Proteomic approaches to drive advances in helminth extracellular vesicle research

Highlights

• The proteomic composition of extracellular vesicles (EVs) is known for 17 helminths.

• No bona fide biomarkers have been described yet for the different EV populations.

• Targeted and untargeted proteomic approaches could help identifying biomarkers.

• Novel proteomic approaches are needed to unravel the mechanisms of EV biogenesis.

Abstract

Helminths can interact with their hosts in many different ways, including through the secretion of soluble molecules (such as lipids, glycans and proteins) and extracellular vesicles (EVs). The field of helminth secreted EVs has significantly advanced in recent years, mainly due to the molecular characterisation of EV proteomes and research highlighting the potential of EVs and their constituent molecules in the diagnosis and control of parasitic infections. Despite these advancements, the lack of appropriate isolation and purification methods is impeding the discovery of suitable biomarkers for the differentiation of helminth EV populations. In the present review we offer our viewpoint on the different proteomic techniques and approaches that have been developed, as well as solutions to common pitfalls and challenges that could be applied to advance the study of helminth EVs.

Introduction

The field of extracellular vesicles (EVs) secreted by helminths has gained significant momentum since the first report in 2012 (Marcilla et al., 2012). Parasitic helminths from different phyla (Platyhelminthes and Nematoda) have been shown to secrete at least two populations of EVs (120k and 15k, also termed small exosome-like EVs and microvesicles, respectively) that contain numerous proteins and genetic material and that are able to interact with their hosts in many different ways (Eichenberger et al., 2018b; Tritten and Geary, 2018). Indeed, EVs from parasitic trematodes and nematodes have been the focus of studies immunomodulation and vaccination since they contain immune-regulatory properties that can be disrupted via vaccine-mediated approaches (Drurey et al., 2020; Kifle et al., 2020a).

So far, the proteomic composition from 17 helminth parasites (including 6 nematode, 6 trematode and 5 cestode species) have been reported in a total of 27 studies, with substantial variability in the number of proteins identified (reviewed in (Sotillo et al., 2020)). In general, most of the studies have focused on analysing the full proteomic content present in EVs, although a few studies, particularly in trematode-derived EVs, have also analysed the different compartments (membrane and internal lumen) of EVs using a combination of mild-digestion with trypsin and the separation of membrane from luminal EV proteins with detergents and centrifugation approaches (Cwiklinski et al., 2015; Kifle et al., 2020b). Most of these studies have identified protein vaccine and diagnostic candidates, particularly in schistosomes (Sotillo et al., 2016; Zhu et al., 2016); however, as we will discuss later in this review, no phylum- or class-specific biomarkers have been characterised for the different helminth EV populations yet.

The diversity in the methodology used for the isolation of EVs as well as in the techniques applied for proteomic analysis has led some authors to propose that the development of standardised purification and analysis methodologies, as well as a more consistent reporting of data, would facilitate comparison between datasets, simplify future research and drive the helminth EV agenda forward (Sotillo et al., 2020). In addition, despite advances in sample processing and mass spectrometry technologies during the last decade (i.e. targeted methods and data-independent acquisition (DIA) approaches), these have not been applied yet to the helminth EV field. Furthermore, while most of the studies employ shotgun LC–MS/MS as a technique for peptide and protein identification, there is a great disparity in sample processing, with some authors performing protein separation (i.e. 1D or 2D electrophoresis) prior to mass spectrometry while others analyse the EV proteome without prior fractionation (Zamanian et al., 2015; Sotillo et al., 2016; Tzelos et al., 2016; Zhu et al., 2016; Siles-Lucas, 2017; Eichenberger et al., 2018c, 2018a; Harischandra et al., 2018; Davis et al., 2019; Nicolao et al., 2019).

In the present review we focus on the different proteomic techniques that can be applied for biomarker discovery, current challenges and potential solutions, as well as novel mass spectrometry technologies that can be used to advance the field of helminth EVs (Fig. 1). These novel approaches and technologies will be of great value to deepen our knowledge in EV biogenesis and find potential biomarkers in the near future.