Staphylococcus aureus infections pose a potential threat to livestock production and public health. A novel strategy is needed to control S. aureus infection due to its adaptive evolution to antibiotics. Autophagy plays a key role in degrading bacteria for innate immune cells. In order to promote S. aureus clearance via TLR induced autophagy pathway, the domain fusion TLR2-4 with the extracellular domain of TLR2, specific recognizing S. aureus, and transmembrane and intracellular domains of TLR4 is assembled, then the goats expressing TLR2-4 is generated. TLR2-4 substantially augments the removal of S. aureus within macrophages by elevating autophagy level. Phosphorylated JNK/ERK1/2 promote LC3-puncta in TLR2-4 macrophages during S. aureus-induced autophagy via MyD88-mediated the TAK1 signaling cascade. Meantime, the TRIF-dependent TBK1-TFEB-OPTN signaling is involved in TLR2-4-triggered autophagy after S. aureus challenge. Moreover, the transcript of ATG5 and ATG12 is significantly increased via cAMP-PKA-NF-kB signaling, which facilitates S. aureus-induced autophagy in TLR2-4 macrophages. Overall, the novel receptor TLR2-4 enhances the autophagy-dependent clearance of S. aureus in macrophages via TAK1/TBK1-JNK/ERK, TBK1-TFEB-OPTN and cAMP-PKA-NF-kB-ATGs signaling pathways, which provide an alternative approach to resistant against S. aureus infection.

For the RNA-seq alignment, purified sequence data was aligned to reference goat genome downloaded from Ensembl website (https://asia.ensembl.org/index.html) by HISAT2. Read count matrices were obtained using Feature Counts. Differentially expressed genes were accessed using R package DESeq2, with a false discovery rate less than 0.05 and ∣log2foldchange∣> 2 or 3. Principal component analysis was also performed by DESeq2. Both GO and KEGG functional enrichment analysis or visualization were finished by R package ClusterProfiler or ClueGO, a plug-in of Cytoscape. Additionally, autophagy related gene sets were obtained from Molecular Signatures Database (MSigDB) (http://www.broadinstitute.org).