RNA interference in parasitic nematodes – from genome to control
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Tzelos2015.docx (4.030Mb)
Date
04/07/2015Author
Tzelos, Thomas
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Abstract
Teladorsagia circumcincta is a parasitic nematode which is a major cause of ovine
parasitic gastroenteritis in temperate climatic regions. The parasite has developed
resistance to the major anthelmintic drug classes and this challenges its future
control. Vaccination is a potential alternative control method since sheep are able to
develop protective immunity against this parasite. Although potential vaccine
candidates have been revealed, the increasing gene datasets suggest that vaccinetarget
selection may be aided by screening methods such as RNAi. This is a reverse
genetic mechanism that causes highly specific gene silencing which was initially
described and applied to defining gene function in Caenorhabditis elegans.
Nevertheless, its application was more difficult than anticipated in parasitic
nematodes because of the inconsistency of the silencing effect. In the unsuccessful
cases, did the dsRNA penetrate the parasite and activate the RNAi pathway? Thus
far, there are no internal controls that indicate the activation of the pathway. Are the
RNAi pathway genes constantly transcribed or are they ‘switched on’ in response to
the dsRNA exposure? The initial aim of the study was to determine potential marker
genes in the RNAi pathway that could indicate the activation of the pathway in C.
elegans. After the exposure to dsRNA from two target genes, the transcript levels of
three candidate marker genes (Ce-dcr-1, Ce-ego-1 and Ce-rsd-3) were examined and
showed that exposure to dsRNA has no effect on the transcript levels of these genes
making them inappropriate markers for the activation of the RNAi pathway. The two
target-genes were Ce-cpr-4 and Ce-sod-4 which had been proven to be consistently
susceptible and refractory to RNAi, respectively. Another aim of the project was to
develop an RNAi platform in T. circumcincta for use as a screening method for
potential vaccine candidates. The targets selected for the in vitro RNAi included: five
members of the Activation-associated Secreted Proteins (ASPs); a Macrophage
migration Inhibitory Factor-like (Tci-mif-1) and a Surface Associated Antigen gene
(Tci-saa-1), all of which have been associated with vaccine-induced protective
immunity. The selection of the ASPs was based on a bioinformatic and
transcriptomic analysis of the ASPs in T. circumcincta. The results showed
successful knock-down only for three out of five ASP targets after 1 hour of soaking
in gene-specific double stranded RNA (dsRNA) which illustrates the inconsistency
and the target specificity of RNAi in T. circumcincta which has been observed in the
past with other parasitic nematodes. Inconsistencies were also observed within the
successful ASP targets with the results not being reproducible after several
successful experiments. Potential reasons for the inconsistencies were examined with
the duration of larval storage being a critical factor. Larvae stored for a short or long
period of time were susceptible and refractory to RNAi, respectively. Experiments
were also conducted to investigate how the ASPs relate to extracellular microvesicles
(EMVs). These vesicles are considered to play an important role in the intercellular
communication between parasites and their hosts, and thus represent potentially
useful vaccine and/or drug targets. Transmission electron microscopy (TEM)
confirmed that EMVs are excreted / secreted by the parasite and the proteomic
analysis revealed several types of proteins within the vesicles such as: ASPs, Actins,
Metallopeptidases, and RAB proteins. A comparative analysis of EMVs, EMV-free
ES (Excretory / Secretory) and total ES products showed that approximately 35% of
the proteins found in the vesicles could also be identified in EMV-free ES and in
total ES products, whilst the remaining 65% were present only in EMVs.