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  1. Evaluation Summary:

    This study is of interest to animal geneticists studying molecular breeding for infection resistance. The authors provide substantial new insight into the generation of a goat expressing domain fusion receptor TLR2-4, and clarify the mechanism to effectively eliminate S. aureus in macrophages from the gene-edited goat. Taken together, their findings suggest that TLR2-4 knock-in goats can serve as a good animal model for disease resistance breeding.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    Wang et al. construct the novel TLR2-4 receptor by fusing the extracellular domain of TLR2 and the transmembrane and intracellular domains of TLR4, which could specifically recognize S. aureus and activate the autophagy pathway. This study indicated that macrophages derived from TLR2-4 transgenic goats could produce a strong autophagy response for the clearance of S. aureus. Detailed transcriptomic analysis and biochemical experiments showed that the TLR2-4-mediated enhanced autophagy process was mainly completed through the TAK1/TBK1-JNK/ERK, TBK1-TFEB-OPTN and cAMP-PKA-NF-κB-ATGs signaling pathways. The improved autophagy level via TLR2-4 could effectively improve resistance to S. aureus infection in dairy ruminants, which might provide new insight into further improving animal health and welfare, reducing the use of antibiotics, and ensuring human food safety.

    The work of this paper is original and logical. However, only the macrophages derived from one TLR2-4 transgenic goat have been used for the mechanism study, the lack of trait determination in TLR2-4 transgenic goat dramatically reduced the value of this work in molecular breeding.

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  3. Reviewer #2 (Public Review):

    Wang et al. investigated the mechanism of eliminating S. aureus in macrophages by combining TLR2 to recognize S. aureus and TLR4 to activate autophagy to regulate the autophagy pathway. They found that TLR2-4 could enhance autophagy-dependent elimination of S. aureus by activating JNK/ERK signaling and increase the expression of autophagy-related genes (ATGs) by cAMP signaling. They show that TLR2-4 can also activate the TBK1-TFEB-OPTN signaling to regulate S. aureus-induced autophagy. Interestingly, the data also indicate that the PI3K-AKT-FoxO1 signaling was barely involved in regulating S. aureus-induced autophagy in TLR2-4 macrophages. S. aureus is a major pathogen causing mastitis in dairy milch animals including goats. These data add in an interesting way to the ongoing discussion on whether S. aureus could be extremely eliminated in goats.

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  4. Reviewer #3 (Public Review):

    To improve the resistance against S. aureus infection, Wang et al. generated TLR2-4 domain-fusion transgenic goat using CRISPR/Cas9 gene-editing tool. They find that TLR2-4 could recognize S. aureus and enhance the elimination of S. aureus by promoting the autophagy pathway in macrophages. Evidence of the autophagy-dependent clearance of S. aureus is shown meticulously using several methods. The conversion of LC3-I to LC3-II is up-regulated in TLR2-4 macrophages with S. aureus treatment and more autophagosomes are observed in TLR2-4 macrophages than that in WT macrophages after being stimulated by S. aureus under transmission electron microscopy and laser scanning confocal microscopy. TLR2-4 activates the TBK1-TFEB-OPTN signaling to enhance S. aureus-induced autophagy and triggers the TAK1/TBK1-JNK/ERK signaling to promote the autophagy level to effectively eliminate S. aureus in macrophages. Interestingly, they show that JNK and ERK signaling decreased the expression of the autophagy-related genes ATG5 and ATG12. By performing ELISA and qPCR analysis of TLR2-4 and WT macrophages with S. aureus infection, the authors find that cAMP-PKA-NF-kB signaling enhanced the expression of ATG5 and ATG12 to enhance autophagy levels in TLR2-4 macrophages.

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