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Hamilton, Hazel Jean (2019) the Leish Niche: the Secretome of Leishmania and Its Role in Parasite Virulence

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Hamilton, Hazel Jean (2019) the Leish Niche: the Secretome of Leishmania and Its Role in Parasite Virulence Hamilton, Hazel Jean (2019) The Leish niche: the secretome of Leishmania and its role in parasite virulence. PhD thesis. https://theses.gla.ac.uk/41158/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] The Leish Niche: The secretome of Leishmania and its role in parasite virulence Hazel Jean Hamilton BSc (Hons), MRes Submitted in fulfilment of the requirement for the Degree of Doctor of Philosophy Institute of Infection, Immunity and Inflammation College of Medical, Veterinary and Life Sciences University of Glasgow April 2019 II Abstract Leishmaniasis is a neglected tropical parasitic disease that causes several debilitating manifestations. No commercially available vaccine exists against this disease, and treatment strategies are far from ideal with the emergence of resistance, coupled with toxic side effects of many of the drugs available. Rational drug design relies on knowledge of the cell biology of the parasite and the interplay between the parasite and its hosts. Production of secreted proteins, the secretome, has become a known strategy for parasite invasion and persistence in host cells, however, host-parasite interaction is still not well defined. Virulence factors secreted by the parasite mediate the host-parasite interaction and create a niche permissive for parasite proliferation. They therefore represent potential therapeutic targets and vaccine candidates. Here, the use of secretomics was implemented to investigate these virulence factors. Parasite conditioned culture supernatant, containing the secretome, was characterised by morphological, immunochemical and proteomic analyses. Here, we optimised and extended current methods and applied them to the medically relevant amastigote stage. Method development and validation was implemented to extract a reproducible secretome in vitro. Induction of cell stress was managed and cell viability maintained to minimise interference of intracellular proteins. A total of 256 proteins were reproducibly identified in the secretome of promastigotes and 36 proteins were reproducibly identified in the secretome of amastigotes. Analysis of their protein abundance index (emPAI) allowed comparison of the relative abundance of proteins and functions of the secretome throughout the parasite life cycle. Differences in the putative functions of nutrient salvage, protease production and antioxidant activity were observed. Analyses reveal that many proteins lack a signal peptide and as such are thought to be released by nonclassical secretion mechanisms. Several exosome-associated proteins and membrane proteins were also detected in the secretome, suggesting the occurrence of secretion by exosomes or microvesicles. Extended comparative analyses between the secretome of parasites with differing phenotypes allowed us to infer functionality of the secretome in the parasite’s III survival but also variations within the same species which result in differing disease outcomes. Dysregulation in the secretion of various proteins in attenuated parasites implicates these proteins in the virulence of the parasite. An increase in the secretion of pro-inflammatory mediators and destructive proteases by parasites isolated from patients with chronic cutaneous leishmaniasis compared to those from patients with self-healing lesions, indicates the role of the parasite in the chronicity of cutaneous leishmaniasis. Here, we demonstrate an applicable method for the study of the Leishmania mexicana promastigote and amastigote secretome. Results suggest that the secretome plays a role in disease progression and virulence. Proteomic analyses of the secretome, like this study presented here, provide crucial information on the host:parasite interaction for the identification of therapeutic targets and potential vaccine candidates for the provision of safer treatments and new vaccines for eradication of this disease. IV Table of Contents Abstract ................................................................................... II List of Tables ............................................................................... VIII List of Figures ................................................................................ IX Conference Proceedings .................................................................... XI Acknowledgements ......................................................................... XII Author’s Declaration ...................................................................... XIII Abbreviations ............................................................................... XIV Introduction ................................................................... 16 1.1 Leishmaniasis ................................................................. 17 1.1.1 Diagnosis, treatment and vaccination ..................................... 19 1.2 The Leishmania parasite .................................................... 21 1.3 Mechanisms of Leishmania parasite survival ............................. 22 1.3.1 Promastigote survival in the sand fly vector ............................. 22 1.3.2 During transmission from vector to host .................................. 24 1.3.3 Entry to the phagolysosome and differentiation ........................ 25 1.3.4 Amastigote survival .......................................................... 28 1.4 Protein secretion ............................................................. 32 1.4.1 Trypanosomatid secretion .................................................. 33 1.5 Proteomics .................................................................... 36 1.5.1 Gel-based proteomics ....................................................... 36 1.5.2 Gel-free proteomics ......................................................... 37 1.5.3 Quantitative MS-based proteomics ........................................ 39 1.5.4 Secretomics ................................................................... 40 1.6 Summary and Aims ........................................................... 44 Materials & Methods ......................................................... 45 2.1 Materials ....................................................................... 46 2.2 Parasite culture .............................................................. 46 2.3 Alamar blue assay ............................................................ 47 2.4 Secreted protein isolation .................................................. 47 2.5 Whole cell protein lysate collection ....................................... 48 2.6 Protein assay .................................................................. 49 2.7 1D SDS-PAGE .................................................................. 49 2.8 Western blotting.............................................................. 50 2.9 2D electrophoresis ........................................................... 50 2.9.1 Gel casting using the Ettan DALT system ................................. 50 2.9.2 Difference gel electrophoresis (DiGE) sample preparation ............ 51 2.9.3 Isoelectric focussing (1st dimension) ...................................... 51 V 2.9.4 Molecular weight separation, SDS-PAGE (2nd dimension) ............... 52 2.9.5 Gel imaging ................................................................... 52 2.10 Spot Picking and Processing ................................................ 53 2.11 Trypsin digestion ............................................................. 53 2.12 TMT™ Labelling ............................................................... 54 2.13 Peptide/protein identification by nLC-ESI-MS/MS ....................... 55 2.14 Mascot ......................................................................... 56 2.15 Following TMT labelling ..................................................... 57 2.16 Analyses of the L. mexicana secretome .................................. 57 Method development for Leishmania secretomics ...................... 58 3.1 Introduction ................................................................... 59 3.1.1 Axenic cell culture in secretomic studies ................................. 59 3.1.2 Isolating the secretome of Leishmania .................................... 62 3.2 Aims and hypotheses ......................................................... 65 3.3 Results ......................................................................... 66 3.3.1 Evaluation of media for secretome collection ........................... 66 3.3.2 Cell viability is monitored during secretome isolation ................. 68 3.3.3 Supernatant recovery and concentration ................................. 69 3.3.4 Amastigote secreted proteins are degraded by proteolysis ............ 71 3.3.5 Protein quantitation ......................................................... 73 3.3.6 Identification of secretome proteins by mass spectrometry ........... 74 3.3.7 Validation of the secretome by comparison to lysate .................. 84 3.4 Discussion
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