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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2020.103969., This work supports the massive presence of potential immunomodulatory peptides in the human gut metaproteome. These peptides were identified through the MAHMI database as potentially anti-inflammatory, and sixteen of them synthesized for characterize their mechanism of action. From them, peptide HM14 was encrypted in an extracellular protein produced by Bifidobacterium longum, a common member of the human microbiota, and displayed the highest anti-inflammatory capability. Molecular mechanism of action of HM14 pointed to a specific interaction between this immunomodulatory peptide and antigen presenting cells, which resulted in a higher formation of iTreg cells. Moreover, HM14 was effective in decreasing pro-inflammatory parameters in PBMCs isolated from a cohort of Crohns patients. Finally, non-targeted metabolomics confirmed the ability of HM14 to modulate the metabolic activity of PBMCs to fulfil its energy and biosynthetic requirements. Overall, our combined in silico/multiomics approach supports the human gut metaproteome as a source for immunomodulatory peptides., Our work is supported by the Spanish “Programa Estatal de Investigación. Desarrollo e Innovación Orientada a los Retos de la Sociedad” (grants AGL2013-44761-P and AGL2016-78311-R); the Asociación Española Contra el Cancer (“Obtención de péptidos bioactivos contra el Cáncer Colo-Rectal a partir de secuencias genéticas de microbiomas intestinales”, Grant PS-2016), by the Asturias Regional Plan I + D + i for research groups (FYCYT-IDI/2018/000236) and by the Autonomic “Investigadores Emerxentes do Sistema Universitario de Galicia” (Grant EM2014/046). This work was partially supported by the Consellería de Educación. Universidades e Formación Profesional (Xunta de Galicia) under the scope of the strategic funding of ED431C2018/55-GRC Competitive Reference Group. Finally, the authors wish to thank Jaume Morales and Rubén García form Agilent Technologies for technical support., info:eu-repo/semantics/publishedVersion

Lactobacillus genus includes both probiotic and representative strains of the human gut microbiota. Independent studies have reported on the anti-inflammatory properties of different Lactobacillus strains, although we are far from understanding the underlying molecular interplay. In this work we show that a daily administration of Lactobacillus acidophilus DSM20079T (DSM20079) to healthy piglets resulted in plasmatic increases of the anti-inflammatory IL10, whilst IL12 and the pro-inflammatory ratio IL12+TNF/IL10 decreased. The extracellular protein fraction of DSM20079 was identified as the responsible for the crosstalk interaction that elicited these tolerogenic effects. This strain was able to activate innate immune pathways in dendritic cells and to decrease the production of pro-inflammatory cytokines in both CD4+/CD8+ T cell subsets in healthy donors and in Crohns Disease patients. The tolerogenic effect exerted by the extracellular proteins of this strain suggests their potential use as coadjutant for therapeutic applications targeting chronic inflammatory illnesses., Our work is supported by the Spanish “Programa Estatal de Investigación, Desarrollo e Inovación Orientada a los Retos de la Sociedad” (grant AGL2016-78311-R); the Asociación Española Contra el Cancer (“Obtención de péptidos bioactivos contra el Cáncer ColoRectal a partir de secuencias genéticas de microbiomas intestinales”, Grant PS-2016) and by the Asturias Regional Plan I+D+i for research groups (FYCYT-IDI/2018/000236). This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684). SING group thanks CITI (Centro de Investigación, Transferencia e Innovación) from University of Vigo for hosting its IT infrastructure. LJR was supported by the Principado de Asturias, PCTI 2018–2020 (GRUPIN: IDI2018-000237) and FEDER. This work was partially supported by the Consellería de Educación, Universidades e Formación Profesional (Xunta de Galicia) under the scope of the strategic funding of ED431C2018/55-GRC Competitive Reference Group., info:eu-repo/semantics/publishedVersion

Advances in the field of genome sequencing have enabled a comprehensive analysis and annotation of the dynamics of the protein inventory of cells. This has been proven particularly rewarding for microbial cells, for which the majority of proteins are already accessible to analysis through automatic metagenome annotation. The large-scale in silico screening of proteomes and metaproteomes is key to uncover bioactivities of translational, clinical and biotechnological interest, and to help assign functions to certain proteins, such as those predicted as hypothetical. This work introduces a new method for the prediction of the bioactivity potential of proteomes/metaproteomes, supporting the discovery of functionally relevant proteins based on prior knowledge. This methodology complements functional annotation enrichment methods by allowing the assignment of functions to proteins annotated as hypothetical/putative/uncharacterised, as well as and enabling the detection of specific bioactivities and the recovery of proteins from defined taxa. This work shows how the new method can be applied to screen proteome and metaproteome sets to obtain predictions of clinical or biotechnological interest based on reference datasets. Notably, with this methodology, the large information files obtained after DNA sequencing or protein identification experiments can be associated for translational purposes that, in cases such as antibiotic-resistance pathogens or foodborne diseases, may represent changes in how these important and global health burdens are approached in the clinical practice. Finally, the Sequence-based Expert-driven pRoteome bioactivity Prediction EnvironmENT, a public Web service implemented in Scala functional programming style, is introduced as means to ensure broad access to the method as well as to discuss main implementation issues, such as modularity, extensibility and interoperability., This work was supported by the Spanish “Programa Estatal de Investigación, Desarrollo e Inovación Orientada a los Retos de la Sociedad” (grant AGL2013-44039R); the Asociación Española Contra el Cancer (“Obtención de péptidos bioactivos contra el Cáncer Colo-Rectal a partir de secuencias genéticas de microbiomas intestinales”, grant PS-2016). This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684). SING group thanks CITI (Centro de Investigación, Transferencia e Innovación) from University of Vigo for hosting its IT infrastructure.

Trabajo presentado en el 7th International Human Microbiome Congress, IHMC, celebrado en County Kerry (Irlanda) del 28 al 26 de junio de 2018, The human gastrointestinal tract (GIT) is a very complex ecosystem involving a continuous interaction between nutrients, host cells and microorganisms. Recently, we have discovered a new family of immunomodulatory peptides that are encrypted within the sequence of a precise extracellular protein, which is secreted by the lactic acid bacterium Lactobacillus plantarum (Patent WO 2013034795 A1) (1). The genetic information about commensal/probiotic bacteria and gut microbiomes that is present in public repositories is huge, with only a list of 10 million unique bacterial proteins available mainly from the METAHIT and HMP projects (http://gigadb.org/dataset/100064). Our aim is to explore these 10 million unique proteins to obtain information on encrypted peptides which might have immunomodulatory bioactivity on host cells. With this in mind we have developed the MAHMI (Mechanism of Action of the Human Microbiome) database, a unique resource that provides comprehensive information about the sequence of potential immunomodulatory and antiproliferative peptides encrypted in the proteins produced by the human gut microbiota. Fifteen peptides were chosen by its potential immunomodulatory bioactivity. Selected peptides were assayed in vitro at a final concentration of 10 µg/mL using monocyte derived cells (Mo-DCs). Our bioinformatics/in vitro methodology allowed selection of new inmunomodulatory peptides encrypted in the human intestinal bacteria metaproteome. HM14 signaled through an unknown receptor, and imprinted anti-inflammatory traits in immune cells as evidenced by RNASeq and flow cytometry. The mechanisms used by this kind of peptides may be useful for the treatment of inflammatory diseases