conditions showed
the mutant was able to undergo a single doubling cycle immediately after
transferring to myo-inositol-free CDM. However, at 24 hours the cells displayed
abnormal cellular morphology, marked by increasing swelling and extensive
intracellular vacuolation (Figure 3.7A). Rapid loss of cell viability was detected
from 48 hours onward, as shown by propidium iodide staining, with less than 1 %
of the mutant promastigotes viable after four days (Figure 3.7B). Surprisingly,
some of these parasites could be rescued even after culture in myo-inositol-free
medium for seven days, by re-supplying the medium with 200 μM myo-inositol
(data not shown). The recovered parasites retained the ino1-null genotype, verified
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by PCR, suggesting that this population may be able to persist in a quiescent state.
However, all Δino1 promastigotes had lost viability after cultivation in
myo-inositol-free medium for more than two weeks.
A (A) B
(B)
Figure 3.6| Deletion of ino1 results in myo-inositol auxotrophic parasites. (A) L. mexicana wild-type, Δino1, and Δino1+pXG-INO1 promastigotes were inoculated at 1x105 cells/mL in RPMI supplemented with 10 % iFBS. Cell density was measured daily by manual counting using a haemocytometer. Error bars indicate SD of 3 biological replicates. (B) CDM-adapted L. mexicana wild-type and Δino1 promastigotes were inoculated at 1x105 cells/mL in CDM containing various concentrations of myo-inositol. Cell density was measured daily by manual counting using a haemocytometer. Error bars indicate SD of 3 biological replicates.
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(A)
Day 0 Day 1 Day 2
Day 3 Day 4
(B)
Figure 3.7| L. mexicana Δino1 undergo rapid loss of viability without exogenous myo-inositol. L. mexicana Δino1 promastigotes were cultured in myo-inositol-free CDM. (A) Changing cell morphology over four days was monitored by DIC microscopy at 64 x magnification. Loss of cell integrity was marked by increasing swelling and vacuolation. Scale bars = 10 μm. (B) The associated percentage of viable cell (stain-negative) was determined by staining with propidium iodide and manually counting of 300 cells. Error bars indicate SD from three replicate experiments.
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3.5 Loss of INO1 does not affect growth inside macrophages in vitro
To examine how the loss of INO1 affects L. mexicana infectivity in the mammalian hosts, BALB/c bone marrow-derived macrophages (BMDM) were infected with wild-type, Δino1 and Δino1+pXG-INO1 stationary-phase promastigotes and their ability to proliferate within the macrophage hosts monitored by fluorescence microscopy over the course of seven days (see Section 2.4.2 and 2.4.3).
Interestingly, no significant difference in intracellular replication was observed between strains, both in the percent of macrophages infected and the parasite burden over time (Figure 3.8A & B). The continued growth of the ∆ino1 parasites in macrophages could reflect the presence of large intracellular pools of myo-inositol in the parasites used in these infections, which had been cultivated in standard RPMI containing 10 % iFBS medium (containing 200 µM myo-inositol)
(see Section 4.2). Alternatively, since the macrophages were cultivated in RPMI supplemented with 10 % iFBS during the infection period, myo-inositol may also be internalised by infected macrophages and subsequently salvaged by intracellular
∆ino1 amastigotes.
To determine whether the L. mexicana amastigotes rely on intracellular pools of myo-inositol acquired during axenic growth or can actively scavenge the substrate while inside the host cells, stationary-phase parasites were pre-incubated in myo-inositol-free CDM for 24 hours prior to macrophage infection.
Pre-incubation in myo-inositol-free media effectively depletes > 90 % of intracellular pools of myo-inositol (see Section 4.2), but does not compromise cell viability (see Figure 3.7). When subsequent infection studies in BMDMs were
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performed in medium containing myo-inositol (RPMI + 10 % iFBS), the L.
mexicana Δino1 mutant continued to replicate in a manner similar to both wild-type
and add-back strains (Figure 3.9A & B). These experiments suggest that
intracellular amastigotes are capable of salvaging myo-inositol whilst inside the PV,
and the levels present were sufficient for the parasites to not be dependent on de
novo synthesis.
(A) (B)
(C) WT Δino1 Δino1+pXG-INO1
Figure 3.8| Infection and intracellular replication of L. mexicana Δino1 are similar to wild type in macrophages under myo-inositol-replete conditions. BALB/c bone marrow-derived macrophages were infected with L. mexicana wild- type, Δino1, and Δino1+pXG-INO1 stationary phase promastigotes. Assays were conducted in RPMI + 10 % iFBS. (A) The percentage of infected macrophages, and (B) parasite burden was determined by manual counting at day 0, 3, 5, and 7 post- infection. Error bars indicate SEM from 3 independent experiments. (C) Representative images of macrophage infection assay at day 7 observed at 64x magnification. The cells were fixed and then stained with propidium iodide. The outline of a macrophage is marked, and each small punctate is the nucleus of an L. meixcana amastigote within the PV. Scale bars = 10μm.
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(A) (B)
Figure 3.9| L. mexicana Δino1 still replicate in macrophages even after myo- inositol starvation pre-infection. L. mexicana wild-type, Δino1, and Δino1+pXG- INO1 stationary-phase promastigotes were cultured in myo-inositol-free medium for 24 hours prior to BMDM infection. During the 4-hour internalisation stage, BMDMs were also incubated in myo-inositol-free medium. Following infection and washing to remove extracellular parasites, BMDMs were then cultured in RPMI + 10 % iFBS, as for the standard infection assay. (A) The percentage of infected macrophages and (B) parasite burden was determined by microscopy at day 0, 3, 5, and 7 post-infection. Error bars indicate SEM from 3 independent experiments.
The current model of nutrient distribution inside the PV proposes that molecules such as myo-inositol may reach the intracellular compartment via various transport mechanisms (reviewed in Section 1.6.1). These may be delivered directly from the macrophage’s extracellular environment or by salvaging host cell materials. Therefore, to investigate whether Δino1 proliferation inside the macrophage hosts is dependent on exogenous myo-inositol, parasites were pre-starved of myo-inositol and then used to infect BMDMs which were cultivated in myo-inositol-free medium. While both wild-type and add-back strains proliferated within myo-inositol-starved BMDMs, Δino1 parasites failed to replicate and were effectively cleared by the host cells at day 7 post-infection
(Figure 3.10A & B). The macrophages remain adhered to the glass covers at the end of the infection assay, indicating their viability were not affected by
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myo-inositol starvation. These results demonstrate that myo-inositol derived
directly from BMDMs is likely insufficient to sustain the growth of L. mexicana
Δino1 whilst in the PV, and that the intracellular growth of this mutant is dependent
on myo-inositol in the extracellular milieu.
(A) (B)
Figure 3.10| L. mexicana Δino1 are unable to replicate in macrophages when the medium is myo-inositol depleted. BALB/c bone marrow-derived macrophages were infected with L. mexicana wild-type, Δino1, and Δino1+pXG-INO1 promastigotes. Parasites were pre-starved for a shorter course of 4 hours (compared to Figure 3.9), however, the subsequent seven-day infection assays were entirely conducted in myo-inositol-free CDM. (A) Percentage of infected macrophages and (B) parasite burden was determined by microscopy at day 0, 3, 5, and 7 post-infection. Error bars = SEM from three experiments.
3.6 L. mexicana Δino1 are avirulent in the BALB/c model of cutaneous leishmaniasis
To investigate the role of INO1 in vivo, experimental cutaneous infection of
susceptible BALB/c mice was performed using L. mexicana wild type, Δino1, and
Δino1+pXG-INO1 (see Section 2.5.1). Lesion formation was monitored and
recorded weekly following score criteria previously described (see Section 2.5.2).
Both wild-type and Δino1+pXG-INO1 parasites were found to induce large
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granulomatous lesions in these mice over a 12-week period, and mice were subsequently euthanised (Figure 3.11). In striking contrast, the Δino1 mutant was unable to induce any detectable lesions over a 30-week period.
Figure 3.11| L. mexicana Δino1 are avirulent in susceptible mice. BALB/c mice were infected by intradermal injection with L. mexicana wild-type, Δino1, and Δino1+pXG-INO1 promastigotes. Lesion formation was scored weekly as per
Mitchell and Handman (1983). Error bars indicate SEM, n=5.
In order to determine whether some Δino1 mutant parasites persisted asymptomatically in infected BLAB/c mice, skin tissue at and around the infection site, as well as in the draining (inguinal) lymph node were harvested 30 weeks post-infection in order to recover and quantify any viable parasites (see Section
2.5.3 & 2.5.4). While no parasites were recovered from the initial site of infection, low numbers of parasites were isolated from the draining lymph nodes (an average of ~100 cells per lymph node) (Figure 3.12). These parasites were verified to retain the ino1 null genotype by PCR, and were still resistant to both puromycin and bleocin. Collectively, these data indicate that exogenous myo-inositol level in the lesion microenvironment is insufficient to sustain intracellular parasite growth and/or that the combination of myo-inositol restriction coupled with enhanced host microbicidal processes, contributes to the severe loss of Δino1 virulence in vivo.
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(A) (B)
Figure 3.12| A subpopulation of Δino1 persist in the lymph node during mouse infection. BALB/c mice were infected by intradermal injection with L. mexicana, Δino1, wild-type, and Δino1+pXG-INO1 promastigotes. Amastigotes were extracted from (A) the lesion site and (B) inguinal lymph node, after reaching a lesion score of 4 (for wild type and Δino1+pXG-INO1) or after 30 weeks post- infection (for Δino1). Cell counts were determined by microscopy. Error bars indicate SEM, n = 4 technical replicates from five mice.
3.7 Discussion
The key findings of this chapter confirm that INO1 is required for de novo synthesis
of myo-inositol in L. mexicana and that loss of this enzyme leads to myo-inositol
auxotrophy. While exogenous myo-inositol is required for growth of ∆ino1
promastigotes in vitro, this mutant was able to establish infections and proliferate
inside primary BMDMs. This indicates that L. mexicana amastigotes have access
to myo-inositol during intracellular stages. In marked contrast, the ∆ino1 mutant
was unable to attain normal growth or induce cutaneous lesions in susceptible
mouse models, highlighting significant differences between nutrient levels in vivo
and in cultured macrophages. These results are discussed in more details below.
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3.7.1 INO1 localisation and the glycosomal compartmentalisation of glucose 6-phosphate
Analysis of the subcellular localisation of INO1-GFP fusion proteins in L. mexicana promastigotes indicated that INO1 is primarily or exclusively expressed in the cytosol. This localisation is consistent with the absence of any identifiable organelle targeting motifs in this protein and conforms to the localisation previously reported in other eukaryotes. This is of interest given that in Leishmania and other trypanosomatids, several enzymes involved in the synthesis and catabolism of Glc6P, the substrate for INO1, are primarily localised to the peroxisome-like organelles called glycosomes (Michels et al., 2006). These enzymes include hexokinase (Mottram & Coombs, 1988) and fructose
1,6-bisphosphatase, a key gluconeogenic enzyme responsible for converting fructose 1,6-bisphosphate (Fru1,6P2) to fructose 6-phosphate (Fru6P) (Naderer et al., 2006). While little is known about the regulation of these different reactions, there is accumulating evidence that many of the Leishmania and trypanosome enzymes involved in glycolysis lack conventional allosteric regulation (Nwagwu
& Opperdoes, 1982; Cronin & Tipton, 1985; López et al., 2002). The compartmentalisation of these enzymes to the glycosome is therefore thought to represent an alternative mechanism for regulating glycolytic and gluconeogenic fluxes via controlling ATP/ADP and Pi levels (Hannaert et al., 2003; Michels et al.,
2006), and to prevent the toxic accumulation of glycolytic intermediates (Haanstra et al., 2008).
On the other hand, other Glc6P utilising enzymes such as Glc6P dehydrogenase or Glc6P isomerase are located in the cytosol (Mottram & Coombs,
1988), or both the cytosol and glycosome (Nyame et al., 1994). The differential
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localisation of enzymes involved in the synthesis of Glc6P and conversion of this
hexose phosphate to other intermediates suggests there is active flux of Glc6P
across the glycosome membrane. However, previous studies have indicated that
the membrane of glycosomes, like peroxisomes, is poorly permeable (Moyersoen
et al., 2004), and therefore, exchanging of specific metabolites between the cytosol
and the organelles likely involve membrane transporters or pores. So far, little is
known regarding the identities of these shuttling systems in trypanosomatids. In T.
brucei, two classes of transporter systems, ATP-binding cassettes (ABC) and
mitochondrial carrier protein family (MCP), have been identified based on
homology study against mammalian and yeast peroxisomes (Colasante, et al., 2006;
Yernaux et al., 2006). Two of the ABC transporters, GAT1 and GAT3, also have
corresponding orthologues that have recently been isolated from L. donovani
glycosomes (Jamdhade et al., 2015). However, doubts have also been raised
regarding the economics and energy efficiency of using ABC transporters for the
mass movement of glycolytic intermediates such as Glc6P (Michels et al., 2006).
Indeed, evidence of rapid equilibrium between glycosomal and cytosolic Glc6P is
supported by analysis of mannogen synthesis in Leishmania. Mannogen, a major
carbohydrate storage found in Leishmania comprised of mannose polymers derived
from Glc6P, is found to accumulate rapidly in the cytosol when glucose is in excess
of energy requirement (Sernee et al., 2006). Altogether, these findings suggest
Glc6P is effectively shuttled out from the glycosome by an as-yet-uncharacterised
transporter system and/or pore where it can then be utilised by INO1 and other
enzymes found in the cytosol.
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3.7.2 The essentiality of ino1 in Leishmania compared to other protozoan parasites ino1 is an essential gene in the protozoan parasites, T. brucei and P. falciparum, and cannot be disrupted under standard culture conditions, even when exogenous myo-inositol is available (Martin & Smith, 2005; 2006a; Macrae et al., 2014). The essentiality of ino1 in these organisms suggests that de novo synthesised myo-inositol and/or Ino3P is channelled into essential downstream pathways that differ from those that utilise scavenged myo-inositol. As reviewed in Section 1.6.2, studies on myo-inositol metabolism in T. brucei and P. falciparum have described how endogenous and exogenous myo-inositol are differentially utilised in synthesising two subpools of PI (Martin & Smith 2006a; Macrae et al., 2014).
Based on these findings, a hypothetical model has been postulated (Figure 1.8), in which PI generated from de novo myo-inositol is preferentially utilised for GPI synthesis in the ER, while myo-inositol acquired through extracellular salvage is directed towards synthesising bulk PI in the Golgi complex.
In contrast, ino1 was not found to be essential in L. mexicana [this study and Ilg (2002)], as long as the parasites have access to an exogenous source of myo-inositol. This is similar to the situation in many other prokaryotic and eukaryotic organisms (Dean-Johnson & Henry, 1989; Movahedzadeh et al, 2004;
Fischbach et al., 2006). The difference between Leishmania and the other protozoan parasites could reflect differences in the compartmentalisation of myo-inositol and/or GPI biosynthesis between these divergent eukaryotes, or the lack of essentiality of GPI biosynthesis in Leishmania. The latter possibility is suggested by the finding that a number of Leishmania mutants have now been
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generated (∆dpms, DIG1, ∆ads1) that lack all or most GPI and GPI-anchored
macromolecules (reviewed in Section 1.5.2). In contrast, mutants with defects in
GPI biosynthesis in T. brucei and P. falciparum are lethal (Nagamune et al., 2000;
Chang, et al., 2002; Bushell et al., 2017). Further experiments will be needed to
determine whether newly synthesised myo-inositol is preferentially channelled into
GPI biosynthesis, while salvaged myo-inositol can be incorporated into all inositol
lipids.
3.7.3 The role of ino1 in Leishmania growth and infectivity
The phenotypes identified for the ino1 mutant generated in this study replicate
some of the findings of previous work by Ilg (2002). This work showed that the
loss of INO1 in L. mexicana leads to the development of myo-inositol auxotrophy.
However, in contrast to the findings of Ilg (2002), in vitro promastigote growth of
Δino1 was fully restored to that of wild-type levels in the presence of sufficient
exogenous myo-inositol. Furthermore, complementation of the Δino1 mutant with
an episomal construct fully removed the reliance on exogenous myo-inositol, as
reflected by the full complementation of growth within macrophages and virulence
in BALB/c mice. While the ∆ino1 mutant failed to develop lesions in susceptible
mouse models, a small but persistent population of viable Δino1 was isolated from
the draining lymph node. Other Leishmania mutants have also been shown to
persist in mice without eliciting significant pathology (Späth et al., 2003; Huynh et
al., 2006; Naderer et al., 2006). These observations suggest that the lymph nodes
may constitute a privileged host niche containing higher nutrient concentrations
than in the large avascular granulomas induced by wild-type parasites. This is
supported by previous studies on mice models, which have demonstrated that
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myo-inositol can vary significantly in levels between different tissue types (Berry et al., 2003; Chau et al., 2005). However, the levels present in granuloma and lymphatic tissues remain uncharacterised, and will be of significant interest for a future study. Moreover, while it is likely that macrophages remain the major host cell for this population of persistent parasites, it is possible that they may also reside in other cell types, such as fibroblasts, that provide additional nutrient supplementation (Bogdan et al., 2000).
Finally, this study provides additional insight into the nutrient composition of
Leishmania PV. Although this compartment is a major site of protein, glycan and lipid degradation, the resultant amino acids, sugars and fatty acids generated by these processes are thought to be rapidly exported to the cytoplasm of the host cell
(Burchmore & Barrett, 2001). Nutrient levels in the lumen of the PV are thus likely to be dependent on the rate of delivery of cargo via different endocytic/lysosomal and autophagic pathways (McConville et al. 2007; Pinheiro et al., 2009; Ndjamen et al., 2010; Canton et al., 2012; Canton & Kima, 2012), as well as the rate of transport of low molecular nutrients across the PV membrane. Based on the intracellular growth of the ∆ino1 mutant in BMDM under different culture conditions, the transfer of host-synthesised myo-inositol in the cytoplasm to the lumen of the PV appears to be low. Although extracellular metabolites could also be delivered by endocytic/lysosomal pathways, previous studies with other
Leishmania sugar auxotrophs indicate that the bulk uptake of exogenous sugars is insufficient to sustain parasite growth in vivo (Naderer et al., 2006; 2008).
Collectively, these results emphasise the importance of de novo myo-inositol synthesis for Leishmania pathogenesis.
87 37
88
CHAPTER 4 Defining the Molecular Basis of myo-Inositol Starvation in L. mexicana
4.1 Introduction
In Chapter 3, gene knockout studies demonstrated that the first enzyme in de novo myo-inositol biosynthesis is essential for L. mexicana growth in myo-inositol-free medium, as well as infectivity in macrophages and susceptible mouse models. The loss of virulence of this mutant indicates that the PV in which the amastigotes reside in the macrophage host is either deficient in myo-inositol, or that de novo synthesised myo-inositol is used for production of essential myo-inositol-containing metabolites. The latter could include the bulk phospholipid, PI, or derived GPI glycolipids and complex phosphoinositides.
These molecules perform a plethora of cellular functions which are vital for parasite survival and virulence (reviewed in Section 1.5).
