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

Abstract

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 recurring, upstream motif associated with the stichosome, the main secretory organ of this worm, and can be used to predict secreted proteins across experimentally less tractable T. spiralis life cycle stages.

Keywords: ES; Trichinella; helminth; proteomics; secretions.

Figure 1

Venn diagram showing the proportion of the proteome and ESPs annotated. The number of NCBI Conserved Domain Search annotated proteins (6,176 total/314 ESPs), and KEGG Ontology annotated proteins (3,661 total/190 ESPs), and proteins not annotated by these approaches (7,928 total/84 ESPs).

Figure 2

Combined excerpt of KEGG Pathways to illustrate ESPs that may be involved in catabolism of polysaccharide storages to lactate and TCA intermediates. The diagram illustrates the presence (blue with T. spiralis Gene_Stable_ID) and absence (red) of proteins (with KEGG Enzyme ID and Name) in the T. spiralisexcretory/secretory proteins (ESPs). Metabolites are displayed in black, with arrows indicating anticipated flux direction. Proteins pertinent to the key stages Polysaccharide metabolism, Glycolysis, Malate dismutation and the TCA cycle have been indicated by differential background colours.

Figure 3

Alignment and phylogenetic analysis of T. spiralis NUDT9 homologs with their human counterparts. (A) Phylogenetic relationship of NUDT9 homologs. T. spiralis secreted homologues are highlighted in green with human NUDT9 and TRPM2 NUDT9 homology domain boxed). The evolutionary tree was calculated using the maximum likelihood method implemented by the PhyML plugin for Geneious Prime. Proportions after 500 bootstraps are shown at each node. (B) Alignment of 13 T. spiralis NUDT9 homologues performed using ClustalW. Residues conforming to the nudix motif are highlighted in blue. Conserved residues are coloured red. Residues with similar physiochemical properties are coloured yellow when >70% in each column.

Figure 4

Distribution of WoLF PSORT predicted excretory/secretory (ES) protein localisations. Pie chart of top predicted subcellular localisations for ES proteins by WoLF PSORT as a percentage of the ESPs. Dual localisations indicated by “/” represent proteins expected to shuttle between two compartments.

Figure 5

Summary of top Homer2 de novo motif search hit enriched upstream of excretory/secretory proteins (ESPs). The PouStich motif sequence is displayed as a Hidden Markov model logo. Beneath is the percentage of PouStich motif occurrence in the 1000bp upstream of the transcription start site (TSS) of ESPs compared to the “Background” of all T. spiralis protein coding genes and the P-value associated with the enrichment. The position of motif occurrence in distance from the TSS in both sample populations is indicated with +/- Standard deviation. The frequency of which the motif occurs multiple times in one upstream region is represented by the Multiplicity. The table also identifies the nearest known motif database hit and nearest C. elegans homolog motif as an alignment.

Figure 6

Pou domain containing octamer motif binding proteins. (A) MUSCLE alignment of the Human Pou2f3 with the closest (C) elegans and T. spiralis BLASTp hits. Amino acid shading highlights similarity. In the red box is the Pou domain and in the blue box is the homeobox domain, the two essential conserved regions of Pou-domain containing octamer motif binding proteins. (B) Phylogenetic tree from alignment of the T. spiralis and (C) elegans Pou domains. The tree was constructed with Phyml from aligned Pou domains across all sequences shown above using the Whelan and Goldman substitution model. The Pou domain type is annotated adjacent to the tree groupings. T. spiralis genes are included in brackets next to the gene name, while only (C) elegans gene names are provided.

Figure 7

Venn diagram showing the proportion of the ESPs annotated with motifs. 398 ESPs were identified in this study, of which 143 contained a SignalP6.0 detected Secretion Signal, and 127 ESPs contained a Poutich motif upstream of the transcription start site. The 61 ESPs that contain a Secretion Signal and PouStich motif are a subset of high confidence predicted ESPs.