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Characterising the association of CCR5 with Leishmania donovani infection and the protein composition of the Leishmania-containing intracellular phagolysosome

Saunders, Christopher (2016) Characterising the association of CCR5 with Leishmania donovani infection and the protein composition of the Leishmania-containing intracellular phagolysosome. PhD thesis, University of York.

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Abstract

Leishmania parasites infect macrophages where they reside within phagolysosomal compartments but the mechanisms of entry and intracellular survival are not fully understood. The chemokine receptor CCR5 may facilitate L. donovani infection but whether this is through roles in individual macrophages or in coordinating the immune response is unclear. We aimed to determine the association between L. donovani LV9 amastigotes and CCR5 both in vivo and in vitro using an established infection model. We also performed comparative proteomics on isolated phagolysosomal compartments from cells infected with live and heat-inactivated (HI) parasites to better characterise the compartment and determine whether CCR5 was associated with it. Parasite burden between mice and macrophages either possessing or lacking CCR5 did not differ. However, CCR5-/- mice had a transient decrease in leukocyte recruitment to hepatic granulomas. In vitro, we observed a dose- and contact-dependent Leishmania-specific reduction in CCR5 cell surface expression during a short time-course of infection. CCR5 decreases were maintained when using the antagonist ligand Met-RANTES. When combined with reductions seen in CCR5 mRNA levels, the cell surface decrease was likely caused by gene down-regulation rather than pure receptor internalisation. Leishmania parasites were made magnetisable and then isolated from infected cells; we found that CCR5 was not associated with these compartments. Combined use of iTRAQ-labelled LC-MS/MS and mathematical techniques identified a number of protein groups differentially expressed on phagolysosomes isolated from cells infected with live or HI parasites, including those related to actin dynamics, phagosome maturation and intracellular signalling. Overall, these findings demonstrate that CCR5 has a subtle role in the immune response to L. donovani infection and give new insights into how the live parasite exploits its phagolysosomal compartment to fine-tune the host cell’s response to infection.

Item Type: Thesis (PhD)
Academic Units: The University of York > Biology (York)
Depositing User: Mr Christopher Saunders
Date Deposited: 12 Apr 2017 14:42
Last Modified: 12 Apr 2017 14:42
URI: http://etheses.whiterose.ac.uk/id/eprint/16340

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