While the cellular effects of myo-inositol starvation are poorly understood in
Leishmania and other pathogenic organisms, they have been extensively described in the model organism Saccharomyces cerevisiae. Early studies of myo-inositol auxotrophic mutants of S. cerevisiae led to the description of “inositol-less death”, when these cells were transferred to myo-inositol-free culture media (Culbertson &
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Henry, 1975; Henry et al., 1977). Inositol-less death was associated with a more
rapid and precipitous loss of viability than is normally associated with nutrient
starvation in other types of auxotrophs (Culbertson & Henry, 1975; Henry et al.,
1975; Boer et al., 2008). Other studies also highlighted the effects of exogenous
myo-inositol on the synthesis and metabolism of, not only myo-inositol-containing
lipids, but also other lipid species, such as phosphatidylcholine (PC) and
triacylglycerol (TG) (Loewen et al., 2004; Gaspar et al., 2006; 2011). The role of
myo-inositol metabolites in multiple processes was further emphasised by
genome-wide expression analyses, which identified over 100 genes that are
significantly affected by the presence of myo-inositol (Santiag & Mamoun, 2003;
Jesch et al., 2005; 2006). A sophisticated myo-inositol-sensing system has since
been uncovered in yeast that involves the monitoring of the intracellular
phosphatidic acid (PA) levels to triggers downstream responses that enable the cell
to regulate INO1 expression (Loewen et al., 2004; Jesch et al., 2005).
In this chapter, the underlying metabolic effects of myo-inositol starvation on
L. mexicana were investigated using the Δino1 strain. Changes in the major polar
metabolites and lipids were determined using metabolomic and lipidomic
approaches. In addition, phosphoinositide-specific GFP fusion probes were
employed to analyse the dynamics of the major phosphorylated PI species during
myo-inositol starvation.
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4.2 The de novo myo-inositol synthesis pathway is abolished in Δino1
To assess how the deletion of ino1 affects Leishmania at a metabolic level and to better understand how the parasites respond when deprived of exogenous myo-inositol, L. mexicana wild-type and Δino1 mid-log phase promastigotes were cultivated in CDM + 1 % BSA with or without supplementation with 200 µM myo-inositol over the course of 24 hours. Parasites were simultaneously labelled with 13C-U-glucose to measure the rate of synthesis of myo-inositol via the INO1 pathway (see Section 2.7). Parasites were harvested over a range of time points and polar metabolites were analysed by GC-MS to quantitate intracellular levels and 13C-enrichment of Ino3P and myo-inositol. These analyses showed that intracellular levels of Ino3P remained relatively high (7 to 11 nmol per 4×107 promastigotes) in both wild type and Δino1, irrespective of the presence or absence of exogenous myo-inositol in the medium (Figure 4.1A). In contrast, intracellular levels of free myo-inositol changed dramatically depending on the availability of myo-inositol in the medium. Suspension of wild type or ∆ino1 promastigotes in fresh medium containing 200 µM myo-inositol resulted in an initial increase in intracellular levels, which then progressively decreased with time (Figure 4.1A).
In contrast, suspension of both lines in myo-inositol-free medium led to a precipitous drop in intracellular myo-inositol levels (from 4 to 0.02 nmol per 4×107 promastigotes), suggesting rapid utilisation and/or secretion of intracellular pools when exogenous levels of myo-inositol are low.
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(A)
(B)
(C)
Figure 4.1| The de novo myo-inositol synthesis pathway is abolished in Δino1. L. mexicana wild-type and Δino1 mid-log phase promastigotes were cultured in CDM supplemented with either 200 µM inositol (+ino) or no myo-inositol (-ino), and labelled with 13C-U-glucose over 24 hours. Parasites (4×107 cell-equivalent) were harvested and quenched at set time points and (A) intracellular levels and (B) percentage of 13C-labelling of Ino3P and myo-inositol were measured by GC-MS. (C) The levels of free myo-inositol synthesised de novo at the 24-hour time point were calculated (total abundance multiplied by percentage 13C-labelled).
Strikingly, suspension of wild-type parasites in myo-inositol-free medium
containing 13C-U-glucose led to a marked increase in 13C-enrichment of both Ino3P
and myo-inositol, reaching 19 % and 40 %, respectively, by 24 hours. (Figure 4.1B).
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This reflected an almost two-fold increase in de novo myo-inositol biosynthesis compared to myo-inositol-replete condition (Figure 4.1C). The lower percentage of 13C-labelling of Ino3P compared to myo-inositol suggests that there may be two pools of Ino3P; one that may be generated directly from the labelled glucose via the INO1 pathway and dephosphorylated to form myo-inositol, and a second poorly/unlabelled pool that may be derived from other pathways, such as the breakdown of complex polyphosphorylated inositol species (Gee et al., 1988;
Majerus, 1992). In contrast to wild-type parasites, suspension of ∆ino1 parasites in myo-inositol-free medium did not result in 13C-labelling of either Ino3P or myo-inositol (Figure 4.1B). These analyses show that the loss of INO1 results in the complete disruption of de novo myo-inositol synthesis (Figure 4.1C).
Interestingly, suspension of wild-type and ∆ino1 promastigotes in myo-inositol-free medium was associated with the initial release of free myo-inositol into the extracellular medium, as determined by GC-MS analysis of the medium supernatant (Figure 4.2). This secreted myo-inositol was progressively labelled with 13C when wild-type promastigotes were fed 13C-U-glucose, indicating that this pool is not due to carry over from the myo-inositol-containing medium in which the parasites were originally cultivated. Moreover, ∆ino1 promastigotes did not secrete detectable levels of myo-inositol over initial background levels, and the latter pool was unlabeled, confirming the disruption of the INO1 pathway in these parasites and suggesting that the rapid decrease in the intracellular pool in myo-inositol-starved parasites reflects, at least in part, reverse transport of myo-inositol out of the parasites, presumably via the plasma membrane myo-inositol/H+ symporter (Drew et al., 1995).
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Figure 4.2| De novo myo-inositol is released by wild-type promastigotes. L. mexicana wild-type and Δino1 mid-log phase promastigotes were cultured in CDM supplemented with 200 µM myo-inositol (+ino) or no myo-inositol (-ino), and labelled with 13C-U-glucose over 24 hours. Culture medium samples were collected at set time points and the abundance and 13C-enrichment of myo- inositol in 10 µL samples were measured by GC-MS. Error bars represent SD from three replicates.
4.3 Carbon metabolism of L. mexicana is unregulated in response to myo-inositol
starvation
To investigate whether myo-inositol starvation leads to detectable changes in
central carbon metabolism, 13C-enrichment in a range of intermediates in the
glycolytic, pentose phosphate, glycosomal succinate fermentation and TCA
pathways in 13C-U-glucose-fed wild-type and ∆ino1 promastigotes was quantitated
by GC-MS. The results showed that 13C was rapidly incorporated into
intermediates in both upper and lower glycolysis (including alanine, which is
directly connected to pyruvate production) (Figure 4.3), reflecting the highly
glycolytic metabolism of these parasite stages (Saunders et al., 2011). Conversely,
the kinetics of 13C-enrichment of intermediates in the mitochondrial TCA cycle
were slower, consistent with this being an important but slow flux pathway.
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Surprisingly, no significant differences were observed in the labelling kinetics in either wild-type or ∆ino1 promastigotes in the presence or absence of myo-inositol.
This is unexpected considering cellular myo-inositol was mostly depleted after 10 hours and rapid cell death occurs from 24 hours onward (Figure 3.7).
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Glycolytic and TCA intermediates
Amino acids
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Figure 4.3| Metabolic fluxes of central carbon metabolism are unaffected by myo-inositol starvation. L. mexicana wild-type and Δino1 mid-log phase promastigotes were incubated in CDM either supplemented with 200 µM myo- inositol (+ino) or no myo-inositol (-ino), and labelled with 13C-U-glucose over 24 hours. Parasites (4×107 cell-equivalent) were harvested and quenched at set time points, and the percentage of 13C-enrichment of selected intracellular metabolites were measured by GC-MS.
As an alternative measure of the metabolic and physiological state of myo-inositol-replete and inositol-starved promastigotes, the rate of uptake and secretion of key carbon sources and metabolic end-products was measured.
Consistent with the 13C-U-glucose labelling studies, wild-type and ∆ino1 promastigotes were found to utilise glucose at similar rates to produce the same major metabolic end-products succinate, malate, alanine, and proline (Figure 4.4)
(Cazzulo et al., 1985; Saunders et al., 2011; Westrop et al., 2015). Both parasite lines also utilised a variety of different amino acids (aspartate, methionine, leucine, serine, and tryptophan) at similar rates in the presence or absence of myo-inositol.
These results suggest that wild-type and ∆ino1 parasites are unable to sense myo-inositol depletion or starvation, respectively, and enter into a protective metabolically quiescent state. The absence of a sensing mechanism may underlie the precipitous loss of viability seen beyond 24 hours of starvation
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Glycolytic and TCA intermediates
Amino acids
Figure 4.4| Uptake and secretion of metabolites by L. mexicana are unaffected by myo-inositol starvation. L. mexicana wild-type and Δino1 mid-log phase promastigotes were cultured in CDM supplemented with either 200 µM myo- inositol (+ino) or no myo-inositol (-ino) for 24 hours. Culture medium samples were collected at set time points, the levels of key polar metabolites in 10 µL samples were measured by GC-MS. Error bars represent SD from three replicates.
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4.4 myo-Inositol starvation leads to depletion of specific inositol lipids myo-Inositol is required for the synthesis of several abundant membrane lipids in
Leishmania, including PI, IPC, GIPLs, and phosphoinositides (reviewed in Section
1.5). To investigate the consequences of myo-inositol limitation or starvation on the steady-state levels of these lipids, L. mexicana wild-type and Δino1 promastigotes were incubated in CDM with or without 200 µM myo-inositol over three days (in which cell density doubled approximately every eight hours during log-phase). Lipids were extracted and analysed by high-performance thin-layer chromatography (HPTLC) (see Section 2.8.1). The results showed that myo-inositol limitation placed on wild type had no detectable effect on the steady state levels of all inositol lipids (Figure 4.5), suggesting that de novo synthesis of myo-inositol is sufficient to sustain normal synthesis of these lipids. In contrast, suspension of Δino1 promastigotes in myo-inositol-free medium led to the progressive depletion of PI and PI phosphates (PIP; a mixture of mainly PI3P and
PI4P) from 24 hours onward (Figure 4.5). Interestingly, the levels of the major
GIPL species (iM2, iM3 and iM4) remained unchanged over the three days, suggesting that these glycolipids have very low rates of turnover compared to other bulk lipids such as PI.
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PC PE PI IPC
GIPL iM2 PIP GIPL iM3 GIPL iM4
Origin
Day 0 1 2 3 1 2 3 0 1 2 3 1 2 3
Inositol +ino -ino +ino -ino
Cell type Wild type Δino1 Figure 4.5| PI and PIP species are depleted in Δino1 in the absence of exogenous myo-inositol. L. mexicana wild-type and Δino1 mid-log phase promastigotes were incubated in CDM with 200 µM myo-inositol (+ino) or no myo-inositol (-ino). Lipid samples were collected at day 0, 1, 2, and 3, extracted then analysed on HPTLC that was subsequently stained with orcinol and heat charred for visualisation (see Section 2.8.1). Identification was based on the lipid reference described by Ralton & McConville (1998). Abbreviations used: GIPL, glycoinositol phospholipid; Ino, myo-inositol; IPC, inositol phosphorylceramide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate.
In a complementary approach to dissecting changes in promastigote lipid
composition during myo-inositol limitation/starvation, parasite lipids were
analysed by supercritical fluid chromatography coupled to tandem mass
spectrometry (SFC-QTOF-MS). Wild-type and Δino1 mid-log phase
promastigotes were incubated under myo-inositol-replete and -depleted conditions.
Cells were harvested and total lipids were extracted after 36 hours and analysed by
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SFC-QTOF-MS in both positive and negative ionisation modes (see Section 2.9.2).
The raw data were processed using MS-DIAL (see Section 2.9.3), and a total of
3357 features were identified with positive ionisation mode. Dramatic differences were observed in the response of wild-type and Δino1 promastigotes to myo-inositol limitation/starvation. These are depicted in volcano plots which plot p-values and fold-changes due to the effects of myo-inositol limitation (Figure 4.6).
While the lipid composition of wild-type strain showed virtually no change as a consequence of myo-inositol deprivation, substantial increases and decreases of lipids were displayed by Δino1. After Benjamini-Hochberg adjustment to reduce false discovery, approximately 50 % of the identified features (1686) were found to have undergone significant changes in myo-inositol-starved Δino1 promastigotes, whereas only three features were observed under the same perturbation in the wild-type cells. Similarly, 990 features were identified using negative ionisation mode, of which 377 were found to show significantly changes in myo-inositol-starved Δino1 promastigotes, while none were changed in wild type.
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WT
Δino1
Figure 4.6| myo-inositol starvation induces profound changes in Δino1 lipid composition. L. mexicana wild-type and Δino1 mid-log phase promastigotes were incubated in CDM supplemented with 200 µM myo-inositol (+ino) or no myo-inositol (-ino). Lipid samples were extracted after 36 hours and analysed by SFC-QTOF-MS under positive ion mode. Data were median normalised and
projected onto volcano plots with -log10 of raw p-value against log2 of fold change (FC). Dotted lines reflect filter criteria (FC ± 2, p-value ≤ 0.05), while violet dots represent significant features from three experimental replicates.
Over one hundred lipid species (112 species in total) that changed only in
∆ino1 parasites during myo-inositol starvation were definitively identified based
on MS/MS information (Figure 4.7). As expected, the levels of all detected PI
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molecular species were reduced in myo-inositol-starved Δino1 promastigotes, while the levels of the major IPC and GIPL species were only modestly affected
(Figure 4.8), further highlighting differential rates of turnover of these inositol lipid classes. Interestingly, the steady-state levels of several other lipid classes also changed during the starvation (Figure 4.9). In particular, levels of the major molecular species of phosphatidic acid (PA), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) increased dramatically in the myo-inositol-starved
∆ino1 parasites. The levels of ceramides, the major precursors for IPC, were also increased. In contrast, levels of plasmenyl-PE decreased during inositol starvation.
Specific changes in the fatty acid composition of neutral diacylglycerol s (DG) and triacylglycerols (TG) were also observed. In particular, myo-inositol starvation led to the accumulation of TG species with long chain, saturated fatty acids and reductions in unsaturated species (Figure 4.9).
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Figure 4.7| Identification of significant lipid species during myo-inositol starvation based on MS/MS information. An example of total ion chromatograph of Δino1 sample acquired from SFC-QTOF-MS under positive ionisation mode shows lipid classes are distributed in defined regions based on retention time. Initial lipid identification was performed by MS-DIAL based on its internal in silico MS/MS spectra libraries. This was followed by manual curation and confirmation based on the signature fragment ions that matched the lipid species. The species shown are the ones that displayed the most statistically significant differences between the two culture conditions of each lipid class.
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Figure 4.8| PI species are depleted during myo-inositol starvation while other inositol lipids are less affected. L. mexicana Δino1 mid-log phase promastigotes were incubated in CDM supplemented with 200 µM myo-inositol (+ino) or no myo- inositol (-ino). Lipid samples were extracted after 36 hours and analysed by SFC- QTOF-MS. Data were median normalised and lipid species were identified based on MS/MS information. Non-statistically significant species are marked by n.s. Error bars represent SD of triplicates. Abbreviations used: GIPL, glycoinositol phospholipid; IPC, inositol phosphorylceramide; PI, phosphatidylinositol.
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Figure 4.9| A diverse range of lipid species are affected by myo-inositol conditions. L. mexicana Δino1 mid-log phase promastigotes were incubated in CDM supplemented with 200 µM myo-inositol (+ino) or no myo-inositol (-ino). Lipid samples were extracted after 36 hours and analysed by SFC-QTOF-MS. Data were median normalised and lipid species were identified based on MS/MS information. Only statistically significant species (P-value < 0.05, BH adjusted t- test) with confirmed identifications are shown are shown. Error bars = SD of triplicates. Abbreviation used: DG, diacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; TG, triacylglycerol.
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4.5 PI3P is disrupted in L. mexicana during myo-inositol starvation
As shown above, myo-inositol starvation of ∆ino1 promastigotes leads to depletion of steady state levels of phosphoinositides, such as PIP. PIP comprises at least two species, PI3P and PI4P which, in other eukaryotes, have distinct cellular localisations and functions (reviewed in Section 1.5.3). To further define the potential role of these phosphoinositide species, as well as their dynamics during inositol starvation, wild-type and ∆ino1 promastigotes were transfected with GFP fusion proteins containing phosphoinositide-binding peptides, which allow direct visualisation of different PI pools in live cells by fluorescence microscopy.
GFP-fusion proteins that bound specifically to PI3P, PI4P, and PI(4,5)P2 were generated by coupling GFP to the FYVE domain of Hrs (2×FYVE), the Pleckstrin homology (PH) domain of oxysterol binding protein (OSBP), and the PH domain of phospholipase C-δ1 (PLCD1), respectively (see Section 2.3.8). Expression of each of these GFP fusion proteins over 36 hours in the presence or absence of myo-inositol was confirmed by immunoblotting (Figure 4.10). Interestingly, both the 2×FYVE- and OSBP PH-fused GFP probes appeared to be degraded in vivo, as shown by the appearance of a low molecular weight band that had the same apparent molecular weight as GFP (27 kD). The selective degradation of these proteins is likely to be the result of their recruitment to endosomal/lysosomal structures based on fluorescence microscopy. Specifically, long-term labelling (45 min) with the endocytic tracer, FM 4-64 (see Section 2.6.3), showed that the
GFP-2×FYVE probe was localised to the multivesicular tubule (MVT)-lysosome, a prominent tubular organelle that aligns with a quartet of cytoplasmic
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microtubules and constitutes the mature lysosome of Leishmania promastigotes
(Figure 4.11A) (Mullin et al., 2001; Waller & McConville, 2002). The lumen of
the MVT-lysosome is full of microvesicles that are either formed by invagination
of the limiting membrane of the tubule, or by fusion of endosome-derived
multivesiclar bodies (MVB) with the tubule (Mullin et al., 2001; Waller &
McConville, 2002). The strong localisation of the GFP-2×FYVE construct to the
MVT-lysosome and subsequent degradation of the probe to release free GFP
(Figure 4.10), suggests that this probe binds to PI3P on the cytoplasmic leaflet of
the MVT-lysosome or MVB, and that this lipid is subsequently internalised into
the lumen of the MVT-lysosome. In contrast, the GFP-OSBP PH probe was
localised to large punctate structures at the anterior end of promastigotes around
the flagellar pocket (Figure 4.12A), suggesting a localisation to the Golgi or early
endosomes (Field & Carrington, 2009). Short-term labelling (15 min) with FM
4-64, showed almost exact co-localisation, indicating that the GFP-OSBP PH is
binding to PI4P on early endosomes. Finally, the PLCD1 PH-GFP probe was
localised to the plasma membrane of the promastigotes (Figure 4.13A).
