Title page for ETD etd-02182010-185909


Document Type Master's Dissertation
Author Stopforth, Elaine
Email elaines@tuks.co.za
URN etd-02182010-185909
Document Title Proteomic analysis of the humoral antifungal immune response of the soft tick,Ornithodoros savignyi Audouin (1827)
Degree MSc
Department Biochemistry
Supervisor
Advisor Name Title
Prof A W H Neitz Co-Supervisor
Dr A R M Gaspar Supervisor
Keywords
  • Ornithodoros savignyi
  • ticks
  • immune system
  • antifungal drugs
Date 2009-09-02
Availability unrestricted
Abstract

Ticks are blood feeding ectoparasites that ingest large volumes of vertebrate blood. They are the most important arthropods that are capable of transmitting pathogens which cause disease in humans and domestic animals. Ticks are exposed to various microorganisms during feeding as well as in their habitat. They therefore must have a very good immune system to recognize and destroy these microorganisms.

In the present study a micro-broth dilution assay was used to determine whether antifungal activity was present in different tick tissue extracts with or without challenge. The midguts gave the highest inhibition of yeast growth, followed by the salivary glands and then the hemolymph. This was seen with unchallenged tick tissue extracts, as well as tissue extracts collected after yeast challenge (2 hours). Thus all of the tick tissue extracts that was analyzed in this study had antifungal activity.

Proteomics was used to determine whether proteins were differentially expressed in the hemolymph plasma, after a fungal challenge. 2DE was used since proteins are not only separated by molecular mass, but also by their charge. The proteins that were separated on the 2D-gels ranged between 17.5-76 kDa and not all proteins present on the 1D-gels (14-97 kDa) could be seen on the 2D-gels. Ticks were challenged for 2 hours to define the proteins that play a role in the short term innate immune response during a fungal infection. Various proteins were differentially expressed in the hemolymph samples that were collected 2 hours after ticks were injected with saline, -1,3-glucan or yeast (or 72 hours). Injury and fungal challenge play a role in producing proteins that might play a role in the fungal response of the tick. Five spots that were statistically significant in the hemolymph collected 2 hours after ticks were injected with yeast cells were analyzed with MS/MS. No matches were found with MASCOT database searching or with EST searching. This can be due to the limited information that is available on the soft ticks, as only hard tick ESTs heve been published.

It was also attempted to identify hemolymph proteins that might play a role in the recognition of fungi. Hemolymph was incubated with live Candida albicans cells and eluted with buffer. Three protein bands (97, 88 and 26 kDa) were found to be present whether ticks were challenged or unchallenged. These proteins were subjected to MS/MS analysis and database searching was performed revealing no matches to other known proteins.

The antifungal response was found to be present in the soft tick O. savignyi and might play a vital function in the innate immune response during a fungal infection. These proteins may serve as lead molecules that could be used in the development of novel antifungal drugs, as well as in vaccine development.

Copyright 2009, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.

Please cite as follows:

Stopforth, E 2009, Proteomic analysis of the humoral antifungal immune response of the soft tick,Ornithodoros savignyi Audouin (1827), MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-02182010-185909/ >

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