The bovine mammosphere-derived epithelial cell secretome inhibits bacterial growth in vitro and contains peptidoglycan recognition protein 1

• The bovine MDEC secretome possesses antimicrobial activity. • The bovine MDEC secretome reduces the growth of mastitis pathogens in vitro . • The bovine MDEC secretome contains the antimicrobial protein PGLYRP1. Bovine mastitis, caused by bacterial infection of the mammary gland, leads to significant economic losses for the dairy industry because of decreased milk quality and yield. Mastitis is typically treated with conventional antibiotics (ABX) that clear bacteria, but do not address the milk loss associated with infection. Milk from cows being treated with ABX must be discarded leading to acute milk loss, and ABX do not heal milk-producing tissues that are damaged because of infection, leading to a permanent reduction in milk production when compared to pre-mastitis levels. The bovine mammosphere-derived epithelial cell (MDEC) secretome, consisting of all bioactive factors secreted by the cells, contains molecules that directly and indirectly promote tissue repair. This current study shows that the MDEC secretome also inhibits the growth of mastitis-causing bacteria in vitro in planktonic and biofilm assays. Secretome analysis by heat treatment, size fractionization, and extracellular vesicle isolation suggested that soluble proteins of different sizes, or in complexes, drive the anti-bacterial effects. We identified the antimicrobial protein peptidoglycan recognition protein 1 (PGLYRP1) in MDECs and detected it in the secretome. As knock down of PGLYRP1 expression in MDECs was unsuccessful using RNA interference and CRISPR-Cas9 gene editing, a direct link between the antimicrobial activity of the secretome and this protein could not be confirmed. The newly identified antimicrobial activity of the bovine MDEC secretome supplements its tissue reparative features, poising it as a potential adjunct or alternative therapy for bovine mastitis that may reduce acute milk loss and improve long term milk production.