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GFP-2xFYVE GFP-OSBP PH PLCD1 PH-GFP (Expected size: 46.8 kD) (Expected size: 39.3 kD) (Expected size: 47.4 kD) WT Δino1 WT Δino1 WT Δino1 0 36 0 36 0 36 0 36 0 36 0 36
260 26 260 160 160 160 110 110 110 80 80 80 60 60 60 50 50 50 40 40 40
30 30 30 20 20 20
15 15 15
80 80 80 60 60 60 Figure 4.10| Expression of phosphoinositide-specific GFP probes was unaffected during myo-inositol starvation. Western blot of whole cell lysates probed with anti-GFP antibody confirmed L. mexicana wild-type and Δino1 promastigotes transfected with pX-GFP-2×FYVE, pXG-GFP-OSBP PH, or pXG-PLCD1 PH-GFP were able to constitutively express the respective GFP fusion proteins during 36 hours of myo-inositol starvation. Both GFP-2×FYVE and GFP-OSBP PH also displayed degradation to release free GFP. Expected size of GFP is 27 kD. BiP (lower panels) was used as loading control.
myo-Inositol limitation for 36 hours had no effect on the intracellular distribution of the three GFP probes expressed in the wild-type promastigotes (Figure 4.11A,
4.12A, 4.13A), consistent with the notion that de novo synthesis is sufficient to maintain the steady state levels of these minor inositol lipids. In contrast, major changes in the localisation of some of the GFP phosphoinositide sensors and the morphology of target organelles were observed when ∆ino1 promastigotes were deprived of myo-inositol. Specifically, myo-inositol starvation was associated with the contraction of the MVT-lysosome to punctate structures, which were clearly separate from the early endosomes proximal to the flagellar pocket, as determined
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by labelling with the endocytic tracer FM 4-64 (Figure 4.11B). However, some of
the GFP-2×FYVE-labelled compartments were also co-labelled with FM 4-64,
confirming ongoing trafficking between the endosome and lysosome compartment
was still intact. These observations suggest that the loss of PI3P may lead to
perturbation in membrane transport between endosome and the MVT-lysosome
and/or interactions with the microtubule quartet that is required to maintain its
unusual tubular structure, leading to tubule retraction. In contrast, the subcellular
distribution of the PI4P probe (GFP-OSBP PH) and the PI(4,5)P2 probe (PLCD1
PH-GFP) did not change after 36 hours of myo-inositol starvation (Figure 4.12B
& 4.13B), even though many of the promastigotes were starting to swell and round
up. Together these results suggest that PI3P may have a more rapid turnover than
other phosphoinositides, and raises the possibility that this phosphoinositide is
required for maintenance of the MVT-lysosome.
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min min before being analysed by fluorescence and DIC microscopy. Green: GFP, red: FM
promastigotes transfected with pX with transfected promastigotes
45
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(B)
Δ
hour 0
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64 64 for
ino1 ino1
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Figure 4.11| Δ 4 MVT tubule; MVT, multivesicular endosomes; K, kinetoplast; E, arefeatures indicated: (B)
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Figure 4.12| Monitoring PI4P in in PI4P Monitoring 4.12| Figure (B) an fluorescence by analysed being before nucleus.N, kinetoplast; E, endosomes; K,
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(A) Wild type PLCD1 PH-GFP Hoechst Merge
F
K
0 0 hour N
F 36 hours
(B) Δino1 PLCD1 PH-GFP Hoechst Merge
N
0 0 hour K F
F 36 hours
Figure 4.13| Monitoring PI(4,5)P2 in L. mexicana using PLCD1-GFP probe during myo-inositol starvation. L. mexicana wild-type (A) and Δino1 (B) promastigotes transfected with pXG-PLCD1-GFP were incubated in myo-inositol-free CDM for 36 hours. Cells were analysed by fluorescence microscopy. Green: GFP, blue: Hoechst. Specific cellular features are indicated: F, flagellar pocket; K, kinetoplast; N, nucleus. Scale bars = 10 μm.
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4.6 Discussion
In this chapter, the essential function of INO1 in de novo myo-inositol biosynthesis
was confirmed using 13C-U-glucose labelling and the consequences of myo-inositol
depletion on cell carbon metabolism, membrane lipid composition, and organelle
biogenesis/maintenance were investigated. The findings highlight the pleiotropic
effects that myo-inositol starvation has on both inositol and non-inositol lipids, as
well as the function of the unusual tubular lysosome of these parasites, which may
contribute to inositol-less death.
4.6.1 INO1 is essential for de novo myo-inositol synthesis
These analyses show that the intracellular pool size of myo-inositol in Leishmania
promastigotes primarily reflects extracellular levels of myo-inositol and that de
novo synthesis accounts for less than 10 % of this pool when promastigotes are
cutlivated under myo-inositol-replete condition. Following transfer to
myo-inositol-free medium, intracellular pools of myo-inositol decrease rapidly,
likely as a result of secretion and, to a lesser extent, incorporation of intracellular
pools of myo-inositol into PI synthesis. The finding that myo-inositol is secreted
was surprising, and may reflect reverse transport via the myo-inositol/H+ symporter,
which transports myo-inositol down a concentration gradient (Drew et al., 1995).
Thus removal of extracellular myo-inositol after extended growth on
myo-inositol-containing medium would lead to a significant inside-out gradient
that would drive secretion. Whether myo-inositol secretion also occurs to the same
extent in intracellular amastigotes is unknown. These stages reside in acidified
compartments which would promote myo-inositol uptake via the proton gradient
and prevent reverse transport. Amastigotes would thus be expected to be more
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efficient at scavenging myo-inositol and less likely to passively shed this nutrient, which may be crucial for survival within the low-sugar milieu of the PV.
Based on the 13C-U-glucose labelling studies and quantitation of ino3P and myo-inositol levels, glucose flux into de novo myo-inositol biosynthesis is up-regulated by approximately two-fold when exogenous myo-inositol levels are low, and intracellular pools of myo-inositol are depleted by about 90 %. The fact that wild-type Leishmania promastigotes can grow normally and maintain steady state levels of inositol lipids demonstrates that de novo synthesis can sustain all the myo-inositol needs of dividing parasites. Interestingly, the level of 13C-enrichment in Ino3P, the precursor for myo-inositol, lagged behind myo-inositol, which suggests the presence of at least two pools of Ino3P; a highly enriched pool that is primarily used for myo-inositol synthesis, and a significant unlabelled/low enrichment pool that accounts for the low level of 13C-enrichment overall. The latter pool is likely to be at least 50 % of the total pool and could reflect physical compartmentalisation of these different pools or an alternative pathway of inositol phosphate production. One possibility is that other inositol monophosphates could be generated by successive dephosphorylation of inositol polyphosphates released from phosphoinositides (Gee et al., 1988; Majerus, 1992). These species would be labelled more slowly than the intermediates labelled via the INO1 pathway, as they would need to be first incorporated into PI and downstream phosphoinositides. The inositol monophosphates generated via inositol polyphosphate breakdown will be isomers of Ino3P and may not be resolved by GC-MS.
Together, these analyses show that Leishmania maintain a threshold level of myo-inositol via de novo synthesis, even under myo-inositol-replete conditions, but
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that they can also accumulate high levels of myo-inositol if an extracellular source
of myo-inositol available. Given that the availability of myo-inositol is likely to be
significantly different between the sandfly midgut (as a component of blood meal
and honey dew sugar meal) and the PV inside the mammalian macrophage, it is
possible that changes in the intracellular pools of Ino3P or myo-inositol could
constitute a signal for the nutrient status of these parasites and/or the nutrient
environment. However, as outlined below, the precipitous death of
myo-inositol-starved parasites suggests that Leishmania are unable to directly
detect myo-inositol depletion and activate protective metabolic responses.
4.6.2 Effects of myo-inositol starvation on L. mexicana central carbon metabolism
Depletion of intracellular myo-inositol levels had little effect on central carbon
metabolism 24 hours after removal of exogenous myo-inositol from ∆ino1
promastigotes, as shown by 13C-U-glucose labelling studies. This was somewhat
surprising, considering that intracellular level of myo-inositol was depleted by
more than 95 % after 10 hours of starvation, and the parasites began to lose viability
from 24 hours onward. Although it is possible that any remaining residual pool
may have left sufficient myo-inositol to sustain essential inositol-lipid pathways,
a number of studies in other single-celled eukaryotes have shown that inositol-less
death is associated with catastrophic loss of viability. Early studies on myo-inositol
starvation described an “unbalanced” growth in S. cerevisiae and in the fungi
Neurospora, in which the cells failed to initiate cell-cycle arrest at the onset of
perturbation, continued to multiple briefly then experienced rapidly loss of viability
(Ridgway & Douglas, 1958; Lester & Gross 1959). Subsequent studies found that
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even when they stopped dividing, S. cerevisae myo-inositol auxotrophs appeared to be unable to conserve energy and continued to synthesise macromolecules, such as DNA, RNA, and proteins (Atkinson et al, 1977, Henry et al., 1977). This type of rapid cell death marked by the absence of energy conservation is in contrast to yeast auxotrophs that are completely dependent on exogenous sources of carbon, nitrogen, phosphate, or sulphate, which rapidly transition into a quiescent state when starved of each of these nutrients (Broach, 2012; De Virgilio, 2012). In contrast, it is reminscent of auxotrophs of other essential nutrients that typically rely on de novo synthesis, such as fatty acids, uracil and leucine (Henry, 1973;
Saldanha et al., 2004; Boer et al., 2008). A theory proposed is that although cells have evolved to cope with the fluctuations of essential nutrients that are salvaged from the environment, they have not developed protective mechanisms against essential nutrients that are normally synthesised de novo and therefore rarely limiting (Boer et al., 2008). Thus the presence or absence of direct sensing mechanisms for a particular essential nutrient may determine whether starvation trigger a concerted suppression of metabolic activity and cell-cycle arrest, or not.
This concept of unregulated energy wastage during myo-inositol starvation offers an explanation for the unperturbed glycolytic activity and amino acid metabolism observed in L. mexicana Δino1.
Finally, it should be noted that the isotopic labelling experiments for this thesis were conducted using 100 % 13C-U-glucose in order to clearly assess the effect of ino1 deletion on the myo-inositol biosynthetic pathway. While important information was also gathered on the broader effects this had on glycolysis, pentose phosphate pathway, and TCA cycle, the consequences of the deletion on these pathways can only be fully dissected by more complex labelling designs in future
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studies. Examples of these include labelling using either a pre-defined mixture of
natural and 13C-U-glucose, partially-labelled glucose substrates (such as
1,2-13C-glucose, 1,6-13C-glucose), or other 13C-labelled carbon substrates (Metallo
et al., 2009; Saunders et al., 2011; Jang et al., 2018).
4.6.3 Effects of myo-inositol starvation on L. mexicana lipid composition
While myo-inositol depletion clearly leads to eventual parasite death, it remains
unclear which pathway or pathways of myo-inositol utilisation is the primary cause
of death, or whether there is also a hierarchy of utilisation under starvation
conditions. As expected, myo-inositol starvation was associated with the depletion
of PI and PIP species, as demonstrated by both HPTLC and comprehensive
SFC-MS analysis of total lipids. Other inositol lipids, such as IPC and GIPL, were
depleted at a substantially slower rate. The latter are synthesised in the lumen of
the ER and Golgi and are thought to be transported to the exoplasmic leaflet of the
plasma membrane. These inositol lipids appear to be very stable components of the
plasma membrane and their slow turnover suggests that they are not likely to be
responsible for inositol-less death. In support of this conclusion, a number of
studies have shown that neither GIPLs nor IPC are essential for Leishmania growth
in axenic culture or macrophages (Garami et al, 2001; Naderer & McConville, 2002;
Zhang et al., 2003; Zufferey et al., 2003; Denny et al., 2004).
These observations suggest that inositol-less death is likely to be associated
with depletion of PI or PIP species. Interesting, SFC-MS analysis showed that
myo-inositol starvation was also associated with changes in cellular levels of other
classes of neutral (ceramide, DG, TG) and non-inositol containing phospholipids
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(PA, PC, PE). Strikingly, some of the most dramatic changes were associated with specific PC/PE molecular species, which could reflect specific effects on phospholipid synthesis in different organelles, such as the ER, mitochondrion, or lysosome.
Furthermore, the accumulation of ceramide in myo-inositol-starved ∆ino1 promastigotes may reflect global changes in phospholipid synthesis. In particular,
Leishmania are unusual in using sphingolipids (derived from ceramides) as a major source of ethanolamine for PE and PC biosynthesis (Zhang et al., 2007). Changes in PI levels could directly impact on ceramide/sphingolipid levels by affecting the rate of synthesis of IPC in the Golgi, with possible downstream consequences on the synthesis of other phospholipids. Further studies using stable isotope labelled precursors (13C-ethanolamine, 13C-choline, and 13C-serine) would be of interest to determine the turnover and synthesis of different phospholipid species in myo-inositol-starved cells.
The accumulation of PA in myo-inositol-starved ∆ino1 promastigotes provides further evidence for general dysregulation of phospholipid synthesis. PAs are the common precursors for PE, PC, and PI, and a sudden cessation of PI synthesis could result in increased flux into other phospholipids, as well as creating a build-up of upstream precursors, such as PA. Changes in PA is also thought to regulate INO1 expression in yeast by affecting the nuclear translocation of transcriptional repressor (Loewen et al., 2004; Jesch et al., 2005; Henry et al., 2012), although there is no evidence that such a mechanism exists in Leishmania.
On the other hand, plasmeny-PE levels decreased during myo-inositol starvation. While the precise cause for this decrease is currently unknown,
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plasmenyl-PE is known to play a role in regulating a number of cellular membrane
functions such fusion, fission, regulating membrane-associated proteins, and
protection against oxidative damage (Glaser & Gross, 1994; Thai et al., 2001;
Murphy 2001).
Profound changes have also been detected in the neutral lipids, DG and TG,
but they appear less uniform in response to myo-inositol starvation. A co-ordinated
and inter-dependent relationship between TG and PI metabolism has previously
been established (Gaspar et al., 2006; 2011). Changes to neutral lipid homeostasis
are also known biomarkers for cellular stress and adaptive responses in Leishmania
(Kloehn et al., 2015; Bouazizi-Ben et al., 2017). The enrichment of saturated TG
species and reduction in unsaturated species observed in Δino1 will likely have a
significant impact on the integrity of not only plasma membrane, but also
organelles such as ER (Volmer, et al., 2013). This may reflect a number of stress
responses reported to be activated during myo-inositol starvation, the most
well-established being the unfolded protein response that occurs in the ER (Chang
et al, 2002; 2004).
While the direct detection and quantitation of individual phosphoinositide
species remains challenging, the recent development of protein phosphoinositide
sensors has greatly facilitated attempts to follow the localisation and dynamics of
this multi-functional family of lipids. The results showed the expression of
different phosphoinositide probes that specifically recognise PI3P (GFP-2×FYVE),
PI4P (GFP-OSBP PH), and PI(4,5)P2 (PLCD1 PH-GFP) in Leishmania
promastigotes highlights marked differences in the subcellular localisation of these
lipids. In particular, PI3P appears to be primarily targeted to the cytoplasmic leaflet
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of the unusual MVT-lysosome, while PI4P is targeted primarily to early endosomes and PI(4,5)P2 to the plasma membrane and flagellar pocket. These localisations are broadly consistent with the known functions of these phosphoinositides in membrane trafficking and signalling pathways in other eukaryotes. Further, the finding that the GFP-2×FYVE sensor is largely degraded to GFP in live promastigotes suggests that PI3P is packaged into internally budding vesicles that deliver other cargo, such as bound proteins, to the lumen of the lysosome where they are degraded. As shown previously, delivery of GFP-fusion proteins to the
MVT-lysosome results in very inefficient degradation of the protease-resistant
GFP moiety due to the relatively high pH and low hydrolytic capacity of this compartment (Vince et al., 2011).
In this study these biosensors were used to monitor changes in the levels of specific phosphoinositides during myo-inositol starvation as well as the impact of the starvation on organelle structure and biogenesis. Strikingly, myo-inositol depletion was shown to have a profound effect on the stability of the
MVT-lysosome, which contracted to punctate organelles after 36 hours of perturbation. In marked contrast, the localisation of other phosphoinositide biosensors did not change. It remains to be determined whether PI3P is turned over more rapidly than other phosphoinositides. However, these data suggest that PI3P is involved in regulating membrane traffic between the endosome and
MVT-lysosome and/or other functions required for maintenance of this organelle, such as interactions with the microtubule quartet that runs from the flagellar pocket to the posterior end of promastigotes. Perturbation in MVT-lysosome could therefore underlie, in part, the loss of viability of myo-inositol-starved parasites.
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CHAPTER 5 Characterisation of L. mexicana myo-Inositol Monophosphatase (IMPase) and its Role in Pathogenesis
5.1 Introduction
myo-Inositol monophosphatase (IMPase) catalyses the second step of the de novo myo-inositol synthesis pathway. Specifically, the enzyme removes the phosphate group from myo-inositol 3-phosphate (Ino3P) to generate free myo-inositol that can subsequently be used to synthesise PI and other myo-inositol-containing lipids, such as GPI-associated glycolipids and phosphoinositides (Atack et al., 1995; Michell, 2008). In other eukaryotic organisms, IMPase may also be involved in the regeneration of myo-inositol from inositol 1,4,5-trisphosphate (IP3), which is either released as a secondary messenger during phosphoinositide signalling, or from the dephosphorylation of other inositol polyphosphates (Gee et al., 1988; Pollack et al., 1994; Atack et al.,
1995) (Figure 5.1). This dual role of IMPase in both synthesis and recycling of myo-inositol-containing molecules is important in maintaining cellular myo-inositol homeostasis.
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Figure 5.1| Schematics of phosphoinositide and inositol phosphate synthesis and recycling. myo-Inositol monophosphatase (IMPase) is situated at the key junction point between de novo myo-inositol synthesis from glucose 6-phosphate (Glc6P) and recycling of inositol 3-phosphate (Ino3P) generated from the breakdown of phosphoinositides and inositol polyphosphates. Abbreviations used: DAG, diacylglycerol; Ino, myo-inositol; INO1, myo-inositol 3-phosphate synthase; IP3, inositol 1,4,5-trisphosphate; IP4, inositol 1,3,4,5-tetrakisphosphate; IP5, inositol 1,3,4,5,6-pentakisphosphate; IPPase, myo-inositol polyphosphate phosphatase; PI, phosphatidylinositol; PIP, PI 4-phosphate; PIP2, PI 4,5- bisphosphate; PLC, phospholipase C.
IMPase was first purified from bovine and rat tissues (Hallcher & Sherman,
1980; Takimoto et al., 1985; Gee et al., 1988), and the enzyme and its homologues
have since been reported in several prokaryotic and eukaryotic organisms
(McAllister et al., 1992; Gillaspy et al., 1995; Chen & Roberts, 1998; Chen &
Roberts, 2000; Nigou et al., 2002). Multiple IMP isoforms can often be identified
in a single organism and may be present as monomers, or as protein complexes in
the form of dimers (e.g. in mammals and M. tuberculosis) (Gee et al., 1988; Brown
et al., 2007; Ohnishi et al., 2007) or tetramers (e.g. in the thermophile Thermotoga
maritima) (Chen & Roberts, 1999). Phylogenetic analyses have shown that the
sequences of the IMPases are highly conserved across evolutionally divergent
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groups (McAllister et al., 1992; Wreggett 1992; Gillaspy et al., 1995), although the primary amino acid sequences share very little overall similarity with any other known proteins (Atack et al., 1995). However, crystallography studies have since revealed that the secondary structural topology and tertiary structure of the catalytic core of IMPase resemble that of other magnesium-dependent, lithium-sensitive phosphatases, including fructose 1,6-bisphosphatase (FBPase) and inositol polyphosphate phosphatase (Zhang et al., 1993; York et al., 1995).
