TLR4 governs strain-specific transcriptional responses of bovine mammary epithelial cells challenged by Staphylococcus aureus and its small colony variant

The role of Toll-like receptor 4 (TLR4) in regulating bovine mammary epithelial transcriptional responses to S. aureus SCV challenge remains poorly understood. Using CRISPR-Cas9-generated TLR4-knockout (TLR4-KO) MAC-T cells combined with RNA sequencing, global transcriptional responses of wild-type (WT) and TLR4-KO MAC-T cells infected with S. aureus SCV Heba3231 and its isogenic parental strain (PS) were compared.

The WT cells mounted highly divergent S. aureus strain-specific responses, with only 0.5% overlap in differentially expressed genes between PS and SCV conditions. The PS infection induced apoptotic and HIF-1 signalling pathways, whereas SCV infection promoted lipid metabolic reprogramming, accompanied by suppression of epithelial barrier integrity genes. TLR4 disruption dramatically amplified transcriptional dysregulation, with a 192-fold increase for the PS (1,921 DEGs) and a 6.6-fold increase for SCV (2,279 DEGs) relative to WT infected controls.