Biology subjects

Selkirk, M. E.

Publications and source records attributed to Selkirk, M. E..

5 recordsLinked to original sources

bioRxiv2023

Identification of proteins that bind extracellular microRNAs secreted by the parasitic nematode Trichinella spiralis

Small non-coding RNAs such as microRNAs (miRNAs) are conserved across eukaryotes and play key roles in regulating gene expression. In many organisms, miRNAs are also secreted from cells, often encased within vesicles such as exosomes and sometimes extravesicular. The mechanisms of miRNA secretion, how they are stabilised outside of cells and their functional importance are poorly understood. Recently we characterised the parasitic nematode Trichinella spiralis as a model to study miRNA secretion. T. spiralis larvae secrete abundant miRNAs which are largely extravesicular. Here, we investigated how T. spiralis miRNAs might remain stable outside of cells. Using proteomics, we identified two RNA binding proteins secreted by T. spiralis larvae and characterised their RNA binding properties. One, a homologue of the known RNA binding protein KSRP, binds miRNA in a selective and sequence-specific fashion. Another protein, which is likely a novel RNA binding protein, binds non-selectively to miRNA. Our results suggest a possible mechanism for miRNA secretion by T. spiralis and may have relevance for understanding the biology of extracellular miRNA more widely.

biochemistry↗

bioRxiv2022

Gene editing in the nematode parasite Nippostrongylus brasiliensis using extracellular vesicles to deliver active Cas9/guide RNA complexes

Despite recent advances, animal-parasitic nematodes have thus far been largely refractory to genetic manipulation. We describe here a new approach providing proof of principle that CRISPR/Cas9-mediated gene editing of parasitic nematodes is achievable using vesicular stomatitis virus glycoprotein-pseudotyped extracellular vesicles (EV) for the delivery of Cas9-synthetic guide RNA (RNP) complexes. We demonstrate that EV-delivered RNPs can be used to disrupt a secreted DNase II in Nippostrogylus brasiliensis. Introduction of a repair template encoding multiple stop codons led to measurable reduction in expression of the targeted gene. Altered transcripts corresponding to the edited locus were detected by RT-PCR, demonstrating that vesicles can access cells of tissues actively expressing the gene of interest. These data provide evidence that this technique can be employed for targeted gene editing in N. brasiliensis, making this species genetically tractable for the first time and providing a new platform for genetic analysis of parasitic nematodes. Author SummaryParasitic nematodes have a complex life cycle involving passage through a host organism, which makes them very difficult to manipulate genetically. Recently, a method for deleting, changing or replacing genes (gene editing) has been developed in other organisms which has revolutionised our ability to understand fine details of how these organisms work. It has generally not been possible to adapt this method to parasitic nematodes because delivery of the components is difficult, and this has proved to be a bottleneck in understanding how parasites develop, survive and interact with their host. We show here that the components for gene editing can be introduced into a widely used laboratory model of intestinal nematode infection by encapsulation in membrane-bound vesicles which have been modified to carry a protein which facilitates fusion of the vesicles with parasite cells and delivery of the contents. This resulted in accurate editing of a specific gene by deletion and repair, such that the amount of functional protein produced from that gene was reduced. This system should be applicable to all nematode species, and will facilitate understanding of their complex biology, in addition to defining new targets for control of infection.

molecular biology↗

bioRxiv2022

Large-scale proteomic analysis of T. spiralis muscle-stage ESPs identifies a novel upstream motif for in silico prediction of secreted products

The Trichinella genus contains parasitic nematodes capable of infecting a wide range of hosts including mammals, birds and reptiles. Like other helminths, T. spiralis secretes a complex mixture of bioactive molecules capable of modulating its immediate surroundings and creating a hospitable environment for growth, survival and ultimately transmission. The constitution of these excretory-secretory products (ESPs) changes depending on the tissue niche and the specific stage of parasite development. Unique to T. spiralis is a true intracellular stage wherein larvae develop inside striated myotubes. Remarkably, the parasite larvae do not destroy the host cell but rather reprogram it to support their presence and growth. This transformation is largely mediated through stage-specific secretions released into the host cell cytoplasm. In this study, we apply state of the art proteomics and computational approaches to elucidate the composition and functions of muscle-stage T. spiralis ESPs. Moreover, we define a commonly-occurring, upstream motif that we believe is associated with the stichosome, the main secretory organ of this worm, and can thus be used to predict secreted proteins across experimentally less tractable T. spiralis life cycle stages. Author SummaryTrichinella spiralis is the only helminth parasite with a true intracellular stage. Newborn larvae penetrate the intestinal wall of the host, enter the circulation and preferentially infect muscle cells. Remarkably, they do not destroy the host cell but rather initiate a series of modulatory events that transform it into a nurse cell complex, a collagenated cyst that can persist for years. Each stage of T. spiralis development is guided by host-targeted secretions released by the worm directly into its immediate environment, mediating events such as immunoregulation, cell cycle control and angiogenesis. As such, these worm effectors hold therapeutic potential for chronic and autoimmune diseases. The composition of excretory-secretory products (ESPs) changes according to what the worm needs to accomplish and what tissue niche it is occupying at the time, with many deriving from the stichosome, the worms dedicated secretory organ. In this study, we characterise ESPs of muscle-stage T. spiralis larvae using proteomic and bioinformatic approaches and we define a regulatory motif associated with stichosome-derived proteins.