Little is known about the role of IMPase in the metabolism and virulence of microbial pathogens. Recent studies have shown that T. brucei expresses two
IMPase isoforms, TbIMPase1 and TbIMPase2, neither of which are essential for the survival of the mammalian infective bloodstream forms when deleted alone
(Cestari & Stuart, 2015; Cestari et al., 2016). However, the TbIMPase2-null mutant grew more slowly than wild type parasites in vitro and exhibited a delayed virulence phenotype in mouse models. Similarly, disruption of one of the IMPase homologues in Mycobacterium tuberculosis, one of the few bacteria to have a de novo pathway for myo-inositol synthesis, also resulted in loss of virulence in macrophages (Movahedzadeh et al., 2010).
As shown in Chapter 3 and 4, INO1 is essential for virulence of L. mexicana in macrophages and susceptible animal models. To confirm whether downstream enzymes in the de novo myo-inositol biosynthesis pathway are also essential for virulence, studies were initiated to (1) analyse candidate Leishmania IMPase proteins using bioinformatic approaches, (2) investigate their localisation using epitope tagging, and (3) characterise the virulence phenotype of IMPase deletion mutants created by homologous gene knockout approaches.
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5.2 L. mexicana encodes two putative IMPase isoforms
The L. mexicana genome contains two open reading frames, LMXM.17.1390 and
LMXM.15.0880 (Rogers et al., 2011), which are annotated as encoding putative
IMPase isoforms and are referred to here as IMP1 and IMP2, respectively.
Alignment of protein sequences (see Section 2.1.1) indicated that IMP2 shared 33
% amino acid sequence identity with IMP1. Interestingly, IMP2 contained five
additional peptide extensions or inserts (including a 124 amino acid region from
residues 177 to 300) which were absent in IMP1 (Figure 5.2). Consequently, IMP1
is predicted to have a molecular weight of 31 kD, while IMP2 is predicted to have
a molecular weight of 49.6 kD.
Lmex IMP1 -----MTQ------PSLTEDELDDALGLAIRAANTAAFIIN------30 Lmex IMP2 MSISVLSTPAPASSSVDTGEVSLIEALGVAIEAADKGSRIIWQSVEKRRIAVAAAATFNT 60 :: . * .* :***:**.**:..: ** Lmex IMP1 -SAIDERTNSIVEAQTRNHPNDLMTQYEKQCKEEVLNILRVGT------PSYAILSD 80 Lmex IMP2 ASALAAGGAASVECEDKTSATNLVTQYSSQCRETITRILRQYSEEVQREKPHLRFAFLTE 120 **: : **.: :. .:*:***..**:* : .*** : :*:*:: Lmex IMP1 GMHSEAVLGDGPTWIVGPIDGTISFEHGLFDFCVSIALALRKEPILGVVCAPRLQE---- 136 Lmex IMP2 ELNPNTLLSDDYTWVVDPVNGATSFSHGLPDCCISIGLTYRKQPVLAVVFTPFISSGVRL 180 :: :::*.*. **:*.*::*: **.*** * *:**.*: **:*:*.** :* :.. Lmex IMP1 ------Lmex IMP2 TVAAATVLNVAQQQQQHQRQHQQQLVMSPTLSVTAPSISAAMCSSVGATPLTPMDVSKTK 240
Lmex IMP1 ------Lmex IMP2 MLDEHGTAAPPTTALPPTPPTPPFPSLRTATAMAAASASPHSVPIITHTTPSVVPECNGE 300
Lmex IMP1 VFTAVKNRGAFSNGQRIHVSLVHSLKQSVVLLHQNCT------RSDV 177 Lmex IMP2 LFTAIKGFGAFVNGRQVRVNAKVVPTTSVVVFNHPCGVMLAAAEAASPDNAAICRRKCDA 360 :***:*. *** **::::*. . ***:::: * :.*. Lmex IMP1 AVKSMTAMQAELAKLPVQGLRCNGSAALDMCLVAAGRAELFWEAGVNPWNVAAGVIIVRE 237 Lmex IMP2 AVDCSMAMREELLRLPVTALRCYGSCAATLAQVAAGRVDAYLEPAGKAWDVCAGSLLVTE 420 **.. **: ** :*** .*** **.* :. *****.: : * . : *:*.** ::* * Lmex IMP1 AGGIVHDVENTDGFDLTRR-GVCCGCSLDVTKHGVELSLKHNYCSSVLNTSS 288 Lmex IMP2 AGGVVCNMLGRP-LDMAHGTAIVAAATQEMADLMTEKCVRHGFGQYWLVESE 471 ***:* :: . :*::: .: ...: :::. .* .::*.: . * *.
Figure 5.2| Sequence alignment of L. mexicana IMP1 and IMP2. Sequence alignment was performed using Clustal Omega (Sievers et al., 2011). Consensus key: (.), weak residue conservation; (:), strong residue conservation; (*), fully conserved residue. The inserts/extensions in IMP2 are highlighted in grey. The following protein sequence accession numbers were used: L. mexicana XP_003874006.1 and XP_003873614.1.
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IMP1 and IMP2 are highly conserved in terms of protein sequence and synteny across all of the sequenced Leishmania genomes. Each of the genes exhibited > 88 % sequence identity between the major species infecting humans, namely L. mexicana, L. major, L. donovani, and L. infantum, and between 74 %
(for IMP2) and 80 % (for IMP1) with the more divergent species, L. braziliensis.
Significant sequence identity was also observed with the previously characterised
IMPase genes from T. brucei (44 to 55 % identity for IMP1 and IMP2, respectively)
(Figure 5.3 and 5.4), highlighting the high degree of sequence conservation across these evolutionarily divergent protists. Intriguingly, a number of key residues which are thought to be involved in substrate and/or cofactor binding in mammalian IMPase (Gill et al., 2005; Singh et al., 2012; Ferruz et al., 2016), are altered in both Leishmania IMP1 and IMP2. For IMP1, the most significant change was the substitution of aspartate at position 97 for a glycine (Figure 5.3), a key residue in coordinating the positioning of two of the three magnesium ions found in the binding pocket (Ferruz et al., 2016). The switch to glycine would significantly reduce polarity within the binding site, potentially affecting the activity of IMP1. Interestingly, this change has occurred in all Leishmania species.
In contrast, in the T. brucei IMP1 homologue, the equivalent aspartate is replaced by a serine residue, while in T. cruzi, this residue is conserved as aspartate. On the other hand, substitutions of the key residues found in IMP2 all occurred with amino acids of similar chemical properties, and would therefore be less likely to have a profound effect on the enzyme function (Figure 5.4).
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Lmex IMP1 45 TRNHPNDLMTQYEKQCKEEVLNILRVGTPSYAILSDGMH---S-EAVLGDGPTWIVGPID 100 Lmaj IMP1 45 TRNNPNDLVTQCEKHCKEEVLNILRVGTPSYAILSDDMH---S-EAVLGDGPTWIVGPID 100 Ldon IMP1 44 IRNNPNDLVTQYEKQCKEEVLNILRVGTPSYAILSDDVH---S-EAVLDDGPTWIVGPID 99 Linf IMP1 44 IRNNPNDLVTQYEKQCKEEVLNILRVGTPSYAILSDAVH---S-EAVLDDGPTWIVGPID 99 Lbra IMP1 49 TKTNTADLVTPCEKQCREEVLNILRAGTPSYAILSGEMH---R-EAVLGDGPTWIVGPID 104 Tbru IMPase2 42 TRENGEGLITQYDKQCEEEIIAILRLGAPSYDIISEELH---S-DTVLTDAPTWMVSTIN 97 Tcru IMPase2 42 TKVNSVDLVTQYDKQCEEEILTILRTGTPQYDILSEETY---S-DVGLSDKPTWVVDPID 97 Hsap IMPA1 35 LKSSPVDLVTATDQKVEKMLISSIKEKYPSHSFIGEESVAAGE-KSILTDNPTWIIDPID 93 Hsap IMPA2 46 TKTSAADLVTETDHLVEDLIISELRERFPSHRFIAEEAAASGA-KCVLTHSPTWIIDPID 104 Scer INM1 38 TGSRSVDIVTAIDKQVEKLIWESVKTQYPTFKFIGEESYVKG--ETVITDDPTFIIDPID 95 Scer INM2 40 DKANGVDLVTALDKQIESIIKENLTAKYPSFKFIGEETYVKG--VTKITNGPTFIVDPID 97 Mtub IMPA 35 VRKKGNDFATEVDLAIERQVVAALVAA-TGIEVHGEEFGGP-----AVDSRWVWVLDPID 88 Mtub SUHB 48 AKSSPTDPVTVVDTDTERLLRDRLAQLRPGDPILGEEGGGPADVTATPSDRVTWVLDPID 107 . * : . : : . . .:::. *:
Lmex IMP1 207 MCLVAAGRAELFWEAGVNPWNVAAGVIIVREAGGIVHDVENT-DGFDL------TRRGVC 259 Lmaj IMP1 207 MCLVAAGRAELFWKAGVNPWNVAAGVIIVREAGGVVHDVEST-DSFDL------TCRSVC 259 Ldon IMP1 206 MCLVAAGRAELFWEAGVNPWNVAAGVIIVREAGGVVHDVEST-DAFDF------TRRGVC 258 Linf IMP1 206 MCLVAAGRAELFWEAGVNPWNVAAGVIIVREAGGVVHDVEST-DAFDF------TRRGVC 258 Lbra IMP1 211 MCLVAAGRAELCWGVGVNPWDVAAGVIMVREAGGVVLDAESA-DTFDL------TRRGVC 263 Tbru IMPase2 204 MCFIATGRAELFLKVGVHPWDVAAATVIVREAGGVVHDIDNL-NILDL------TTCTVC 256 Tcru IMPase2 204 MCFVASGRAELYFEVGIYAWDIAAASIIVREAGGVVHNIDDA-QSLDL------MSRGFC 256 Hsap IMPA1 200 MCLVATGGADAYYEMGIHCWDVAGAGIIVTEAGGVLMDVTG--GPFDL------MSRRVI 251 Hsap IMPA2 211 LCHLASGAADAYYQFGLHCWDLAAATVIIREAGGIVIDTSG--GPLDL------MACRVV 262 Scer INM1 211 MAYIAMGYLDSYWDGGCYSWDVCAGWCILKEVGGRVVGANPGEWSIDVDNRTYLAVRGTI 270 Scer INM2 211 ICYVASGMLDAYWEGGCWAWDVCAGWCILEEAGGIMVGGNCGEWNIPLDRRCYLAIRGGC 270 Mtub IMPA 196 LVFVADGILGGAISFGGHVWDHAAGVALVRAAGGVVTDLAGQPWTPAS------RSAL 247 Mtub SUHB 215 LCMVAAGRLDAYYEHGVQVWDCAAGALIAAEAGARVLLSTPRAGGAGL------VVVAAA 268 : :* * * *: ... : .*. :
Figure 5.3| Sequence alignment of L. mexicana IMP1 with known and putative
IMP1 homologue from other Leishmania and Trypanosoma (annotated as IMPase2) species, as well as human, yeast and Mycobacterium IMPase. The alignment output has been abridged to focus on the key amino acid residues situated in the binding pocket, which are highlighted in grey (L. mexicana residue 80, 97, 99, 100, and 227). Sequence alignment was performed using Clustral Omega. Consensus key: (.), weak residue conservation; (:), strong residue conservation; (*), fully conserved residue. The following protein sequence accession numbers were used: L. mexicana XP_003874006.1, L. major XP_001682398.1, L. donovani XP_003860006.1, L. infantum XP_001464808.1, L. braziliensis XP_001563919.1, T. brucei XP_844941.1, T. cruzi XP_816379.1, H. sapiens NP_001138350.1 and NP_055029.1, S. cerevisiae NP_011912.1 and NP_010573.3, and M. tuberculosis NP_216120.1 and NP_217217.1.
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Lmex IMP2 108 REKP-----HLRFAFLTEELNPNT----LLSDDYTWVVDPVNGATSFSHGLPDCCISIGL 158 Lmaj IMP2 108 REKP-----HLRFAFLTEELDPDT----PLSDDYTWVVDPIDGATSFLHGLPDCCISIGL 158 Ldon IMP2 108 REKP-----HLRFAFLTEELNPGT----PLSDDYTWVVDPIDGAASFVHGLPDCCISIGL 158 Linf IMP2 108 REKP-----HLRFAFLTEELNPGT----PLSDDYTWVVDPIDGAASFVHGLPDCCISIGL 158 Lbra IMP2 61 REKP-----HLRFAFLT-ELNPDT----PLSDDYTWVIDPIDGAISFLHGLPDCCISIGL 110 Tbru IMPase1 105 NQKRGAGYTSFRFGFITEEICPDK----QLEDVPTWIVDPIDGTMSFVHGSCDCCVSIGL 160 Tcru IMPase1 103 DEKRNEPSEAFSFQFITEELTPDT----PLTDEPTWIVDPIDGTMSFVHGGCDCCISIGL 158 Hsap IMPA1 62 ------YPSHSFIGEESVAAG-EKSILTDNPTWIIDPIDGTTNFVHRFPFVAVSIGF 111 Hsap IMPA2 73 ------FPSHRFIAEEAAASG-AKCVLTHSPTWIIDPIDGTCNFVHRFPTVAVSIGF 122 Scer INM1 65 ------YPTFKFIGEESYVKG-ET-VITDDPTFIIDPIDGTTNFVHDFPFSCTSLGL 113 Scer INM2 67 ------YPSFKFIGEETYVKG-VT-KITNGPTFIVDPIDGTTNFIHGYPYSCTSLGL 115 Mtub IMPA 61 ------ATGIEVHGEEFGGP-----AVDSRWVWVLDPIDGTINYAAGSPLAAILLGL 106 Mtub SUHB 75 ------RPGDPILGEEGGGPADVTATPSDRVTWVLDPIDGTVNFVYGIPAYAVSIGA 125 . * .:::**::*: .: . :*
Lmex IMP2 392 QVAAGRVDAYLEPAGKAWDVCAGSLLVTEAGGVVCNMLGRPLDMAHGTAIVAAATQEMAD 451 Lmaj IMP2 387 QVAAGRVDAYLEPAGKAWNVCAGSLLVTEAGGVVCNMLGRPLDIAHDTTIVAAATQEMAD 446 Ldon IMP2 394 QVAAGRVDAYLEPAGKAWDVCAGSLLVTEAGGVVCNMLGRPLDMAHDTTIVAAATQEMAD 453 Linf IMP2 394 QVAAGRVDAYLEPAGKAWDVCAGSLLVTEAGGVVCNMLGRPLDMAHDTTIVAAATQEMAD 453 Lbra IMP2 336 HVAAGRVDAYLEPAGKIWNVCAGSLLVAEAGGVVYNMLGRPLDMAHDATIVAAATLEMAE 395 Tbru IMPase1 289 FVASGRIDLYMEPSGKIWDVCAGNLLVTEAGGVVKNIWGDEFEMERTTTIIAGANEKLVS 348 Tcru IMPase1 287 QVAAGRIDLYMEPAGMVWDVCAGSLLITEAGGVVKNMWGGDFGLEGTTTIIAAANETLAD 346 Hsap IMPA1 202 LVATGGADAYYEMGIHCWDVAGAGIIVTEAGGVLMDVTGGPFDLMSRRVIAAN-NRILAE 260 Hsap IMPA2 213 HLASGAADAYYQFGLHCWDLAAATVIIREAGGIVIDTSGGPLDLMACRVVAAS-TREMAM 271 Scer INM1 213 YIAMGYLDSYWDGGCYSWDVCAGWCILKEVGGRVVGANPGEWSIDVDNRTYLA-VRG--- 268 Scer INM2 213 YVASGMLDAYWEGGCWAWDVCAGWCILEEAGGIMVGGNCGEWNIPLDRRCYLA-IRG--- 268 Mtub IMPA 198 FVADGILGGAISFGGHVWDHAAGVALVRAAGGVVTDLAGQPWTPASRSALAGP--PRVHA 255 Mtub SUHB 217 MVAAGRLDAYYEHGVQVWDCAAGALIAAEAGARVLLSTPRAGGAG--LVVVAA-APGIAD 273 :* * . . . *: ... : .*. :
Figure 5.4| Sequence alignment of L. mexicana IMP2 with known and putative IMP2 homologue from other Leishmania and Trypanosoma (annotated as IMPase1) species, as well as human, yeast and Mycobacterium IMPase. The alignment output has been abridged to focus on the key amino acid residues situated in the binding pocket, which are highlighted in grey (L. mexicana residue 120, 137, 139, 140, and 410). Sequence alignment was performed using Clustral Omega. Consensus key: (.), weak residue conservation; (:), strong residue conservation; (*), fully conserved residue. The following protein sequence accession numbers were used: L. mexicana XP_003873614.1, L. major XP_001682002.1, L. donovani XP_003859618.1, L. infantum XP_001464427.1, L. braziliensis XP_001563539.1, T. brucei XP_827004.1, T. cruzi XP_811529.1, H. sapiens NP_001138350.1 and NP_055029.1, S. cerevisiae NP_011912.1 and NP_010573.3, and M. tuberculosis NP_216120.1 and NP_217217.1.
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5.3 L. mexicana IMP1 and IMP2 are co-localised to the ER
5.3.1 Bioinformatic prediction of localisation
Bioinformatic analysis of the Leishmania IMPase sequences using PSORTII
(Nakai & Horton, 1999) did not reveal any canonical sorting/localisation motifs in
these proteins. IMP2 was predicted to have one [between residue 169 to 186, based
on Phobius (Käll et al., 2007)] or two transmembrane domains [between 51 to 72
and 167 to 188 based on CCTOP (Dobson et al., 2015)], although other algorithms
predicted no transmembrane domains [DAS-TMfilter (Cserzo et al., 2004) and
TMHMM (Krogh et al., 2001)].
5.3.2 Localisation by GFP-fusion proteins
To determine the subcellular localisation of L. mexicana IMP1 and IMP2,
constructs encoding IMP1 or IMP2 fused to either N- or C-terminal GFP were
transfected into L. mexicana promastigotes (see Section 2.3.4). In contrast to the
IMP1-GFP fusion proteins, the N-terminal GFP-IMP1 fusion protein and both
GFP-IMP2 fusion proteins were poorly expressed in promastigotes (Figure 5.5),
resulting in poor signals when the transfectants were analysed by fluorescence
microscopy. The detection of GFP reactive bands with a lower molecular weight
corresponding to GFP alone (27 kD) in the transfectants expressing IMP1-GFP and
GPF-IMP2, also indicated substantial proteolytic degradation of these fusion
proteins, which created additional interference that could potentially mask the true
locations of the intact proteins.
