Pyrimidine Metabolic Dysregulation Following pyrR Knockout Attenuates the Anti-Biofilm, Anti-Inflammatory, and Antioxidant Activities of Lacticaseibacillus casei Cell-Free Supernatant

Pathogenic infections caused by intestinal microecological imbalance pose a critical threat to livestock breeding and human health. Multiple probiotic functions of Lacticaseibacillus casei (L. casei), an important probiotic, are modulated by pyrR-mediated pyrimidine metabolism. This study used L. casei isolated from the intestines of Min pigs (a local pig breed from northeastern China) and its pyrR gene knockout mutant (named L. casei ΔpyrR, ΔpyrR) to investigate their antibacterial, anti-inflammatory, and antioxidant capacities. The results showed that the mutant exhibited significantly attenuated effects compared with the wild-type in disrupting pre-formed biofilms, damaging bacterial morphology, and downregulating the expression of related genes (biofilm and flagellar genes) in Staphylococcus aureus (S. aureus) and Salmonella typhimurium (S. typhimurium). The mutant-derived supernatant also exhibited impaired anti-inflammatory effects in S. typhimurium-challenged IPEC-J2 cells: it was less effective at restraining pathogen-induced IL-1β upregulation and boosting the production of host-derived TGF-β and IL-10, accompanied by altered regulation of the host STING signaling pathway. However, existing data cannot fully distinguish whether STING alteration corresponds to suppressed inflammation or impaired immunity. Additionally, pyrimidine metabolism in L. casei suppresses pathogen-induced reactive oxygen species (ROS) accumulation. These findings are consistent with pyrR-mediated pyrimidine synthesis contributing to the probiotic functions of L. casei, providing an experimental basis for optimizing probiotic preparations and substituting antibiotics.