Abstract
The parasitic nematode intestine is responsible for nutrient digestion and absorption, and many other processes essential for reproduction and survival, making it a valuable target for anthelmintic drug treatment. However, nematodes display extreme biological diversity (including occupying distinct trophic habitats), resulting in limited knowledge of intestinal cell/protein functions of fundamental or adaptive significance. We developed a perfusion model for isolating intestinal proteins in Ascaris suum (a parasite of humans and swine), allowing for the identification of over 1000 intestinal A. suum proteins (using mass spectrometry), which were assigned to several different intestinal cell compartments (intestinal tissue, the integral and peripheral intestinal membranes, and the intestinal lumen). A multi-omics analysis approach identified a large diversity of biological functions across intestinal compartments, based on both functional enrichment analysis (identifying terms related to detoxification, proteolysis, and host-parasite interactions) and regulatory binding sequence analysis to identify putatively active compartment-specific transcription factors (identifying many related to intestinal sex differentiation or lifespan regulation). Orthologs of A. suum proteins in 15 other nematodes species, five host species, and two outgroups were identified and analyzed. Different cellular compartments demonstrated markedly different levels of protein conservation; e.g. integral intestinal membrane proteins were the most conserved among nematodes (up to 96% conservation), whereas intestinal lumen proteins were the most diverse (only 6% conservation across all nematodes, and 71% with no host orthologs). Finally, this integrated multi-omics analysis identified conserved nematode-specific intestinal proteins likely performing essential functions (including V-type ATPases and ABC transporters), which may serve as promising anthelmintic drug or vaccine targets in future research. Collectively, the findings provide valuable new insights on conserved and adaptive features of nematode intestinal cells, membranes and the intestinal lumen, and potential targets for parasite treatment and control.
Highlights
From the ‡The Genome Institute, Washington University in St Louis, Missouri 63108; §Department of Cell Biology & Physiology and Department of Medicine, Washington University School of Medicine, St
This simple approach lends high confidence that proteins obtained in this perfusate were located in the intestinal lumen (IL), with the exception of protein contaminants that likely originated from pseudocoelomic fluid (PF) during collection
The integral intestinal membrane (IIM; 5k-50k pellet) protein set had the potential to contain proteins destined for the peripheral intestinal membrane (PIM), as well as the lumen, the PF, the basal intestinal membrane and other proteins associated with organellar membranes requiring exclusion of certain protein categories, so these potential sources of contamination were removed, resulting in a set of 81 proteins
Summary
The integral intestinal membrane (IIM) protein set represents expected transmembrane proteins on either the basal or apical intestinal membranes This compartment included the proteins in the P2 pellet fraction (which enriches for TM-domain containing proteins), while excluding [1] the proteins found in either of the perfusate samples, in order to reduce contamination from the peripheral membrane and the intestinal lumen [2] proteins without predicted transmembrane domains according to annotation by Phobius [30], and [3] proteins annotated with ‘cellular compartment’ Gene Ontology terms [23] related to the endoplasmic reticulum, mitochondria, Golgi apparatus, and nucleus, in order to reduce contamination from proteins embedded in these organelles rather than the external cellular membrane, which is the target of interest (GO:0005783, GO: 0005789, GO:0005739, GO:0005741, GO:0000139, GO:0005840, GO:0005740, GO:0005635, GO:0016459). The alignments of protein sequence against the PDB template were applied as restraints of the target proteins related to the templates, with other parameters set to default
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