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IMP1 IMP2 N C N C
260 160 110 80 GFP-fused IMP2 (77 kD)
60 GFP-fused IMP1 (58 kD) 50 40
30 20
80 60 BiP
Figure 5.5| Episomal expression of GFP-fused IMPase is inconsistent in L. mexicana promastigotes. Western blot of cell protein lysates from L. mexicana promastigotes transfected with GFP vector constructs of IMP1 or IMP2 fused to N- or C-terminal GFP, probed with anti-GFP mouse antibody. BiP was used as loading control.
5.3.3 Localisation by HA-tagging
As addition of GFP (27 kD), could interfere with the normal processing and/or folding of the IMPase, fusion proteins containing the smaller triple haemagglutinin
(3×HA) tag (with a predicted MW of 3 kD) were also generated. After parasites were transfected with plasmids expressing either IMP1 or IMP2 tagged with N- or
C-terminal 3×HA (see Section 2.1.7), high levels of expression of both N- and
C-terminally tagged IMP1 and N-terminally tagged IMP2 were achieved as shown by immunoblotting (Figure 5.6).
Both N- and C-terminal HA-tagged IMP1 exhibited a reticulate or punctate distribution after fixation of the promastigotes with paraformaldehyde and labelling with anti-HA antibody (see Section 2.6.2). The reticulate distribution was
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often associated with the nuclear envelop or the cell periphery, and overlapped
significantly with antibody directed against the major ER chaperone protein BiP
(Figure 5.7A). Areas of non-overlap were also noted for more diffuse staining,
which could reflect low level distribution in the cytosol. Similar distributions were
observed for both N- and C-terminally tagged IMP2 proteins (Figure 5.7B).
Collectively, these results suggest that IMP1 and IMP2 are primarily co-localised
to the ER of L. mexicana promastigotes although some protein could be located in
the cytosol or other structures.
IMP1 IMP2
N C N C 260 160 110 80 60 50 HA-fused IMP2 (53 kD) 40 HA-fused IMP1 (35 kD) 30 20
15
80 60 BiP Figure 5.6| L. mexicana promastigotes transfected with episomal constructs expressed HA-tagged IMP1 and IMP2 recombinant proteins. Immunoblotting of cell protein lysates probed with anti-HA mouse antibody confirmed L. mexicana promastigotes transfected with pXG-3xHA-IMP1 (N) or pX-IMP1-3xHA (C) (left) or pXG-3xHA-IMP2 (N) or pX-IMP2-3xHA (C) (right) expressed the corresponding recombinant IMP1 or IMP2 fused to N- or C-terminal 3xHA epitope. Anti-BiP staining was used as loading control.
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Merge
(Continued next on page)
K
N
oechst
H
BiP
HA
3xHA terminal - C
3xHA terminal - N
(A) (A) IMP1
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. . Cells were fixed
promastigotes were were promastigotes
igotes igotes were transfected with
, , respectively
Merge
L. mexicana L.
(B) (B)
.
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promast
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and analysed by immunofluorescence
terminal 3
e bars = 10 e = bars μm
-
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-
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×
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stained stained with Hoechst,
terminal 3 terminal
-
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-
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-
BiP
to express IMP2 fused
[nucleus (N) and kinetoplast (K)] [nucleuskinetoplast and (N)
fused to an to fused
HA
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-
HA HA and anti
-
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-
, Blue: Hoechst , Blue:
(ER)
HA to express IMP1 IMP1 express to HA
×
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-
-
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IMP1 IMP1 and IMP2 are associated with the promastigote ER.
HA
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-
-
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B
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with with paraformaldehyde Figure 5.7| pXG transfected with pXG microscopy. Green:
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5.4 L. mexicana imp1 is not essential in vitro
In order to investigate the role these putative IMPase enzymes play in L. mexicana growth and virulence, attempts were made to delete the imp1 and imp2 genes by homologous recombination and replacement of the imp genes with drug resistance cassettes. A set of linear knockout constructs encoding puromycin and bleocin resistance cassettes were generated (see Section 2.1.8) and sequentially transfected into L. mexicana promastigotes to target opening reading frames (ORF)
LmxM.17.1390 (Figure 5.8) and LmxM.15.0880.
5’ UTR PURO 3’ UTR
LMXM.17.1380 LMXM.17.1390 LMXM.17.1400
LMXM.17.1380 LMXM.17.1390 LMXM.17.1400
5’ UTR BLEO 3’ UTR Figure 5.8| Targeted deletion of myo-inositol monophosphatase 1 (imp1) in L. mexicana. Linear knockout constructs, consisting of puromycin (f) and bleocin (BLEO) resistance cassettes flanked by approximately 1 kb of imp1 (LMXM.17.1390) UTR regions, were sequentially used to transfect wild-type L. mexicana by electroporation. Successful replacement of imp1 loci by homologous recombination and the resulting null mutants were selected by gaining of resistance to both antibiotics and confirmed via PCR analysis.
Following two rounds of transfection and clonal selection by limiting dilution in the presence of selection drugs (see Section 2.3.3), PCR with diagnostic primers
(Table 5.1) were used to verify the deletion of imp1 gene and correct integration of the resistance cassettes in the drug-resistant clones that were recovered. One of the clones isolated using this approach (Figure 5.9) was designated as Δimp1.
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In contrast, drug-resistant parasites recovered after transfection with the linear
constructs targeting imp2 were found to still possess the imp2 ORF by PCR.
Several attempts were made to optimise the linear constructs by altering the
flanking UTR sequences to better target the gene, and multiple transfections were
conducted in order to overcome possible trisomic alleles. Promastigotes were also
transfected with an episome expressing a copy of imp2, prior to attempting deletion
of the chromosomal copies, to minimise selection of parasites with amplified
genomic loci of imp2 and to facilitate deletion of imp2 in the event that the gene
proved essential for parasite viability. However, none of these strategies were
successful in generating an imp2-null mutant.
Table 5.1| PCR primers for diagnosing imp1 and imp2 deletion and complementation. Bracketed ID indicate primers used for verifying imp2 deletion, and provided the same function as their imp1 counterparts (see Figure 5.9). Primer ID Sequence imp1 ORF Fwd A CTACCCGGGATGACGCAGCCCTCCCT imp1 ORF Rev B CGAGGATCCTCAAGATGACGTGTTCAACACC imp2 ORF Fwd [A] CTACCCGGGATGAGCATCAGCGTCCTGT imp2 ORF Rev [B] CGAGGATCCTCACTCGCTCTCGACCAA
imp1 5’ Flank Fwd C CGTCTGTCAAGCCTCCGAT imp2 5’ Flank Fwd [C] TTCGAACTGCCCGACAAGGA PURO Rev D CGTGGGCTTGTACTCGGTC BLEO Rev E GCACTGGTCAACTTGGCC pXG 5’ Flank Fwd F GCGTGCACATCATCAACTGT
Subsequent studies therefore focused on the characterisation of the imp1
mutant. In order to definitively attribute any phenotypes observed in the mutant
cell line to the loss of imp1, an add-back cell line was also created by introducing
the vector construct pXG-IMP1 (see Section 2.1.9) into Δimp1 in order to restore
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IMP1 expression. The presence of imp1 in the add-back strain was verified by PCR
(Figure 5.9).
C D/E
LMXM.17.1380 5’ UTR PURO or BLEO 3’ UTR LMXM.17.1400
LMXM.17.1380 imp1 LMXM.17.1400
A B F Δimp1 WT Δimp1 +pXG-IMP1 B
O P B E O P B E O P B E pXG-IMP1
5000 4000 3000 2500 2000 1500 Expected sizes: 1000 O = 885 bp 800 600 P = 1494 bp B = 1428 bp E = 1021 bp
Figure 5.9| Δimp1 and episomal add-back mutants display the expected genotype. PCR was performed on L. mexicana wild type, Δimp1, and Δimp1+pXG- IMP1 add-back cell lines to confirm presence or absence of imp1 ORF (O) using primers A and B (Table 5.1), integration of puromycin resistance cassette (P) using primers C and D, integration of bleocin resistance cassette (B) using primers C and E, and episome pXG-IMP1 (E) using primers B and F.
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5.5 The L. mexicana Δimp1 mutant retains myo-inositol prototrophy but exhibits a distinct growth defect in vitro
To first determine whether the loss of imp1 resulted in myo-inositol auxotrophy,
wild-type, ∆imp1 and add-back (Δimp1+pXG-IMP1) cell lines were cultivated in
complete RPMI medium containing 10 % iFBS, and in CDM containing various
concentrations of myo-inositol. Surprisingly, the Δimp1 exhibited a distinct slow
growth phenotype, compared to wild type promastigotes in rich medium and failed
to reach the same maximum cell density as wild type (Figure 5.10A). This growth
phenotype was complemented by re-expression of imp1 in the mutant. The
difference in growth rate between wild type and ∆imp1 promastigotes was also
observed in CDM containing 0 to 1 mM myo-inositol (Figure 5.10B), suggesting
that the loss of imp1 does not lead to myo-inositol auxotrophy but does cause a
modest growth defect in vitro.
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A (A) B
(B)
Figure 5.10| Deletion of imp1 results in slower promastigote growth that is not rescued by addition of exogenous myo-inositol. (A) Growth of L. mexicana wild type, Δimp1, and episomal add-back (Δimp1+pXG-IMP1) promastigotes was established at 1x105 cells/mL in RPMI supplemented with 10 % iFBS on day 0. Cell density was measured daily. Error bars = SD of 3 replicate experiments; (B) CDM- adapted L. mexicana wild-type and Δimp1 promastigotes were inoculated from 1x105 cells/mL in CDM with various levels of myo-inositol. Cell density was determined daily. Error bars = SD of 3 replicate experiments.
5.6 L. mexicana Δimp1 is unable to survive in
ex vivo infected macrophages or cause disease in the mouse model
In order to investigate whether the deletion of imp1 also affected L. mexicana
infectivity of the mammalian hosts, in vitro infection assays of BMDMs were
performed using wild-type, Δimp1 and Δimp1+pXG-IMP1 stationary-phase
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promastigotes under standard assay conditions (see Section 2.4.2 and 2.4.3). Both
wild-type and add-back parasites underwent one or more cell divisions within
macrophages over the course of seven days, while Δimp1 parasites did not replicate
within macrophages (Figure 5.10A) and were progressively cleared over the same
period (Figure 5.10B).
To investigate whether the loss of imp1 would also lead to loss of virulence
in vivo, susceptible BALB/c mice were inoculated with wild-type, Δimp1, and
add-back stationary-phase promastigotes by subcutaneous injection (see Section
2.5.1). Lesion development was followed over 30 weeks (see Section 2.5.2). While
both wild-type and add-back strains developed large lesions within 10-15 weeks
and had to be euthanised, none of the mice infected with the ∆imp1 strain
developed lesions over the 30 weeks (Figure 5.11A).
Finally, in order to determine whether any Δimp1 parasites survived in
infected tissues and were capable of establishing low level chronic infections,
tissue from the site of inoculation and draining lymph node were harvested after
∆imp1-infected mice were culled at week 30, and parasite burden were assessed by
limiting dilution (see Section 2.5.3 & 2.5.4). No parasites were recovered from
skin or lymph node tissue of mice infected with Δimp1 (Figure 5.11B & C), while
parasite burdens corresponding to more than 108 and 106 parasites were observed
in the skin and lymph node, respectively, of mice infected with wild-type and the
add-back cell line.
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(A) (B)
(C) Wild type Δimp1 Δimp1+pXG-IMP1
Figure 5.11| L. mexicana Δimp1 parasites are unable to replicate in macrophages in vitro. BALB/c bone marrow-derived macrophages were infected with L. mexicana wild-type, Δimp1, and Δimp1+pXG-IMP1 promastigotes. Cell assays were conducted in RPMI supplemented with 10 % iFBS, while (A) percentage of infected macrophages and (B) parasite burden (parasites per infected macrophage) were determined by microscopy at day 0, 3, 5, and 7 post-infection. Error bars indicate SEM from 3 independent experiments. (C) Representative images of microphage infection assay at day 7 observed at 64x magnification. The cells were fixed with methanol and stained with propidium iodide. The outline of a macrophage is marked, and each small punctate is the nucleus of an L. mexicana amastigote within the PV. Scale bars = 10μm.
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(A)
(B) (C)
Figure 5.12| L. mexicana Δimp1 are unable to generate lesions in a susceptible mouse model of infection and are completely cleared by the host. BALB/c mice were infected by subcutaneous injection with L. mexicana wild-type, Δimp1, and Δimp1+pXG-IMP1 promastigotes. (A) Lesion formation was scored weekly. Error bars indicate SEM, n=5. Amastigotes were subsequently extracted from (B) the inoculation site and (C) inguinal draining lymph node after reaching a lesion score of 4 (for wild type and add-back) or after 30 weeks post-infection (for Δimp1). Cell counts were determined by microscopy (inoculation site) and limiting dilution (lymph node). Error bars indicate SEM, n = 4 technical replicates from five mice.
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5.7 Discussion
As shown in Chapter 3 & 4, the first enzyme in the pathway for de novo myo-inositol synthesis in Leishmania is essential for virulence in macrophages and susceptible animal models. Unexpectedly, the results from this chapter show that the deletion of imp1 resulted in only a modest growth defect in vitro that this was not due to myo-inositol auxotrophy. Notwithstanding the most modest growth phenotype in culture, this mutant exhibited a dramatic loss of virulence phenotype in animal models. On the other hand, attempts to delete the second imp2 gene using homologous recombination were unsuccessful. Together, these findings suggest that IMP1 and IMP2 may have redundant functions in regulating the levels of myo-inositol-3-phosphate and/or that they may function in other metabolic pathways. These findings are discussed in more details in the following sections.
5.7.1 Comparison between the two L. mexicana IMPase isoforms, IMP1 and IMP2
The genomes of L. mexicana and other human-infective species of Leishmania are all predicted to encode two putative IMPase isoforms. Protein sequence alignment revealed that IMP1 and IMP2 share high levels of sequence conservation within common domains, but that IMP2 differs markedly from IMP1 in containing five significant inserts and extensions. Leishmania proteins commonly have additional peptide inserts or C/N-terminal extensions that are not found in homologous proteins from other species (Souza et al., 1992; Williams et al., 2003; Zhang et al.,
2016). While the functional significance of these modifications is generally unknown, they could reflect novel allosteric or protein-protein dependent regulatory mechanisms. There is increasing evidence that Leishmania are more
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dependent on post-transcriptional regulation than most other eukaryotes by virtue
of lacking conventional transcription factors (Brandau et al., 1995; Morales et al.,
2010), which is likely to have led to the evolution of new post-transcriptional
regulatory mechanisms. Interestingly, similar sequence differences are also found
between the two IMPase isoforms of T. brucei and T. cruzi, suggesting the
diversification of these isoforms occurred before the evolutionary divergence of
Leishmania and Trypanosoma.
Importantly, both IMP1 and IMP2 retain residues around the catalytic pocket
that are highly conserved in all IMPase from across eukaryotic and prokaryotic
phylogenetic domains. The only divergence in sequence conservation in the
catalytic pocket occurred in IMP1 which has an aspartate-to-glycine substitution at
the position responsible for coordinating the position of the Mg2+ cofactors. This
change was conserved in IMP1 sequences from all Leishmania species analysed.
Interestingly, while substitution at the corresponding position is absent in T. cruzi,
an aspartate-to-serine mutation is also found in T. brucei TbIMPase2 (the
Leishmania IMP1 homologue) (Cestari et al. (2016). However, due to the retention
of the polar hydroxyl group, this change is likely to be less disruptive. TbIMPase2
has been shown to have IMPase activity, but only when in complex with
TbIMPase1 (the Leishmania IMP2 homologue) (personal communication, Prof.
Terry Smith, St Andrews University, Scotland). While this finding suggests that
the substitution of aspartate for serine does not result in loss of activity, these
changes may have occurred in the context of dimerised substrate binding site. As
attempts to directly measure the activity of the Leishmania IMPases have been
unsuccessful to date (see Chapter 6), it remains unclear whether the substitution
of aspartate to glycine in Leishmania IMP1 alters enzyme activity.
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HA-tagging of the two L. mexicana IMPase proteins in conjunction with immunofluorescence imaging revealed that both IMP1 and IMP2 were associated with extensive reticulate structures that largely co-localised with the ER marker,
BiP. Further studies will be needed to confirm whether all of IMP1 and IMP2 is localised to the ER or whether these enzymes are also partially distributed in the cytoplasm, as indicated by incomplete localisation with BiP. Interestingly,
TbIMPase1 in T. brucei has been reported to be ER-localised (Martin & Smith,
2006a). The ER localisation of IMPase in L. mexicana and T. brucei is consistent with the proposed compartmentalised model of de novo myo-inositol biosynthesis
(see Section 1.6.2). In this model, de novo synthesised Ino3P is selectively used for the biosynthesis of ER-located PI pools that are subsequently used for GPI biosynthesis. While it remains unclear how Ino3P synthesised by cytoplasmic
INO1 is channelled into the ER for PI synthesis, the localisation of both IMP1 and
IMP2 to the ER raises the possibility that these enzyme could form a complex with
ER-located PI synthase (and INO1) to channel myo-inositol into PI synthesis.
Further studies will be needed to determine whether the IMPases and the PI synthase are located on the cytoplasmic or luminal faces of the ER. Localisation of the PI synthase in the lumen of the ER would require the presence of an additional myo-inositol/inositol phosphate transporter to import cytoplasmic pools of Ino3P or myo-inositol into this compartment. The possibility that the Leishmania IMPase may form a complex on the ER membrane is supported by the finding that recombinant TbIMPase1 and TbIMPase2 form a functionally active hetero-oligomeric complex in vitro (Prof. Terry Smith lab, unpublished data; also discussed in more detail in Section 6.7.4), and studies in other organisms which have shown that IMPase readily undergoes oligomerisation (Gee et al., 1988; Chen
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& Roberts, 1999; Brown et al., 2007; Ohnishi et al., 2007). Finally, the lack of
predicted transmembrane regions in IMP1 and weak transmembrane domain
predictions for IMP2, suggest any IMPase complex may involve additional
interactions with other membrane-associated proteins.
5.7.2 The role of imp1 in Leishmania proliferation and virulence
Notwithstanding the importance of IMPase in myo-inositol metabolism,
surprisingly few studies have investigated whether these enzymes are essential for
the virulence of pathogenic organisms. Genetic knockout of TbIMPase2
(homologue of IMP1) in T. brucei bloodstream stages results in a slightly reduced
growth rate in vitro, and delayed onset of parasitaemia in mice, although infection
with this mutant was still ultimately fatal (Cestari et al., 2016). On the other hand,
disruption of TbIMPase1 (homologue of IMP2) had no effect on the growth of the
bloodstream forms (Cestari & Stuart, 2015). As deletion of either TbIMPase1 or
TbIMPase2 is expected to lead to a dramatic drop in IMPase activity, based on in
vitro studies showing that the two IMPases form a hetero-oligomeric complex, it
is likely that T. brucei bloodstream forms can salvage most of their myo-inositol
requirements from the serum. On the other hand, M. tuberculosis and other
actinomycetes are also able to synthesise myo-inositol de novo using a
eukaryotic-like pathway. The myo-inositol synthesised by this pathway is used to
generate PI and a complex array of cell wall lipoglycans (Jackson et al., 2000), as
well as the major redox thiol, mycothiol (Sareen et al., 2003). Interestingly, three
out of the four M. tuberculosis IMPase homologues (impA, suhB, cysQ) were found
to be non-essential for myo-inositol synthesis, while the isoform encoded by impC
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was crucial for cell viability (Movahedzadeh et al., 2010). Whether these different isoforms are involved in synthesising myo-inositol for different secondary metabolites is currently unknown.
