Shotgun Proteomics of Ascidians Tunic Gives New Insights on Host–Microbe Interactions by Revealing Diverse Antimicrobial Peptides

Abstract

Ascidians are marine invertebrates associated with diverse microbial communities, embedded in their tunic, conferring special ecological and biotechnological relevance to these model organisms used in evolutionary and developmental studies. Next-generation sequencing tools have increased the knowledge of ascidians’ associated organisms and their products, but proteomic studies are still scarce. Hence, we explored the tunic of three ascidian species using a shotgun proteomics approach. Proteins extracted from the tunic of Ciona sp., Molgula sp., and Microcosmus sp. were processed using a nano LC-MS/MS system (Ultimate 3000 liquid chromatography system coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer). Raw data was searched against UniProtKB – the Universal Protein Resource Knowledgebase (Bacteria and Metazoa section) using Proteome Discoverer software. The resulting proteins were merged with a non-redundant Antimicrobial Peptides (AMPs) database and analysed with MaxQuant freeware. Overall, 337 metazoan and 106 bacterial proteins were identified being mainly involved in basal metabolism, cytoskeletal and catalytic functions. 37 AMPs were identified, most of them attributed to eukaryotic origin apart from bacteriocins. These results and the presence of “Biosynthesis of antibiotics” as one of the most highlighted pathways revealed the tunic as a very active tissue in terms of bioactive compounds production, giving insights on the interactions between host and associated organisms. Although the present work constitutes an exploratory study, the approach employed revealed high potential for high-throughput characterization and biodiscovery of the ascidians’ tunic and its microbiome.

Influence of Dietary Supplementation with an Amino Acid Mixture on Inflammatory Markers, Immune Status and Serum Proteome in LPS-Challenged Weaned Piglets

“…The protein identification and quantitation were performed by nano LC-MS/MS as previously described [ 18 ]. This equipment is composed of an Ultimate 3000 liquid chromatography system coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany).…”

“…The mass spectrometer was operated in data-dependent (dd) positive acquisition mode alternating between a full scan ( m/z 380–1580) and subsequent higher-energy C-trap dissociation MS/MS of the 10 most intense peaks from full scan (normalised collision energy of 27%), as previously described [ 18 ]. ESI spray voltage was 1.9 kV.…”

“…The raw data were processed using Proteome Discoverer 2.4.0.305 software (Thermo Scientific) and matched with the UniProt database for the Sus scrofa Proteome 2020_01. The Sequest HT search engine was used to identify tryptic peptides, as described by Matos et al [ 18 ]. The ion mass tolerance was 10 ppm for precursor ions and 0.02 Da for fragment ions.…”

Reflections on the Use of an Invertebrate Chordate Model System for Studies of Gut Microbial Immune Interactions

“…Moreover, metaproteomic and metabolomics may help identify active proteins, enzymatic pathways, and metabolites used for mediating interactions between host and microbes within the gut; recent examples focus on the tunic of three ascidian species, including Ciona sp. (146,147) (Figure 7). Subsequently derived hypotheses can be tested via experimental approaches, if isolation of the identified components is possible, and combined into functional assays.…”

Symbiotic Associations in Ascidians: Relevance for Functional Innovation and Bioactive Potential

“…Studies applying integrative and multivariate approaches regarding ascidians’ microbiome have been published in recent years [ 49 ]. The application of transcriptomic and proteomic approaches in such studies is still scarce in comparison with the previously mentioned methods ( Figure 4 ) [ 105 , 160 , 161 ].…”