11 research outputs found
Characterisation of secreted exosomes from the intestinal nematode Heligmosomoides polygyrus
The parasite secretome has been shown to play a key role in both pathogenicity and
the regulation of host defence, allowing pathogens, such as helminths, to establish a
chronic infection within the host. The recently discovered presence of extracellular
vesicles within parasite-derived excretory-secretory products introduces a new
mechanism of potential cross-species communication. Extracellular vesicles (EVs),
such as exosomes, facilitate cellular communication through the transfer of small
RNAs, lipids and proteins between cells and organisms across all three kingdoms of
life. In addition to their roles in normal physiology, EVs also transport molecules
from pathogens to hosts, presenting parasite antigens and transferring infectious
agents.
Here, I examine secreted vesicles from the murine gastrointestinal nematode
Heligmosomoides polygyrus, and their potential role in the host-helminth
interactions. Transmission electron microscopy reveals vesicle-like structures of 50-
100 nM in the ultracentrifuged secretory product, and potential evidence of multi-vesicular
bodies in the worm intestine. This, coupled with information from the
exoproteome, helped support the hypothesis that exosomes originate from the
parasite intestinal tract.
I have completed a series of studies looking at the fundamental properties of
exosome-cell interactions, providing comparative studies between mammalian and
H. polygyrus-derived exosomes. I have determined some of the key factors
influencing exosome uptake, including time of incubation, cell type and exosome
origin. Through microarray analysis of H. polygyrus exosome-treated small
intestinal epithelial cells, we see significant gene expression changes, including those
involved in the regulation of signalling and the immune response, such as DUSP1
(dual-specificity phosphatase) and IL1RL1 (the receptor for IL-33). The modest
reduction of inflammatory cytokine responses by exosomes in small intestinal cell
lines was amplified in immune cells, such as macrophages. Exosomes can
significantly reduce expression of classical activation markers, as well as
inflammatory cytokine production in the macrophage cell line RAW 264.7, and this
is further supported by similar responses in bone marrow-derived macrophages.
Owing to their suppressive nature, I demonstrate that immunization of mice
with an exosome/alum conjugate generates significant protection from a subsequent
H. polygyrus larval challenge, as seen through a reduction in egg counts and worm
burden.
I have investigated the role of the IL33 receptor (IL-33R); a key molecule
associated with parasitic resistance that is suppressed by exosomes in type-2
associated immune responses. Uptake of H. polygyrus-derived exosomes by
alternatively activated macrophages caused the suppression of type 2
cytokine/protein release and the reduction of key genes associated with this
phenotype. In addition, there was also significant repression of both transcript and
surface T1/ST2, a subunit of the IL-33R). Using a model of lung inflammation, in
vivo studies demonstrate that, in both prophylactic and co-administration
experiments, exosomes modulate the innate cellular response. This is represented by
changes in the number of innate lymphoid cells (ILCs), bronchoalveolar lavage
eosinophils and type-2 cytokine output. In this system, the expression of T1/ST2 on
type 2 ILCs was also significantly reduced.
I have extended the investigation on exosome-IL-33R responses by using
T1/ST2 knockout mice. Despite generating strong antibody responses, vaccination
against exosomes could not protect T1/ST2 knockout mice against a subsequent
infection.
This work suggests that exosomes secreted by nematodes could mediate the
transfer and uptake of parasite products into host cells, establishing cross-species
communication to suppress the host ‘danger’ or inflammatory response
Identification of Newly Synthesized Proteins by Echinococcus granulosus Protoscoleces upon Induction of Strobilation
The proteins responsible for the key molecular events leading to the structural changes between the developmental stages of Echinococcus granulosus remain unknown. In this work, azidohomoalanine (AHA)-specific labeling was used to identify proteins expressed by E. granulosus protoscoleces (PSCs) upon the induction of strobilar development.The in vitro incorporation of AHA with different tags into newly synthesized proteins (NSPs) by PSCs was analyzed using SDS-PAGE and confocal microscopy. The LC-MS/MS analysis of AHA-labeled NSPs by PSCs undergoing strobilation allowed for the identification of 365 proteins, of which 75 were differentially expressed in comparison between the presence or absence of strobilation stimuli and 51 were expressed exclusively in either condition. These proteins were mainly involved in metabolic, regulatory and signaling processes.After the controlled-labeling of proteins during the induction of strobilar development, we identified modifications in protein expression. The changes in the metabolism and the activation of control and signaling pathways may be important for the correct parasite development and be target for further studies