In summary, IMP1 is dispensable for growth in rich medium (although required for optimal growth), but is crucial for intracellular growth in macrophages and pathogenicity in BALB/c mice. Surprisingly, the modest growth defect of
Δimp1 promastigotes in vitro is independent of the availability of exogenous myo-inositol. These results strongly suggest that IMP2 can sustain sufficient flux through the myo-inositol biosynthetic pathway under myo-inositol starvation conditions to maintain parasite growth. Whether, or to what extent, IMP1 forms a complex with IMP2 and is required for maximum IMPase activity remains to be determined. Alternatively, IMP1 could be involved in other metabolic pathways or perform non-metabolic functions.
5.7.3 The essentiality of imp2
Several attempts were also made to generate a L. mexicana mutant that lacks imp2.
Integration of drug selection markers into the native imp2 chromosomal loci was confirmed by PCR analysis. However, all clones recovered were subsequently shown to have retained an additional chromosomal copy of the imp2 gene.
Attempts to delete the chromosomal alleles of imp2 after first transfecting parasites with a plasmid expressing imp2 were also unsuccessful. This difficulty in deleting all alleles of certain genes in Leishmania using sequential homologous gene replacement has previously been described (Cruz et al., 1993), and one of the key obstacles is the remarkable ability of these parasite to undergo and tolerate aneuploidy, or changes in chromosome copy number. Indeed, sequencing of L.
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mexicana genome has indicated that chromosome 15 (which contains the imp2
gene) displays a higher tendency to be trisomic (Rogers et al., 2011). In support of
this notion, our group has recently shown that it is possible to delete imp2 using a
newly developed CRISPR/Cas9 system (Beneke et al., 2017). Preliminary PCR
analysis has confirmed the imp2-null genotype of the mutants isolated from clonal
selection. This observation highlights the much greater efficiency of CRISPR/Cas9,
which is capable of targeting all the alleles simultaneously (Ran et al., 2013;
Beneke et al., 2017) and also shows that imp2 is not essential for L. mexicana (at
least in the presence of exogenous myo-inositol). Further experiments are needed
to determine whether the deletion has any phenotypic consequences.
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CHAPTER 6 Functional Analysis of L. mexicana Δimp1
6.1 Introduction
Eukaryotic cells express one or more IMPase enzymes which are involved in de novo myo-inositol biosynthesis and/or regulation of mono-phosphorylated myo-inositol generated during catabolism of phosphoinositides. However, there is increasing evidence that IMPase can have broader substrate specificities beyond inositol phosphates. In higher plants (Arabidoposis and Actinidia deliciosa), members of the IMPase family have been shown to catalyse the dephosphorylation of L-galactose 1-phosphate to L-galactose, one of the final steps in ascorbic acid biosynthesis (Laing et al., 2004). An IMPase with similar activity has been isolated from mammalian brain tissues (Parthasarathy et al., 1997). In the archaea,
Methanococcus jannaschii, and other thermophilic organisms, proteins with high homology to IMPase have been shown to catalyse the dephosphorylation of fructose 1,6-bisphosphate to fructose 6-phosphate, and to replace the canonical enzyme, fructose 1,6-bisphosphatase that normally catalyses the last committed step in gluconeogenesis (Stec et al., 2000). The archaeal enzymes can also dephosphorylate NADP(H) and inositol bisphosphates, supporting a broad substrate specificity (Fukuda et al., 2007). Similarly, the M. tuberculosis IMPase homologue, CysQ, possesses both IMPase and FBPase activity (Gu et al., 2006),
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and can also dephosphorylate 3’-phosphoadenosine 5’-phosphate and
3’-phosphoadenosine 5’-phosphosulfate, the key metabolic intermediates of the
sulphate assimilation pathway (Hatzios et al., 2008).
In Chapter 5, deletion of the L. mexicana IMPase gene, imp1, did not result
in myo-inositol auxotrophy, indicating that loss of IMP1 can be compensated for
by IMP2 and/or that IMP1 is not involved in de novo myo-inositol synthesis.
Interestingly, ∆imp1 mutant promastigotes exhibited a modest growth defect in rich
culture medium, but were severely attenuated in their capacity to survive in
macrophages or to establish infections in susceptible mice. This growth and
virulence phenotype was completely reversed by complementation of the mutant
with a plasmid expressions imp1. These data suggest that IMP1 is largely redundant
in promastigote stages but that this enzyme fulfils an important function in
amastigote stages. In this chapter, we have further investigated the metabolic
phenotype of the ∆imp1 mutant and the capacity of this mutant to differentiate to
amastigotes.
6.2 The Δimp1 mutant is able to synthesise myo-inositol
To investigate whether deletion of imp1 led to a defect in de novo myo-inositol
synthesis, wild-type and ∆imp1 mutant promastigotes were cultivated in the
presence of 13C-U-glucose for 24 hours and incorporation of 13C into Ino3P and
free myo-inositol was monitored over time by GC-MS (see Section 2.7).
Promastigotes were initially cultivated in complete RPMI supplemented with 10 %
iFBS (contains an equivalent of 200 µM myo-inositol) to mid-log phase, then
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resuspended in CDM supplemented with or without 200 µM myo-inositol for the labelling experiments. Similar to what was previously observed in both wild-type and ∆ino1 promastigotes (see Section 4.2), intracellular levels of myo-inositol remained high (approximately 6 nmol per 4×107 promastigotes) in ∆imp1 promastigotes cultivated under myo-inositol-replete conditions (Figure 6.1A).
13C-labelling in these myo-inositol pools remained below 5 % over the 24-hour labelling period, indicating that these pools are primarily sustained by uptake from the medium (Figure 6.1B). However, the low but detectable levels of
13C-enrichment of myo-inositol in both wild-type and ∆imp1 promastigotes suggested that de novo synthesis continues even when exogenous myo-inositol is available and that this pathway is still active in the ∆imp1 mutant. In support of this conclusion, incorporation of 13C-label derived from 13C-U-glucose into myo-inositol increased markedly (up to 50 % fractional labelling) in both wild-type and ∆imp1 promastigotes suspended in CDM lacking exogenous myo-inositol.
However, cultivating promastigotes in myo-inositol-free medium also resulted in a rapid decrease in the overall size of intracellular myo-inositol pools comparable to what was observed in Section 4.2 (Figure 6.1A). Interestingly, by calculating the proportion of total myo-inositol that is de novo synthesised at the 24-hour time point, the results showed that while both wild type and ∆imp1 increased their intracellular levels of de novo myo-inositol in response to the depletion of exogenous myo-inositol by 79.4 and 53.7 %, respectively, ∆imp1 also maintained a basal level of de novo myo-inositol that is two-fold that of wild-type even when exogenous myo-inositol supply was abundant (Figure 6.1C).
In contrast to myo-inositol, the intracellular levels of Ino3P, the substrate for
IMPase enzymes, remained relatively constant under both myo-inositol-replete and
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myo-inositol-starved conditions in both wild-type and ∆imp1 promastigotes
(Figure 6.1A). However, fractional 13C-enrichment in Ino3P of ∆imp1 also
mirrored that of wild type, which increased steadily under myo-inositol starvation
conditions but was highly repressed in the presence of exogneous myo-inositol
(Figure 6.1B). This further supported the fact that de novo myo-inositol synthesis
remained undisturbed in the absence of imp1.
(A)
(B)
(C)
Figure 6.1| The de novo myo-inositol synthesis pathway remains functional in Δimp1. L. mexicana wild type and Δimp1 were cultivated either in CDM containing 200 µM inositol (+ino) or no myo-inositol (-ino), and labelled with 13C-U-glucose over 24 hours. Parasites (4×107 cell-equivalent) were harvested at indicated time points, and (A) intracellular levels and (B) percentage of 13C-enrichment of Ino3P and myo-inositol were measured by GC-MS. (C) The levels of free myo-inositol synthesised de novo at the 24-hour time point were determined.
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Analysis of the steady-state intracellular concentrations of myo-inositol in wild-type and ∆imp1 promastigotes grown in the presence or absence of exogenous myo-inositol suggests these parasites rapidly accumulate myo-inositol if exogenous concentrations exceed intracellular concentrations. To investigate whether de novo synthesised myo-inositol is exported by both cell lines when exogenous concentrations are low, the levels of 13C-labelled myo-inositol in the culture supernatant of 13C-U-glucose-labelled wild type and ∆imp1 promastigotes were measured by GC-MS. The results showed that both wild-type and Δimp1 promastigotes secrete newly synthesised myo-inositol into the culture media
(Figure 6.2). Interestingly, this extracellular pool appeared to reach a steady state concentration within 2 hours, suggesting that parasites may recycle secreted myo-inositol when it reaches a threshold concentration. The identical kinetics of
13C-labelling of secreted myo-inositol in the medium of wild-type and Δimp1 parasites, provides further support for the conclusion that loss of imp1 has little effect on the synthesis of myo-inositol in promastigote stages.
Figure 6.2| Extracellular myo-inositol abundance and percentage of 13C- enrichment of L. mexicana wild type and Δimp1. L. mexicana wild-type and Δimp1 promastigotes were incubated in CDM supplemented with 200 µM myo- inositol (+ino) or no myo-inositol (-ino), and labelled with 13C-U-glucose over 24 hours. Culture medium samples were collected at indicated time points and the levels and 13C-enrichment of myo-inositol in 10 µL samples were measured by GC- MS. Error bars represent SD from three replicates.
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6.3 Inositol lipid biosynthesis is unchanged in ∆imp1 mutant
To investigate whether IMP1 may have a role in regulating Leishmania inositol
lipid metabolism downstream of de novo myo-inositol synthesis, L. mexicana
wild-type and Δimp1 promastigotes were cultivated in myo-inositol-free CDM or
CDM supplemented with 200 µM myo-inositol. Parasites were harvested at various
time points and lipids extracted and analysed by high-performance thin-layer
chromatography (HPTLC) (see Section 2.8.1). As shown in Figure 6.3, levels of
the bulk inositol lipids (PI and IPC), phosphoinositides (PIP), and free GPI
glycolipids (iM2, iM3 and iM4) were expressed at similar levels in both parasite
lines, regardless of the presence or absence of exogenous myo-inositol. These
results contrast with the situation in the ∆ino1 mutant, where myo-inositol
starvation resulted in the rapid depletion of bulk PI and phosphoinositides (see
Section 4.4). Therefore, IMP1 does not seem to be directly involved in regulating
the intracellular levels of these inositol lipids.
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PC PE PI IPC
GIPL iM2 PIP GIPL iM3 GIPL iM4 Origin
Day 0 1 2 3 1 2 3 0 1 2 3 1 2 3
Inositol +ino -ino +ino -ino
Cell type Wild type Δimp1 Figure 6.3| Lipid composition of Δimp1 is unaffected by the absence of exogenous myo-inositol. L. mexicana wild-type and Δimp1 promastigotes were cultivated in CDM with 200 µM myo-inositol (+ino) or no myo-inositol (-ino). Cells were harvested at day 0, 1, 2, and 3, and the extracted lipid samples were run on high-performance thin-layer chromatography (HPTLC) before being stained with orcinol and heat charred for visualisation. Identification was based the lipid reference described by Ralton & McConville (1998). Abbreviations used: GIPL, glycoinositol phospholipid; Ino, myo-inositol; IPC, inositol phosphorylceramide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate.
6.4 Changes in Δimp1 promastigote central
carbon metabolism are subtle
To further examine whether the loss of imp1 results in detectable changes in central carbon metabolism, wild type and ∆imp1 promastigotes were labelled with
13C-U-glucose in CDM with or without exogenous myo-inositol (200 µM).
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Parasites were sampled and metabolically quenched at various time points over 24
hours and polar metabolites were extracted and analysed by GC-MS (see Section
2.8). The analysis showed 13C-U-glucose is taken up and the carbon backbone
rapidly incorporated into intermediates of central carbon metabolism, including
metabolites of glycolysis, the succinate fermentation pathway, and the TCA cycle
(Figure 6.4). Strikingly, the kinetics of labelling for most intermediates in central
carbon metabolism were essentially indistinguishable between wild-type and
∆imp1 promastigotes, and within each of the lines grown in the presence or absence
of myo-inositol. No significant differences was also observed in the intracellular
levels of these metabolites.
On the other hand, significant and reproducible differences were observed in
the rate of labelling of serine and glycine (Figure 6.4). The rate of de novo
synthesis of these amino acids is normally low in wild-type parasites, but was
elevated in ∆imp1 promastigotes. Serine and glycine are synthesised from the
glycolytic intermediate, 3-phosphoglycerate (3PG), and the more rapid labelling of
these amino acids could reflect increased flux through lower glycolysis or reduced
uptake of exogenous serine and/or glycine.
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Glycolytic and TCA intermediates
Amino acids
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Figure 6.4| Intracellular 13C-enrichment of key carbon metabolites show modest differences between wild type and Δimp1. L. mexicana wild-type and Δimp1 promastigotes were incubated in complete defined medium supplemented with 200 µM myo-inositol (+ino) or myo-inositol-free medium (-ino), and labelled with 13C-glucose over 24 hours. Promastigotes (4×107 cell-equivalent) were harvested at indicated time points and the 13C-enrichment of key polar intracellular metabolites measured by GC-MS. Abbreviations used: 3PG, 3-phosphoglycerate; Glc6P, glucose 6-phosphate; Fru6P, fructose 6-phosphate; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; Succ-CoA; succinyl- CoA; α kG, α-ketoglutarate.
To further assess the metabolic phenotype of the ∆imp1 mutant, wild-type and
mutant promastigotes were cultivated in CDM with or without myo-inositol for 24
hours, and changes in metabolite levels in the extracellular media were measured
at various time points by GC-MS. Both parasite lines utilised glucose as a major
carbon source, and depleted this substrate with essentially identical kinetics under
both growth conditions (Figure 6.5). As expected, the major secreted end products
of glucose catabolism were succinate (the major product of glycosomal succinate
fermentation) and alanine (derived from transamination of pyruvate). While the
rate of succinate secretion was similar in both lines and under both growth
conditions, the rate of alanine secretion was appreciably slower in the ∆imp1 line.
Decreased alanine secretion could reflect increased flux of pyruvate into other
pathways, such as the TCA cycle. In support of this hypothesis, ∆imp1
promastigotes also secreted lower amounts of malate. Malate is an intermediate in
succinate fermentation and can be secreted or transported from the glycosome to
the mitochondrion to top up TCA cycle intermediates (Saunders et al., 2011).
Interestingly, the rate of uptake of aspartate, serine, and tryptophan were also
decreased in the ∆imp1 mutant. Overall, these results suggest that glucose
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metabolism might be subtly remodelled in the ∆imp1 mutant, with increased flux of glycolytic pyruvate and malate into the TCA cycle. These changes are likely to be linked to the slow growth rate of the ∆imp1 promastigotes, which is not modulated by the availability of myo-inositol.
Glycolytic and TCA intermediates
Amino acids
Figure 6.5| Abundance of key extracellular metabolites from L. mexicana wild type and Δimp1. L. mexicana wild-type and Δimp1 mid-log phase promastigotes were cultured in CDM supplemented with 200 µM myo-inositol (+ino) or no myo- inositol (-ino) for 24 hours. Culture medium samples were collected at set time points and the levels of key polar metabolites in 10 µL samples were measured by GC-MS. Error bars represent SD from three replicates.
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6.5 Changes to lipid composition in Δimp1 are minimal
As loss of imp1 resulted in only modest changes in central carbon metabolism, it
was of interest to determine whether imp1 might have a role in regulating lipid
metabolism. Therefore, wild-type and ∆imp1 promastigotes were cultivated in
CDM containing 200 µM myo-inositol for 36 hours and total lipid extracts were
analysed by supercritical fluid chromatography (SFC) coupled to high mass
resolution QTOF mass spectrometry (see Section 2.8.2). SFC-QTOF-MS analysis
in positive ionisation mode led to the identification of 3351 common features
between the two cell lines. As shown in Figure 6.6, significant differences in total
lipids were observed between wild-type and ∆imp1 promastigote, based on
p-values and fold changes in the volcano plot. After Benjamini-Hochberg
correction for false discovery, 385 of these features were identified to be
statistically significant. An additional 989 common features were identified under
negative ionisation mode, and 16 were found to be significantly different.
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Figure 6.6| Volcano plot of differences in the lipid profile of wild type against Δimp1 under myo-inositol-replete condition. L. mexicana wild-type and Δimp1 promastigotes were incubated in CDM supplemented with 200 µM inositol. Cells were collected after 36 hours and lipids extracted. The samples were then analysed by SFC-QTOF-MS to identify mass spectral features. The volcano plot displays the relationship between fold change and significance of the features identified during pair-wise comparisons of the two cell lines. The points in violet represent the statistically significant features (FC ± 2, p-value ≤ 0.05) from three experimental replicates.
From these two datasets of statistically significant features, 44 unique lipid species were conclusively identified based on MS/MS information (Figure 6.7).
The majority of these lipids were found to be triacylglycerols (TG), and closer examination revealed distinct patterns based on size and degree of unsaturation
(Figure 6.8). Interestingly, the ∆imp1 mutant was found to be enriched in TG species with longer fatty acid chains, at the expense of TGs with shorter fatty acids
(with the conspicuous exception of TG 56:8). Mutant promastigotes were also enriched for TGs with unsaturated fatty acids. Other lipid species found to be elevated in Δimp1 included phosphatidylethanolamine (PE) and plasmenyl-PE, while all the diacylglycerols (DG) identified were of lower abundance in Δimp1.
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Figure 6.7| Identification of significant lipid species between wild type and Δimp1 based on MS/MS information. An example of a total ion chromatograph of Δimp1 sample acquired from SFC-QTOF-MS under positive ionisation mode shows lipid classes are distributed at defined regions based on retention time. Initial lipid identification was performed by MS-DIAL based on its internal in silico MS/MS spectra libraries. This was followed by manual curation and confirmation based on the signature fragment ions that matched the lipid species. The species shown are the ones that displayed the most statistically significant differences between the two cell lines of each lipid class.
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Figure 6.8| Lipid species identified as being significantly different between L. mexicana wild type and Δimp1 under myo-inositol-replete condition. L. mexicana wild-type and Δimp1 mid-log phase promastigotes were incubated in CDM for 36 hours in the presence of 200 µM exogenous myo-inositol. Lipid samples were collected and analysed by SFC-QTOF-MS under both positive and negative ion mode. Data were median normalised and the lipid species were identified based on MS/MS information. Only statistically significant species (p- value < 0.05, BH adjusted t-test) with confirmed identifications are shown. Error bars represent SD from three replicates. Abbreviations used: PE, phosphatidylethanolamine; DG, diacylglycerol; TG, triacylglycerol.
