|Abstract: ||Exploitation of RNA interference (RNAi) has revolutionised work on Caenorhabditis elegans,
and considerable success has been recently reported with plant-parasitic nematodes. It has
proven difficult to transfer this technology to animal parasitic species, and previous attempts in
this laboratory by feeding Nippostrongylus brasiliensis larvae with Escherichia coli expressing
double-stranded RNA (dsRNA) gave no consistent reductions in levels of target transcripts.
The aim of this study was to develop methods for RNAi in N. brasiliensis, a rodent strongylid
nematode which is closely related to gastrointestinal nematodes of humans and livestock, in
order to explore the biological functions of parasite secreted proteins.
In order to promote uptake of exogenous macromolecules such as dsRNA and small interfering
RNA (siRNA) by infective larvae, the process of activation, whereby larvae are induced to
resume feeding and development, was studied in vitro. Activation could be induced solely by
exposure of larvae to elevated temperature (37oC), whereas host serum or glutathione had no
effect. Neither a membrane permeant analogue of cyclic GMP nor muscarinic receptor agonists
promoted activation, suggesting that a cholinergic neural pathway is not involved in the process.
Activation at 37oC could be blocked by inhibitors of phosphatidylinositol 3-kinase, Akt protein
kinase & cytochrome P450. These data indicate that the early signalling events for larval
activation in N. brasiliensis differ substantially from the hookworm Ancylostoma caninum,
most probably acting through thermosensory rather than chemosensory neurons, but that they
may converge downstream of a step dependent on phosphatidylinositol 3-kinase. Stimulation of
protein secretion paralleled resumption of feeding, suggesting that these processes were tightly
linked and regulated by similar pathways during activation.
Temperature-activated larvae were exposed to dsRNA and siRNA via electroporation or
soaking in the presence of a variety of transfection reagents, but RNAi was unsuccessful.
Serotonin was demonstrated to increase the rate of uptake of macromolecules, yet larvae
exposed to exogenous dsRNA in the presence of serotonin still failed to show RNAi-mediated
gene silencing. In addition, RNAi was also observed to be irreproducible in adult N.
brasiliensis using the same methods of delivery. The use of mRNA encoding firefly luciferase
identified uptake into larvae or adult worms as a major impediment in the process.
Venom Allergen Homologue/ASP-Like (VAL) molecules have been identified as major
components of secreted proteins from many species of parasitic nematodes. In this study, eight
N. brasiliensis VALs were identified and sequenced, and all were shown to be present in
products secreted by adult parasites. NbVAL-7 and NbVAL-8 were also detected in secreted
products of infective larvae. Structural similarities to other members of the Pathogenesis-
Related protein superfamily are discussed, as are possible functions for these proteins in N.