1 Vol.:(0123456789) Scientifc Reports | (2021) 11:23798 | https://doi.org/10.1038/s41598-021-03220-z www.nature.com/scientificreports Sequence edition of single domains modulates the fnal immune and antimicrobial potential of a new generation of multidomain recombinant proteins Ramon Roca‑Pinilla 1 , Ravi Holani 2 , Adrià López‑Cano 1 , Cristina Saubi 1 , Ricardo Baltà‑Foix 1 , Eduardo R. Cobo 2 , Elena Garcia‑Fruitós 1,3* & Anna Arís 1,3* Combining several innate immune peptides into a single recombinant antimicrobial and immunomodulatory polypeptide has been recently demonstrated. However, the versatility of the multidomain design, the role that each domain plays and how the sequence edition of the diferent domains afects their fnal protein activity is unknown. Parental multidomain antimicrobial and immunomodulatory protein JAMF1 and several protein variants (JAMF1.2, JAMF2 and AM2) have been designed and recombinantly produced to explore how the tuning of domain sequences afects their immunomodulatory potential in epithelial cells and their antimicrobial capacity against Gram‑positive and Gram‑negative bacteria. The replacement of the sequence of defensin HD5 and phospholipase sPLA 2 by shorter active fragments of both peptides improves the fnal immunomodulatory (IL‑8 secretion) and antimicrobial function of the multidomain protein against antimicrobial‑resistant Klebsiella pneumoniae and Enterococcus spp. Further, the presence of Jun and Fos leucine zippers in multidomain proteins is crucial in preventing toxic efects on producer cells. The generation of antimicrobial proteins based on multidomain polypeptides allows specifc immunomodulatory and antimicrobial functions, which can be easily edited by modifying of each domain sequence. Te continuous rise in drug-resistant microbes is already challenging the treatment of infections and has put health authorities on alert. Antimicrobial resistances (AMRs) are one of the health threats included within the One Health approach, which aims to fnd solutions to the appearance and prevalence of AMRs by coop- eration across all sectors, including human, animal and environmental health 1 . In the search for new antimi- crobials, several strategies have been studied including developing anti-infectious molecules based on natural compounds, such as essential oils or favonoids, probiotics and prebiotics, and bacteriophages, among others 2 . However, most of them present important drawbacks such as low antimicrobial efciencies, challenging and time-consuming procedures for their isolation and/or narrowed antimicrobial spectra, or a combination of several of these handicaps. Taking a new perspective in the development of antimicrobial molecules, the use of active peptides or proteins naturally present in the innate and adaptive immunity ofers a powerful strategy to develop new antimicrobial molecules 3,4 . Among them, there are larger antimicrobial proteins formed by more than 100 amino acids which are ofen lytic enzymes, nutrient-binding proteins, or proteins containing sites that target specifc microbial macromolecules 5 . Te smaller antimicrobial peptides, also known as host defense peptides (HDPs), are cationic, amphiphilic and short peptides synthesized by nearly all multicellular organisms with activity against bacteria, viruses and fungi 6 . So far, HDPs have been intensively explored, proving that they ofer a great potential to treat a wide range of microorganisms, including multidrug-resistant strains, and some OPEN 1 Department of Ruminant Production, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), 08140 Caldes de Montbui, Spain. 2 Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada. 3 These authors contributed equally: Elena Garcia-Fruitós and Anna Arís. * email: elena.garcia@irta.cat; anna.aris@irta.cat