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6.6 L. mexicana Δimp1 is thermosensitive and cannot differentiate to amastigote in vitro
While the loss of imp1 has only a modest effect on promastigote growth and
metabolism, it was of significant interest to test whether ∆imp1 promastigotes can
survive at elevated temperature/low pH and differentiate to amastigotes, which is
essential for pathogenesis in the mammalian host. Axenic differentiation was
induced by resuspending wild-type, Δimp1 and episomal add-back
stationary-phase promastigotes in acidified RPMI medium (pH 5.5) supplemented
with 20 % iFBS for three days at 34 °C (see Section 2.3.4). The wild-type and
add-back strain successfully differentiated into rounded, aflagellated axenic
amastigotes over the three-day period. In contrast, Δimp1 parasites progressively
lost viability, as shown by their increased granularity and vacuolation (Figure 6.9A)
as well as staining with propidium iodide, indicating the cell membranes have
become permeable, which is indicative of compromised parasites (Figure 6.9B).
The ∆imp1 promastigotes also continued to express promastigote markers, such as
the GPI anchored surface protein, GP63 (Medina-Acosta et al., 1989) (Figure 6.10)
indicating incomplete differentiation.
To determine whether the reduced viability of Δimp1 was the result of an
increased sensitivity to elevated temperature and/or lower pH, wild-type and
Δimp1 promastigotes were cultivated in either acidified RPMI medium (pH 5.5) at
27 °C, or in normal RPMI medium (pH 7.4) at 34 °C for three days. Lowering the
pH of the medium alone did not induce amastigote differentiation in any of the
strains and did not affect the viability of the Δimp1 mutant (Figure 6.11A). In
contrast, incubation at 34 °C in pH 7.4 media resulted in the appearance of rounded
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aflagellate cells in all three lines, with intermediate morphology between promastigotes and amastigotes, indicating partial amastigote differentiation.
However, the intermediate forms in the Δimp1 cultures were more granular and vacuolated, and permeable to propidium iodide (Figure 5.11B). Together, these analyses indicate that the loss of imp1 results in increased thermosensitivity and a defect in the capacity for the ∆imp1 mutant to differentiate into amastigotes.
(A) WT Δimp1 Δimp1+pXG-IMP1
(B)
Figure 6.9| L. mexicana Δimp1 cannot differentiate into amastigotes under axenic condition. L. mexicana wild-type, Δimp1, and episomal add-back (Δimp1+pXG-IMP1) stationary-phase promastigotes were transferred to acidified RPMI + 20 % iFBS (pH 5.5) and incubated at 34 °C for three days. Live cells were (A) examined by DIC microscopy; scale bars = 10 μm; and (B) stained with propidium iodide and the percentage of viable cells was determined by fluorescence microscopy (300 cells per experimental replicate, error bars = SD from 3 experiments).
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(A) (B)
Δimp1+ WT Δimp1 pXG-IMP1
kD P A P A P A
260 160 110 80 60 GP63 50 (63kD) 40
30 20
15
50 α-tub
Figure 6.10| L. mexicana Δimp1 retain the promastigote-associated protein marker, GP63. (A) Western blot of cell protein lysates from L. mexicana wild type, Δimp1, and episomal add-back (Δimp1+pXG-IMP1) promastigotes and axenic amastigotes (sampled after three days of differentiation) probed with L3.8 (anti- GP63). α-tubulin was used as a loading control, indicating similar protein load between strains (note the significant differences in intensity between promastigote and amastigote samples were due to selective binding by the antibodies); (B) Signal intensities of GP63 were normalised by loading control for promastigote and amastigote samples.
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(A) WT Δimp1 Δimp1+pXG-IMP1
C, pH 5.5 C, pH
27°
4
.
7
C, pH C, pH
° 34
(B)
Figure 6.11| L. mexicana Δimp1 show reduced viability as a result of increased temperature during axenic amastigote differentiation. L. mexicana wild type, Δimp1 and Δimp1+pXG-IMP1 stationary phase promastigotes were either transferred to RPMI + 20 % iFBS buffered at pH 7.4 with 25 mM HEPES and incubated at 27 °C, or acidified to pH 5.5 and incubated at 34 °C for three days. Live cells were (A) examined by DIC microscopy; scale bars = 10 μm; and stained with propidium iodide before the (B) percentage of viable cells were determined by fluorescence microscopy (300 cells per experimental replicate, error bars = SD from 3 experiments).
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6.7 Discussion
Genetic deletion of imp1 in L. mexicana leads to a loss of virulence in susceptible
mice, although this was not associated with the acquisition of myo-inositol
auxotrophy under defined culture conditions. In this chapter, the results show that
de novo myo-inositol biosynthesis is not disrupted in ∆imp1 promastigotes and that
the normal feedback loops that repress this pathway under myo-inositol-replete
conditions are also unaffected. Broad metabolomics and lipidomics analyses also
failed to identify major changes in central carbon and lipid metabolism, although
subtle changes in amino acid synthesis and triacylglycerol accumulation were
observed. Notwithstanding the absence of a conspicuous biochemical phenotype,
the results show that loss of imp1 is associated with increased thermosensitivity,
which likely underlies the inability of this mutant to survive within macrophages
or induce lesions in mice. These findings are discussed in more detail below.
6.7.1 imp1 and de novo myo-inositol synthesis
IMP1 is predicted to catalyse the second step in de novo myo-inositol biosynthesis.
However, metabolic labelling studies with 13C-U-glucose showed that the
biosynthesis of Ino3P and myo-inositol under myo-inositol-starved conditions was
unaffected in ∆imp1 promastigotes. Significantly, the loss of imp1 was also not
associated with any increase in its putative precursor, Ino3P, or a reduction in the
secretion of myo-inositol, indicating the absence of any significant change in flux
through this pathway. Interestingly, Δimp1 promastigotes maintained higher levels
of de novo myo-inositol than wild type under both myo-inositol-replete and -starved
conditions. While the basis for this modest accumulation of myo-inositol is
unknown, it could reflect reduced surface expression of the H+/myo-inositol
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transporter in this line (and hence secretion of intracellular pools) or reduced rates of utilisation by downstream pathways. Collectively, these results confirm that
IMP1 is not required for de novo myo-inositol biosynthesis, suggesting that IMP2 is able to compensate for loss of IMP1 and/or that IMP1 normally has only a minor role in myo-inositol biosynthesis in promastigotes.
6.7.2 Metabolic consequences of imp1 deletion in L. mexicana
Metabolic profiling of key intermediates of central carbon metabolism and amino acid metabolism did not reveal major changes in these pathways in Δimp1 promastigotes in the presence or absence of myo-inositol. However, modest changes in the metabolism of serine and glycine were observed. In particular, labelling with 13C-U-glucose indicated higher rates of de novo synthesis of these amino acids in ∆imp1, which was accompanied by higher intracellular levels of serine and reduced consumption of exogenous serine from the culture media.
Together, these results suggest the mechanism involved in the uptake of serine is disrupted by the loss of IMP1, leading to a compensatory increase in de novo biosynthesis of serine and glycine. Previous studies have demonstrated that de novo synthesis of serine and glycine in Leishmania is only activated when exogenous amino acids are not available (Saunders et al., 2011). Serine uptake is achieved by a single transport system (dos Santos et al., 2009), and is important for intracellular parasite growth (Scott et al., 2008). While there is some evidence in other organisms that serine uptake may be influenced by changes in myo-inositol
(Lembach & Charalampous, 1967), the precise role of IMPase in this process remains unknown.
Another metabolic change detected in ∆imp1 is the simultaneous reduction in
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alanine being secreted by the promastigotes. Decreased alanine secretion may
reflect increased pyruvate flux being directed into the TCA cycle. This is supported
by decreased secretion of malate produced from succinate fermentation.
Furthermore, both the exogenous uptake of aspartate and the intracellular aspartate
pool are decreased in ∆imp1, which is consistent with the reduced need for amino
groups in the transamination reaction of alanine production, and/or the provision
of carbon skeletons to “top-up” the TCA cycle (due to alternate use of pyruvate
and malate for anapleurotic reactions). In addition, tryptophan uptake was also
significantly reduced in ∆imp1. Tryptophan is predominantly catabolised by
Leishmania through transamination reaction to generate indole-3-lactate, which is
then secreted as a waste product (Westrop et al., 2015). Together with the slower
growth, these results suggest Δimp1 may require more energy to grow. Further
analysis of cell energetics will be of significant interest, and will need to account
for the rate of glucose uptake versus overflow metabolism. The latter in Leishmania
is reflected by the major secreted metabolites: succinate, alanine, as well as acetate
(Saunders et al., 2011; 2014).
Relatively restrained metabolic differences between wild type and Δimp1
were also observed in their steady-state lipid profiles. One of the most prominent
changes found in Δimp1 was the enrichment of triacylglycerols with long fatty acid
chains and higher degree of unsaturation. Triacylglycerols are major components
of lipid bodies and the altered fatty acid compostion of these lipids could reflect
changes in their turnover and exchange with other membranes in the cell. Whether
these changes results in changes in membrane fluidity or intraorgannel membrane
transport (Walther & Farese, 2012; de Kroon et al., 2013; Barbosa & Siniossoglou,
2017) remains to be determined.
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6.7.3 imp1 is required for thermotolerance and amastigote differentiation
It is well established that thermotolerance is critical for Leishmania amastigote differentiation and pathogenicity in the mammalian host. Species-specific differences in thermotolerance may also determine the outcome of infection, such as whether parasites remain in the skin or are capable of infecting internal organs
(i.e. liver and spleen), where they are exposed to elevated temperatures (37 °C compared to 33 °C). Here, we show that the failure of the ∆imp1 mutant to survive in macrophages or induce lesions in susceptible mice likely reflects their increased sensitivity to elevated temperatures and consequent inability to survive as amastigotes. How IMP1 contributes to thermotolerance remains unclear. While the apparent increased sensitivity to temperature stress observed in Δimp1 may be partly attributed to increased membrane fluidity, other more complex factors may also be at play. Phosphoinositide signalling is intimately connected to heat shock responses and thermotolerance in other eukaryotes, by regulating the mobilisation of intracellular calcium pools (Stevenson et al., 1986; Calderwood et al., 1987).
Amastigote differentiation has been shown to be linked to increased influx of Ca2+
(Sarkar & Bhaduri, 1995; Lu et al., 1997; Prasad et al., 2001), a process that is also essential for parasite survive in elevated temperature (Naderer et al., 2011). The accumulation of Ca2+ is likely to active various Ca2+-dependent signalling
2+ pathways; in particular, the Ca dependent phosphatase, calcineurin, has been showen to be vital for L. major to survive under elevated temperature in vitro and to establish infection in mouse models (Naderer et al., 2011).
In other eukaryotes, heat shock is associated with increased expression of heat shock proteins, which provide chaperone function to stabilise other proteins during
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cellular encounter of heat stress (Calderwood et al., 2010). Thermotolerance and
survival of Leishmania parasites in mammalian hosts is also dependent on a
network of heat-shock proteins, (Hübel et al., 1997; Wiesgigl & Clos 2001;
Hombach et al., 2014). However, in contrast to the situation in other eukaryotes,
these proteins are thought to be constitutively expressed throughout Leishmania
life cycle stages lack(Brandau et al., 1995; Morales et al., 2010) and are activated
under stress conditions by phosphorylation, which promotes the formation of large
protein complexes and their interaction with client proteins (Morales et al., 2010).
The exact process of how the heat stress signal is relayed from calcineurin and
other upstream enzymes to the heat shock protein kinases remains to be determined.
The strong link between IMP1 expression and Leishmania thermotolerance
suggests that IMP1 could be involved in regulating the cellular levels of inositol
1,4,5-trisphosphate (IP3) or downstream inositol phosphates generated by heat
shock-induced uptake of calcium and degradation of plasma membrane pools of
PI(4,5)P2 (Stevenson et al., 1986; Calderwood et al., 1987; Liu et al., 2006). In
Leishmania and other trypanosomatids, IP3 binds to the IP3 receptor located on the
acidocalciosomes, the primary reservoir of intracellular Ca2+ (Docampo & Huang,
2015), triggering the efflux of Ca2+ into the cytosol and the activation of the
aforementioned heat shock responses that underlie amastigote differentiation. The
concentration of IP3 and related degradation products are likely to be low and were
not detected in the LC-MS and GC-MS analyses used in the study. In future
experiments, it will be of interest to detect these intermediates using targeted
inositol phosphate analyses during attempted amastigote differentiation.
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6.7.4 Possible links between IMP1 and IMP2 functions
Recent studies on the T. brucei IMPase, TbIMPase1 and TbIMPase2, have shown that the recombinant proteins either have low or no activity against Ino3P when used alone, but form a catalytically active complex when mixed together in a 3:1 ratio (personal communication, Prof. Terry Smith, St Andrews University,
Scotland). Attempts to express and purify the recombinant Leishmania IMP1 and
IMP2 using E. coli and L. tarentolae expression systems were largely unsuccessful.
Low levels of soluble IMP1 and IMP2 were purified, but neither preparation has detectable Ino3P phosphatase activity when used alone or together under a range of in vitro assay conditions. Future attempts to determine if Leishmania IMP1 and
IMP2 have Ino3P phosphatase activity could involve co-expression studies in E. coli or L. tarentolae expression systems, and/or complementation of the T. brucei or yeast IMPase mutants. An intriguing possibility is that IMP2 is the catalytically active IMPase isoform in Leishmania, but that it requires IMP1 for stable expression/activity when cells are stressed, such as during heat shock and amastigote differentiation. Loss of IMP1 would therefore lead to destabilisation of
IMP2 and loss of IMPase activity in amastigotes, accounting for the loss of parasite viability during differentiation. This model also assumes that the H+/myo-inositol symporter is down-regulated in amastigote stages, which has been suggested by previous studies (Vince et al., 2011). Further elucidation of the role of IMP1 and
IMP2 has important implications for understanding the metabolic processes that underlie amastigote differentiation and pathogenesis.
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CHAPTER 7 Final Remarks and Future Directions
Human leishmaniasis is one of the leading infections caused by parasitic protozoa, second only to malaria in terms of morbidity and mortality. It represents a significant public health and economic burden on many of the poorest countries in the world (Okwor & Uzonna, 2016). Current therapeutic options for leishmaniasis are very limited, problematic, and are increasingly being undermined by the emergence of drug-resistant parasite strains. There is therefore an urgent need to search for new drug targets and develop alternative treatment options. This thesis investigated the significance of de novo myo-inositol synthesis in Leishmania and the potential role of the putative INO1 and IMPase enzymes in parasite survival and virulence. The outcomes of this investigation have provided important insights into one of the most pressing questions surrounding Leishmania biology, in identifying factors that are crucial for amastigote proliferation within the phagolysosome.
Consistent with a previous short report by Ilg (2002), this work shows that the loss of INO1 leads to the complete loss of de novo myo-inositol synthesis with concomitant acquisition of myo-inositol auxotrophy. Significantly, disruption of this pathways leads to loss of virulence in macrophages and in susceptible mice, demonstrating that this pathway is important for amastigote growth in macrophages. Importantly, and in contrast to the study by Ilg (2002), this
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phenotype could be fully complemented by ectopic re-expression of INO1 in the
mutant line. Further analysis of the virulence phenotypes showed that loss of
virulence in mice is likely to be due to low levels of myo-inositol in the PV
(extrinsic factors), rather than intrinsic factors such as down-regulation of the
amastigote myo-inositol/H+ symporter or increased dependence on myo-inositol
metabolism per se. Specifically, growth of ∆ino1 parasites in macrophages was not
compromised when macrophages had access to exogenous myo-inositol, but was
severely limited when infected macrophages were cultured in myo-inositol-free
medium.
These findings provide further insights into nutrient levels in Leishmania
lesions and the potential role of granulomas in either controlling or promoting the
growth and spread of different pathogens. Granulomas are organised aggregates of
macrophages that appear to be an ancient innate immune response to both
infectious and non-infectious stimuli that cannot be eradicated by single
macrophages (Pagán and Ramakrishnan 2018). Granulomas are a hallmark of
infection by Leishmania (in both cutaneous and visceral infections), as well as
many other bacterial, fungal and protozoan pathogens. The best characterised
pathogen-induced granulomas are those induced by Mycobacterium tuberculosis,
the cause of tuberculosis. While it is generally thought that granulomas represent
an attempt by the host to wall off intractable infections, there is accumulating
evidence that pathogens such as M. tuberculosis may exploit the constant
recruitment of new macrophages to these structures as a way to expand their
numbers, and that granulomas may be disease promoting (Davis &, Ramakrishnan,
2009; Volkman et al., 2010; Martin et al., 2015). Although granulomas induced by
Leishmania are generally much simpler than those induced by M. tuberculosis (in
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lacking epithelioid macrophages and extensive fibrosis), it is likely that these structures also represent a permissive niche that allows the progressive expansion of Leishmania numbers. In this context, the finding that L. mexicana ∆ino1 amastigotes are completely absent from the site of infection, suggests that granulomas may still help to restrict parasite growth by limiting availability of essential nutrients, such as myo-inositol. Indeed, a study of M. tuberculosis ∆ino1 mutants also found they suffer significant loss of virulence during mice infections
(Movahedzadeh et al., 2004). Consistent with this notion, studies from our laboratory have shown that Leishmania amastigotes enter into a slow growth, metabolically quiescent stage in granulomas, which appears to be developmentally regulated (i.e hardwired) during amastigote differentiation (Saunders et al., 2014;
Kloehn et al., 2015). This stringent response may reduce the overall need for amastigotes to salvage essential nutrients and prevent overgrowth of the PV. It remains to be determined how granulomatous macrophages restrict nutrient levels to intracellular pathogens. One possibility is that these structures lack extensive vascularisation, which would limit nutrient availability in vivo. In support of this notion, viable ∆ino1 parasites were recovered from neighbouring lymph nodes in infected mice, suggesting that these tissues may be exposed to higher levels of exogenous myo-inositol. Alternatively, macrophages can exist in different physiological/metabolic states depending on the immune response of the host, which may determine nutrient availability for the intracellular pathogens (Muraille et al., 2014). These include the highly glycolytic M1 state characteristic of protective inflammatory immune responses or the metabolically less active M2 state in which macrophages are more dependent on oxidative phosphorylation for energy production (Galván-Peña & O'Neill, 2014; Kelly & O'Neill, 2015). In future
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studies, it would be of interest to determine (1) the levels of myo-inositol in
Leishmania-induced granulomatous lesions; (2) the extent to which these levels are
dependent on import via the circulation or are synthesised by the host cells
themselves; and (3) whether levels of myo-inositol are coupled to the immune
status of the granulomas.
