Investigation of the regulation of transcriptional changes in Ancylostoma caninum larvae following serum activation, with a focus on the insulin-like signalling pathway

Datu, Bennett J.D., Loukas, Alex, Cantacessi, Cinzia, O'Donoghue, Peter, and Gasser, Robin B. (2009) Investigation of the regulation of transcriptional changes in Ancylostoma caninum larvae following serum activation, with a focus on the insulin-like signalling pathway. Veterinary Parasitology, 159 (2). pp. 139-148.

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Abstract

The exit from dauer in the free-living nematode Caenorhabditis elegans is under the control of a single amphidial neuron (ASJ) of the insulin-like signalling pathway. Mutations of this pathway have the ability to suppress entry into the dauer stage. It has been postulated that insulin-like signalling plays a significant role in the response to serum stimulation in vitro of the third-stage larvae (L3s) of the canine hookworm Ancylostoma caninum. To test for the possible involvement of the insulin-like signalling cascade in the response to serum stimulation, the effects of two signalling stimulants (8-bromo cGMP and arecoline) and four inhibitors, namely 4,7-phenanthroline, phosphoinositide-3 kinase (PI3K), Akt inhibitor IV and rapamycin on feeding and on levels of selected activation-associated mRNAs in serum-stimulated L3s were explored. L3s of A. caninum were pre-incubated with or without the appropriate inhibitor/agonist. Following serum-stimulation, the feeding activity was assessed. The transcription levels of a number of activation-associated mRNAs linked to particular expressed sequence tags (ESTs) were investigated by reverse transcription, real-time PCR (rtPCR). The treatment of worms with 4,7-phenanthroline completely suppressed feeding and significantly reduced the differential levels of most activation-associated mRNAs, whereas the treatment with cGMP resulted in the resumption of feeding in almost 85% of the L3s and yielded a specific transcriptional profile consistent with that following serum stimulation. The treatment of L3s with arecoline resulted in the resumption of feeding in ∼85% of L3s, but did not result in a transcriptomic profile consistent with activation. A complete reduction in feeding was recorded in the presence of the PI3K inhibitor LY294002 (1 mM) and resulted in a pronounced dampening of differential transcription in response to serum stimulation for the molecules examined. Akt inhibitor IV resulted in a ∼70% reduction in feeding but had almost no effect on the level of any of the activation-associated mRNAs studied. Rapamycin was shown to have a weak effect on feeding, and several of the mRNAs studied exhibited greater than expected transcription following treatment. The complexities of activation-associated transcription could not be addressed using the current approach. A larger number of mRNAs needs to be investigated in order to predict or identify regulatory mechanisms proposed to function in the insulin-like signalling pathway in A. caninum.

Item ID: 28835
Item Type: Article (Research - C1)
ISSN: 1873-2550
Keywords: Ancylostoma caninum; Caenorhabditis elegans; Dauer arrest; insulin-like signalling pathway; real-time polymerase chain reaction rtPCR; serum-activation; transcriptional changes
Date Deposited: 18 Sep 2013 03:46
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1108 Medical Microbiology > 110803 Medical Parasitology @ 100%
SEO Codes: 92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920109 Infectious Diseases @ 100%
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