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Studies on Oligopeptidase B of Leishmania Major

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Studies on Oligopeptidase B of Leishmania Major Studies on Oligopeptidase B of Leishmania major Jane Claire Munday BSc (Hons) MSc A thesis submitted in the fulfillment of the requirement for the degree of Doctor of Philosophy in the Faculty of Veterinary Medicine, University of Glasgow Wellcome Centre for Molecular Parasitology Glasgow Biomedical Research Centre University of Glasgow United Kingdom May 2008 Jane Claire Munday 2008 ii Abstract Peptidases of Leishmania are acknowledged virulence factors. It is hypothesised that peptidases are crucial for the survival of Leishmania in its hosts and that many could be potential targets for new antileishmanial drugs. As such, the investigation of peptidase activity in live Leishmania promastigotes was proposed as a valuable approach by which to increase knowledge on particular peptidases. In order to complete this investigation, it was decided to use short peptidyl fluorogenic substrates, which only fluoresce once the bond linking the peptide to the fluorescent moiety is cleaved. These allow detection of peptidase activity by quantifying the release of the fluorescent moiety. Detection of peptidase activity in live Leishmania using the fluorogenic substrate Bz-R-AMC proved fruitful, enabling study of the activity of the serine peptidase oligopeptidase B (OPB) in live L. major promastigotes. OPB is a member of the Family S9 peptidases, the prolyl-oligopeptidases, which are taxonomically restricted to plants, bacteria and trypanosomatid flagellates. In African and American trypanosomes, OPB has been shown to have important roles: OPB is a virulence factor in Trypanosoma cruzi , mediating entry into host cells, and OPB is released into the serum by African trypanosomes, where it cleaves host blood factors. In this study, the inhibition profile of L. major OPB has been determined and OPB has been localised to the cytosol, the site of hydrolysis of Bz-R-AMC. Immunoprecipitation of OPB confirmed that OPB was the sole peptidase responsible for the hydrolysis of Bz-R-AMC and anti-OPB antibodies were found to inhibit the hydrolysis of Bz-R-AMC. Inhibitors of OPB could also kill Leishmania promastigotes, suggesting OPB could be a valuable drug target. However, genetic manipulation of OPB was successful, with mutants over- expressing OPB and ∆opb null mutants produced. OPB is thus not essential for the growth of promastigote L. major , though the ∆opb null mutants did have a defect in metacyclogenesis, in survival in macrophages and a reduced ability to induce lesions on the footpads of mice. A role in amastigote differentiation or survival in macrophages was also suggested. OPB is thus likely to be a virulence factor, though not essential, and thus not suitable as a primary drug target. iii A number of avenues require further investigation, including the need for re- expression of OPB in the ∆opb null mutants to confirm that lack of OPB is indeed responsible for the phenotypic deficiencies of the null mutants. Other important areas requiring attention are investigation of the role of OPB in amastigote differentiation or survival, investigation of the reported release of OPB by promastigotes, and identification of the physiological substrate of OPB. iv Table of Contents Abstract .......................................................................................................... ii Table of Contents................................................................................................... iv List of Figures ........................................................................................................ ix List of Tables .........................................................................................................xii Acknowledgements...............................................................................................xiii Author’s Declaration..............................................................................................xiv List of Abbreviations.............................................................................................. xv Chapter 1 INTRODUCTION...............................................................................1 1.1 Leishmania .....................................................................................2 1.1.1 Causative agent of leishmaniasis.................................................2 1.1.2 Life cycle ......................................................................................3 1.1.3 Ultrastructure of Leishmania ........................................................8 1.1.3.1 Specialised organelles...............................................................8 1.1.4 Treatment of leishmaniasis ........................................................13 1.1.4.1 Visceral leishmaniasis .............................................................13 1.1.4.2 Cutaneous leishmaniasis.........................................................14 1.1.4.3 Effect of immunosuppression ..................................................15 1.1.4.4 Future treatment options .........................................................15 1.1.5 Potential for vaccination .............................................................16 1.2 Interaction Between Leishmania and Host Cells...........................17 1.2.1 Invasion of macrophages ...........................................................17 1.2.2 Survival of Leishmania ...............................................................18 1.2.2.1 Neutrophils in Leishmania infection.........................................19 1.2.2.2 Leishmania and antigen presentation......................................20 1.2.2.3 Manipulation of dendritic cells .................................................21 1.2.3 Nutrient acquisition by Leishmania .............................................22 1.3 Peptidases....................................................................................24 1.3.1 Peptidase biochemistry ..............................................................24 1.3.2 Types of peptidases...................................................................25 1.3.3 Peptidase inhibitors as drugs .....................................................27 1.3.4 Peptidases as potential drug targets..........................................28 1.3.5 Peptidases in Leishmania ..........................................................30 1.3.5.1 Aspartic peptidases .................................................................30 1.3.5.2 Threonine peptidases..............................................................32 1.3.5.3 Metallo-peptidases ..................................................................32 1.3.5.4 Cysteine peptidases ................................................................34 1.3.5.5 Serine peptidases....................................................................37 1.4 Aims of Project..............................................................................39 Chapter 2 MATERIALS & METHODS..............................................................40 2.1 Cell Culture...................................................................................41 2.1.1 Leishmania spp promastigote culture.........................................41 2.1.2 Growth of Leishmania spp axenic amastigotes..........................41 2.1.3 Growth of THP-1 cells ................................................................42 v 2.1.4 Isolation of L. major metacyclic promastigotes...........................42 2.1.5 Determination of cell densities ...................................................42 2.1.5.1 Promastigote parasites............................................................42 2.1.5.2 Axenic amastigote parasites....................................................43 2.1.5.3 THP-1 cells..............................................................................43 2.1.6 Harvest, lysis and fractionation of cells ......................................43 2.1.7 Differentiation of THP-1 cells......................................................43 2.2 Infectivity of Leishmania spp.........................................................44 2.2.1 Harvest of peritoneal exudate macrophages (PEM)...................44 2.2.2 Infectivity in vitro in PEM ............................................................44 2.2.3 Assessment of infectivity in BALB/c mice...................................45 2.3 Testing the Effectiveness of Anti-Leishmanial Drugs....................45 2.3.1 Alamar blue tests with promastigote cells ..................................45 2.3.2 Testing of amastigotes within PEM ............................................46 2.4 Uptake and Cleavage of Fluorescent Molecules Observed with Fluorescent Plate Reader .............................................................46 2.4.1 Basic peptide-AMC cleavage assay...........................................46 2.4.2 Inhibition of cleavage .................................................................47 2.5 Molecular Biology Techniques......................................................48 2.5.1 Isolation of genomic DNA from L. major .....................................48 2.5.2 Polymerase chain reactions (PCRs) ..........................................49 2.5.3 Oligonucleotides used................................................................49 2.5.4 Cloning of DNA fragments .........................................................51 2.5.4.1 Digestion of DNA with restriction enzymes..............................51 2.5.4.2 DNA gel electrophoresis and gel extraction.............................51 2.5.4.3 Ligations..................................................................................52
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