Biochemical analysis of the ∆ino1 mutant showed that myo-inositol starvation
was associated with the depletion of the bulk phospholipid, PI, as well as derived
phosphoinositides, which may underlie the lethality associated with “inositol-less
death”. In contrast, myo-inositol starvation resulted in little effect on the steady
state levels of other inositol lipids, such as IPC and the major GIPL species,
reflecting their slower rate of turnover in the exoplasmic leaflet of the plasma
membrane. While these results suggest that loss of IPC/GIPLs do not contribute to
inositol-less death in vitro, it remains possible that these lipids contribute to the
virulence of amastigotes in infected tissues. However, genetic studies have shown
that both IPC and GIPLs are expendable for virulence (Garami et al, 2001; Naderer
& McConville, 2002; Zhang et al., 2003; Zufferey et al., 2003; Denny et al., 2004)
suggesting that any depletion of these lipids in vivo is likely to have only a minor
effect on pathogenesis.
One of the most striking results from the lipidomics analysis of
myo-inositol-starved ∆ino1 parasites was the extent to which depletion of inositol
lipids was associated with global changes in the levels of other lipid classes. These
include marked increases in the steady-state of PA, ceramide and specific PE and
PC molecular species, as well as changes in the fatty acid composition of the
neutral DG and TG lipid species. As discussed in Chapter 4, these changes could
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be directly or indirectly linked to loss of PI, which is involved in regulating multiple pathways of lipid biosynthesis (Loewen et al., 2004; Gaspar et al., 2006;
2011). In future studies, it would be of interest to investigate the dynamics of phospholipid/neutral lipid biosynthesis in myo-inositol-starved ∆ino1 parasites using 13C-labelled headgroup precursors (i.e. 13C-serine, 13C-choline, and
13C-ethanolamine). It is possible that disruption of lipid homeostasis during myo-inositol starvation contributes to, or is responsible for, loss of parasite viability.
In this context, it is notable that myo-inositol starvation also impacted on the biogenesis and/or function of specific organelles, such as the MVT-lysosome. This unusual tubular lysosome is unique to the promastigote stages of some trypanosomatids (i.e Leishmania, Crithidia, Herptemonas) and is maintained by physical association with an unusual quartet of microtubules that extend from the flagellar pocket to the posterior end of these parasite stages. Fluorescence microscopy showed that the minor phosphoinositide species, PI3P, is targeted to the MVT-lysosome, and internalised into the intraluminal vesicles, as shown by live cell imaging of L. mexicana wild type and ∆ino1 parasites expressing the
GFP-2×FYVE phosphoinositide sensor. Under myo-inositol starvation conditions, the PI3P-positive organelle contracted to the posterior end of promastigotes, reflecting either changes in membrane transport between the MVT-lysosome and late endosome/multivesicular bodies and/or disruption of the link between the
MVT-lysosome and the microtubule quartet. As degradation of exogenous
(host-derived) and endogenous parasite proteins in the lysosomes is thought to be essential for amastigote survival (Besteiro et al., 2006; 2007), disruption of endosome to lysosome transport in amastigote stages could contribute to loss of virulence of the ∆ino1 parasites. Further studies are needed to define the properties
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of the collapsed MVT-lysosome in ∆ino1 during myo-inositol starvation, including
the rate of delivery of extracellular cargo to this compartment and its capacity to
degrade proteins (Mullin et al., 2001).
In common with yeast inositol auxotrophs, L. mexicana ∆ino1 parasites
appear to be unable to sense and/or respond to a decrease in intracellular pools of
myo-inositol in a way that maintains viability (Ridgway & Douglas, 1958; Lester
& Gross 1959; Henry et al., 1977). This contrasts to the situation when Leishmania
(and yeast) are starved of essential amino acids, purines or vitamins, which triggers
a protective quiescent state (Saldanha et al., 2004; Boer et al., 2008; Carter et al.,
2015). The rapid death phenotype and lack of quiescence displayed by L. mexicana
during myo-inositol starvation, make INO1 an attractive drug target and reduce the
chance of the parasite developing drug resistance.
Studies on the role of the putative IMPase proteins that catalyse the second
step in de novo myo-inositol synthesis highlighted further complexity in the
Leishmania pathway. The Leishmaina genomes encode two putative IMPase
enzymes, IMP1 and IMP2, which appear to contain all of the amino acids residues
needed for IMPase activity. Attempts were made to knock out both genes
separately. However, only imp1 could be successfully deleted, while attempts to
knock out imp2 by sequential replacement of chromosomal alleles with drug
resistance cassettes through homologous recombination were unsuccessful. Failure
to delete genes in Leishmania using this approach can indicate essentiality and/or
poly/aneuploidy at the target loci. The latter possibility has been suggested by
recent attempts to delete imp2 using a newly developed CRISPR/Cas9 system
which were successful (Jiang Nan Zhu and Fleur Sernee, personal communication).
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There is increasing evidence that CRISPR/Cas9 is more efficient than conventional homologous recombination approaches in deleting genes in Leishmania, regardless of the ploidy of the loci. Collectively, these studies suggest that neither IMP1 nor
IMP2 are essential when parasites are expressing the other homologue. Future studies will be directed towards disrupting both genes at the same time using
CRISPR/Cas9 and determining the effects on cell viability and myo-inositol metabolism.
Unexpectedly, loss of imp1 alone resulted in a severe loss of virulence in macrophages and susceptible mice that appeared to be independent of exogenous myo-inositol levels. Significantly, Δimp1 were shown to be highly sensitive to elevated temperatures that normally lead to amastigote differentiation in vitro and to which parasites are exposed in vivo. Again, the increased thermosensitivity of the ∆imp1 mutant in vitro was not dependent on levels of myo-inositol in the medium. It is possible that IMP2 largely compensates for the loss of IMP1 in promastigote stages, but not in amastigotes. Redundancy between different IMPase isoforms has been reported in some other organisms, such as M. tuberculosis and yeast (Lopez et al., 1999; Movahedzadeh et al, 2010).
An alternative hypothesis is that IMP1 fulfils functions unrelated to de novo myo-inositol synthesis. This possibility is supported by the finding that all of the in vitro phenotypes of the ∆imp1 mutant appeared to be independent of the presence or absence of exogenous myo-inositol, in sharp contrast to the phenotype of the
∆ino1 mutant. One possibility is that IMP1 is involved in the catabolism of complex inositol phosphates generated during phosphoinositide signalling.
Specifically, lipase-mediated cleavage of PI(4,5)P2 in response to calcium fluxes
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is associated with the release of IP3, which is subsequently catabolised back to
myo-inositol via the action of multiple inositol phosphatases. The Leishmania
genome encodes a number of putative inositol phosphatases, although none have
been biochemically characterised, and one or more of these steps could be
catalysed by IMP1. A defect in inositol phosphate catabolism would not be
expected to lead to myo-inositol auxotrophy or to be rescued by myo-inositol
supplementation. It is, however, difficult to resolve the different isomers of inositol
phosphate by either GC-Ms or LC-MS, which complicates the detection of this
phenotype. Further studies will be needed to measure fluxes through the de novo
and catabolic myo-inositol-phosphate pathway using chiral LC or GC-MS columns
that allow resolution of these species.
Another possibility is that IMP1 may perform a non-enzymatic function that
is essential for IMP2 activity. Recent studies in T. brucei suggest that the IMPase
proteins in this parasite form hetero-oligomers that are essential for full activity.
Specifically, the T. brucei IMP1 homologue itself appears to have no catalytic
activity, but may function as a prozyme that stimulates the activity of IMP2 through
complex formation. This prozyme paradigm has previously been described in the
polyamine biosynthesis pathway of T. brucei, and more recently in arginine
methylation (Willert et al., 2007; Kafková et al., 2017). Therefore in Leishmania,
IMP2 may require the co-expression of IMP1 for stimulating its activity during
amastigote differentiation. Attempts to test this hypothesis by measuring the
activity of recombinant IMP1 and IMP2 alone or in combination have been
unsuccessful to date, despite the use of two different expression systems (E. coli
and L. tarentolae) and a variety of different assay conditions. Future studies will
attempt to co-express both proteins in E. coli and the use of the L. tarentolae in
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vitro translation expression system. The latter has demonstrated to be very useful for studying the function of hetero-oligomeric protein complexes (Phan et al.,
2009).
Moreover, it is also possible that IMPase catalyses reactions that are independent of myo-inositol metabolism (Stec et al., 2000; Laing et al., 2004; Gu et al., 2006). Specifically, IMPase family members have been shown to catalyse the dephosphorylation of L-galactose 6-phosphate, an intermediate in ascorbic acid catabolism, as well as the dephosphorylation of Fru1,6P2 in place of the canonical gluconeogenic enzyme, FBPase. Interestingly, Leishmania retain the terminal enzymes involved in ascorbic acid synthesis, downstream of the putative
L-galactose 6-phosphate phosphatase (Wilkinson et al., 2005) and this pathway is thought to be essential for intracellular growth and virulence. Similarly, L. major is also dependent on FBPase for virulence, although there is no evidence for a second 'FBPase' activity in the L. major ∆fbp mutant, suggesting that IMPase does not have FBPase activity in vivo.
Another aspect of Leishmania myo-inositol metabolism that requires further resolution is to determine to what extent the metabolic compartmentalisation of PI synthesis described in other protozoan parasites is present in Leishmania (see
Section 1.6.2). The results from this work have demonstrated that both L. mexicana
IMPase isoforms are targeted to the ER, potentially providing a mechanism for directing the flux of de novo myo-inositol into the ER to promote its preferential usage for GPI synthesis. In contrast to the situation in both T. brucei and P. falciparum (Martin & Smith 2005; Martin & Smith 2006a; Macrae et al., 2014),
GPI glycolipids are not essential in Leishmania, making this parasite a useful
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model system for further exploring the role of this compartmentalisation in these
parasites and further opportunities for exploiting unique features of the
biochemistry of this pathway for new therapies.
Finally, since this study has concluded that both ino1 and imp1 are vital for
Leishmania growth and virulence, it will be of significant interest to determine the
potential of these enzymes as drug targets. In the last few decades, IMPase has
generated considerable amount of attention in the field of biological psychology
and psychopharmacology due to in vitro and in vivo evidence suggesting that these
enzymes are the likely targets for lithium and a number of other mood-stabilising
drugs (valproate and carbamazepine) in treating bipolar disorder and schizophrenia
(Berridge et al., 1989; Kofman & Belmaker, 1993; Moore et al., 1999; Williams et
al., 2002; Singh et al., 2013). Likewise, there is emerging interest in targeting INO1
for similar applications (Agam et al., 2002). While the research to determine the
underlying relationship between myo-inositol metabolism and these psychiatric
disorders is still ongoing, they have provided a foundation for drug screening and
development, and more importantly, the potential for drug repurposing. Therefore,
future experiments involving testing the effects of these compounds on Leishmania
parasites and their potential to inhibit infections will be a good starting point.
186
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Appendices
Appendix 1 BSA standards for Bradford colorimetric assay
Table A.1| Bovine serum albumin protein standards. [BSA] (mg/mL) 2mg/mL BSA Milli-Q water Dilution factor stock (µL) (µL) 0 0 200 0 0.05 5 195 40 0.1 10 190 20 0.25 25 175 8 0.4 40 160 5
Figure A.1| Calibration curve of Bradford colorimetric assay based on bovine serum albumin. The graph depicts the correlation between BSA concentration and absorbance at 595 nm; n=2.
229
APPENDICES
Appendix 2 Semi-defined medium (SDM) for Leishmania
SDM-79 is a versatile semi-defined medium for culturing Trypanosomes such as
Leishmania spp. The original recipe was described by Brun & Schönenberger
(1979) and further modifications have been applied. To make up 1 L working SDM,
the powder stock denoted in Table A.2 is dissolved in 985 mL of Milli-Q water.
The solution is adjusted to pH 7.4 and then filter sterilised. 8 mL of sterile 50×
Eagle’s minimal essential medium (MEM) essential amino acid mix and 6 mL 100×
MEM non-essential amino acid mix (Gibco, Thermo Fisher Scientific) are added
to the medium. Finally, 100 mL of heat-inactivated foetal bovine serum (iFBS) is
supplemented.
Table A.2| Composition of semi-defined medium SDM-79 powder stock. Component /L Component /L Medium base Amino acids MEM Powder 7.0 g DL-Alanine 200 mg Medium 199 powder 2.0 g L-Arginine 100 mg Buffers L-Glutamine 300 mg HEPES (sodium salt) 8.0 g DL-Methionine 70 mg MOPS (free acid) 5.0 g L-Phenylalanine 80 mg Sodium bicarbonate 2.0 g L-Proline 600 mg Carbon sources DL-Serine 60 mg Glucose, anhydrous 1.0 g Taurine 160 mg Sodium pyruvate 0.1 g DL-Threonine 350 mg L-Tyrosine 100 mg Trace nutrients p-Aminobenzoic acid 2 mg Folic acid 4 mg D-Glucosamine 50 mg Guanosine 10 mg
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APPENDICES
Appendix 3 Completely defined medium (CDM) for Leishmania
The original recipe of CDM was described by Merlen et al. (1999), and has been further optimised.
Table A.3| Composition of completely defined medium base. Component /988 mL Component /988 mL Inorganic salts Vitamins
Ca(NO3)2 ∙ 4H2O 100 mg p-Aminobenzoic (9 mM, 1,000×) 1 mL KCl 400 mg L-Ascorbic Acid (1 mM, 16,667×) 60 µL
KH2PO4 12 mg Biotin (15 mM, 10,000×) 100 µL
MgSO4∙ 7H2O 100 mg Cyanocobalamin (4 mM, 1,000×) 1 ml NaCl 6.8 g Folic acid (3 mM, 1,000×) 1 ml
NaHCO3 2 g Vitamin mix (1000×) 1 ml
Na2HPO4∙ 12H2O 300 mg Cofactors buffers and detergents Biopterin (0.5 mg/ml, 1,000×) 1 mL HEPES 4.8 g Calciferol (1 mM, 20,000×) 50 µL Tween 80 (10 %) 40 µL Cholesterol (1 mM, 10,000×) 100 µL Nucleotides Glutathione (36 mM, 10,000×) 100 µL Adenine (100 mM, 10,000×) 100 µL Hydroxyproline (170 mM, 1,000×) 1 mL Thymine (4.8 mM, 10,000×) 100 µL Menadione (10 mM, 106×) 1 µL Nucleotide mix (1000×) 1 mL Retinyl acetate (7 mM, 100,000×) 10 µL Amino acids Cysteine (12 mM, 100,000×) 10 µL Cystine 188 mg Glutamine 336 mg MEM essential AA (50x) 20 mL MEM non-essential AA (100×) 30 mL
Nucleotide mix /50 mL Vitamin mix /10 mL Adenosine 11.4 mg D-calcium panthothenate 10 mg Guanosine 4.5 mg Choline chloride 10 mg Hypoxanthine 3.5 mg Folic acid 10 mg Uracil 3.5 mg Nicotinamide 10 mg Xanthine 3.8 mg Pyridoxal hydrochloride 10 mg Riboflavin 1 mg Thiamine hydrochloride 10 mg
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APPENDICES
To make up CDM base, the components listed in Table A.3 are prepared and
top up to 988 mL with Milli-Q water. After adjusting to pH 7.4, the solution is filter
sterilised. When a working CDM is required, a batch is made by following Table
A.4 and supplemented with 10 mL filter sterilised bovine serum albumin (BSA).
Table A.4| Working solution of completely defined medium. All components are filter sterilised beforehand. myo-Inositol is omitted for inositol-free conditions. Component /100 mL CDM base 98.8 mL Haemin (5 mg/mL, 1000x) 100 µL Glucose (600 mM, 100x) 1 mL myo-Inositol (200 mM, 1000x) 100 µL
232
APPENDICES
Appendix 4 GC-MS library for DExSI
Table A.5| List of metabolites and their signature ions detected and identified by GC-MS after TMS derivitisation. RT is retention time. RT (min) Metabolite Ions 5.67435 Pyruvate 174, 175, 176, 177 5.75 Lactate 117, 118, 119 6.23933 L-Alanine 116, 117, 118 7.484 L-Valine 218, 219, 220 8.102 L-Leucine 158, 159, 160, 161, 162, 163 8.348 L-Isoleucine 158, 159, 160, 161, 162, 163 8.422 L-Proline 142, 143, 144, 145, 146 8.497 L-Glycine 248, 249, 250, 251, 252 8.588 Succinic acid 247, 248, 249, 250, 251 8.628 L-nor-leucine 158, 159, 160, 161, 162, 163 8.977 Fumaric acid 245, 246, 247, 248, 249 9.04 L-Serine 218, 219, 220 9.115 pipecolic acid 258, 259, 260, 261, 262, 263, 264 9.309 L-Threonine 320, 321, 322, 323, 324 10.368 Malic acid 335, 336, 337, 338, 339 10.677 L-Aspartic acid 334, 335, 336, 337, 338 10.728 L-Methionine 176, 177, 178, 179, 180 10.77 5-oxo-proline 156, 157, 158, 159, 160 10.82 GABA 304, 305, 306, 307, 308 11.074 L-cysteine 294, 295, 296 11.249 α-ketoglutaric acid 304, 305, 306, 307, 308, 309 11.449 Phosphoenolpyruvate 369, 370, 371, 372 11.644 L-Glutamic acid 348, 349, 350, 351, 352, 353 11.77 L-Phenylalanine 192, 193, 194, 195, 196, 197, 198, 199, 200 11.85 D-Xylose 307, 308, 310, 311 12.13 L-Asparagine 231, 232, 233, 234 12.565 Glycerol 2-phosphate 445, 446, 447, 448 12.731 Putrescine 361, 362, 363, 364, 365 12.754 DHAP 400, 401, 402, 403 12.811 cis-Aconitic acid 375, 376, 377, 378, 379, 380, 381 12.891 Glycerol 3-phosphate 445, 446, 447, 448 13.286 D-3-Phosphoglyceric acid 459, 460, 461, 462
233
APPENDICES
RT (min) Metabolite Ions 13.386 Citrate 465, 466, 467, 468, 469, 470, 471 13.406 L-Ornithine 420, 421, 422, 423, 424, 425 13.784 D-Fructose 307, 308, 309, 310 13.91 D-Mannose 319, 320, 321, 322, 323 13.95 D-Galactose 319, 320, 321, 322, 323 14.001 D-Glucose 319, 320, 321, 322, 323 14.259 L-Lysine 434, 435, 436, 437, 438, 439, 440 14.299 L-Histidine 356, 357, 358, 359, 360, 361, 362 14.419 L-Tyrosine 280, 281, 282, 283, 284, 285, 286, 287, 288 15.037 scyllo-Inositol 318, 319, 320, 321, 322 15.529 myo-Inositol 318, 319, 320, 321, 322 15.621 D-Ribose 5-phosphate 459, 460, 461, 462 15.706 D-Ribulose 5-phosphate 357, 358, 359 16.542 L-tryptophan 202, 203 16.923 D-Fructose 1-phosphate 414, 415, 416, 417 17.023 D-Fructose 6-phosphate 459, 460, 461, 462 17.114 D-Glucose 6-phosphate 357, 358, 359 17.761 D-myo-Inositol 3-phosphate 318, 319, 320, 321, 322 17.83 6-Phosphogluconic acid 471, 472, 473, 474, 475
234
Minerva Access is the Institutional Repository of The University of Melbourne
Author/s: Liu, Tim Chung-Ting
Title: The role of de novo myo-inositol synthesis and matabolism in Leishmania parasites
Date: 2018
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