Proteomics Profiling and Functional Characterization of Caenorhabditis elegans Excreted/Secreted Proteins

Abstract

Excretory-secretory products (ESPs) are first characterized and defined in parasitic nematode proteomics studies as the combination of various biomolecules that are continuously excreted or secreted into the environment throughout the whole life cycle. ESPs are particularly interesting to many scientists as anti-parasitic vaccine candidates and as promising drug targets since large portions of ESPs are active enzymes that potentially function directly at the parasite-host or worm-environment interfaces. However, majority of the parasites lack whole genome sequence knowledge and genome-editing tools. Thus, the number of ESPs identified is limited and many functions of ES proteins cannot be elucidated. Therefore, we use the most studied nematode, Caenorhabditis elegans, as the model to characterize the composition of excreted/secreted proteins with the help of nanoliquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS). We characterized more than 509 excreted/secreted proteins with mix-staged worms, including many metalloproteases, cysteine proteases, and lysozymes. Proteases and proteases inhibitors are a major group in C. elegans ESPs. We performed stable isotope dimethylvlabeling quantitative proteomics and compared C. elegans ESPs on different bacteria diets. Lysozymes are not only enriched in C. elegans ESPs but are also up-regulated in response to pathogen and bacteria. Comparative studies of expression profiles of developmental life stages and pathogen infections elucidate the dynamics in regulating ESP components. We successfully identified stage-specific ESP groups associated with L1, L3, adult, L2 dauer, and postdauer. We demonstrated that proteases activities are down regulated by increased protease inhibitor expressions, while during dauer exit proteases expressions are increased. The comparison between dauer excretome/secretome and RNA-seq dauer expression profiles revealed 91 ESP encoding genes that are highly expressed in dauers. We performed dauer formation assay to these dauer-associated gene mutants. The great prediction rate confirmed that our comparative method is the simplest way to quickly pick out candidates for functional assays. Similarly, we employed this comparative method to pathogeninduced transcriptomes. We reported a group of genes that are associated with Serratia marcescens infection and a group of bacterial pathogens responding genes. We confirmed the roles of C. elegans ESPs in immuoregulation by infection assays with various pathogens. Lysosomes and cysteine protease inhibitor are among the most important genes in innate immune response pathway of C. elegans defending pathogen infection. The recent discovery of a C. elegans sibling species, Caenorhabditis inopinata, allows the deeply comparative study for evolutional interpretation. The excretome/secretome of C. inopinata has not been characterized. We took advantage of the sensitive and highthroughput technique of nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) to directly characterize the protein components of C. inopinata excretome/secretome. Functional annotations reveal several protein families, including C-type lectins, Cathepsin Z, Cathepsin B family, transthyretin, and saposin-like families, suggesting ESPs play critical roles in regulating innate immune response. We compared C. inopinata excretome/secretome with C. elegans. The structures are highly conserved across species, suggesting the sibling species share common mechanism to respond to environmental stimuli.