cell biology↗

bioRxiv2022

Temporal profiling of CD4 T-cell activation and differentiation upon SARS-CoV-2 spike protein immunisation

CD4 T-cells require T-cell receptor (TCR) signalling for their activation and differentiation. Foxp3+ regulatory T-cells (Treg) are dependent on TCR signals for their differentiation and suppressive function. However, it is not fully known how TCR signalling controls the differentiation of polyclonal CD4 T-cells upon antigen recognition at the single-cell level in vivo. In this study, using Nr4a3-Tocky (Timer-of-cell-kinetics-and-activity), which analyses temporal changes of antigen-reactive T-cells following TCR signalling, we investigated T-cell response to Spike protein fragments (S1a, S1b, S2a, and S2b) upon immunisation. We show that S1a and S2a induced the differentiation of PD1hiCXCR5+ T follicular helper (Tfh) cells, which is related to CD4 T-cell immunogenicity. In contrast, S1b induced CD25hiGITRhiPD-1int Treg, which intermittently received TCR signalling. Using Foxp3-Tocky, which analyses Foxp3 transcriptional dynamics, the S1b-reactive Treg sustained Foxp3 transcription over time, which is a hallmark of activated Treg. Foxp3 fate-mapping showed that the S1b-reactive Treg were derived not from pre-existing thymic Treg, suggesting Foxp3 induction in non-Treg cells. Thus, the current study reveals temporally dynamic differentiation of CD4 T-cells and Treg upon immunisation in the polyclonal TCR repertoire.

immunology↗

bioRxiv2020

Taenia larvae possess distinct acetylcholinesterase profiles with implications for host cholinergic signalling

Larvae of the cestodes Taenia solium and Taenia crassiceps infect the central nervous system of humans. Taenia solium larvae in the brain cause neurocysticercosis, the leading cause of adult-acquired epilepsy worldwide. Relatively little is understood about how cestode-derived products modulate host neural and immune signalling. Acetylcholinesterases, a class of enzyme that degrade acetylcholine, are produced by a host of parasitic worms to aid their survival in the host. Acetylcholine is an important signalling molecule in both the human nervous and immune systems, with powerful modulatory effects on the excitability of cortical networks. Therefore, it is important to establish whether cestode derived acetylcholinesterases may alter host neuronal cholinergic signalling. Here we make use of multiple techniques to profile acetylcholinesterase activity in different extracts of both Taenia crassiceps and Taenia solium larvae. We find that the larvae of both species contain substantial acetylcholinesterase activity. However, acetylcholinesterase activity is lower in Taenia solium as compared to Taenia crassiceps larvae. Further, whilst we observed acetylcholinesterase activity in all fractions of Taenia crassiceps larvae, including on the membrane surface and in the excreted/secreted extracts, we could not identify acetylcholinesterases on the membrane surface or in the excreted/secreted extracts of Taenia solium larvae. Finally, using whole-cell patch clamp recordings in rat hippocampal brain slice cultures, we demonstrate that Taenia larval derived acetylcholinesterases can modify neuronal responses to acetylcholine. Together, these findings highlight the possibility that Taenia larval acetylcholinesterases can interfere with cholinergic signalling in the host, potentially contributing to pathogenesis in neurocysticercosis. Author summaryInfection of the human nervous system with larvae of the parasite Taenia solium is a significant cause of acquired epilepsy worldwide. Despite this, the precise cellular and molecular mechanisms underlying epileptogenesis in neurocysticercosis remain unclear. Acetylcholinesterases are a family of enzymes widely produced by helminthic parasites. These enzymes facilitate the breakdown of acetylcholine, which is also a major neurotransmitter in the human nervous system. If T. solium larvae produce acetylcholinesterases, this could potentially disrupt host cholinergic signalling, which may in turn contribute to seizures and epilepsy. We therefore set out to investigate the presence and activity of acetylcholinesterases in T. solium larvae, as well as in Taenia crassiceps larvae, a species commonly used as a model parasite in neurocysticercosis research. We found that both T. crassiceps and T. solium larvae produce acetylcholinesterases with substantial activity. We further demonstrate that the acetylcholinesterase activity in the products of these parasites is sufficient to disrupt cholinergic signalling in an ex vivo brain slice model. This study provides evidence that Taenia larvae produce acetylcholinesterases and that these can interfere with cholinergic signalling in the host and potentially contribute to pathogenesis in neurocysticercosis.

neuroscience↗