Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat generated by CRISPR/Cas9

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-κB 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-κB-ATGs signaling pathways, which provide an alternative approach to resistant against S. aureus infection.


4C).
Further, the transcripts involved in autophagy were clustered ( Figure 4D). 195 241 autophagy-related DEGs were found, including the ATG genes, 141 and 54 of which DEGs 242 were up-regulated and down-regulated, receptively ( Figure 4D). Nine significant functional 243 pathways closely related to autophagy, including endocytosis, phagosome, lysosome and 244 Toll-like receptor signaling pathway, were enriched by KEGG analysis (Figure 4E). These 245 results suggested that TLR signaling pathway, cAMP signaling pathway and MAPK might be 246 involved in S. aureus-induced autophagy in TLR2-4 macrophages.  Figure 5A). Meanwhile,256 compared to WT macrophages, the conversion of LC3-I to LC3-II was prominently increased 257 in TLR2-4 macrophages with S. aureus treatment, and depressed by SP600125 (JNK inhibitor) 258 and PD98059 (ERK inhibitor) ( Figure 5B, Figure 5-source data 2). These results suggested 259 that inhibition of JNK or ERK signaling led to decrease of autophagy level induced by S. 260 aureus. As shown in figure 5C, the clearance of S. aureus was decreased more dramatically in 261 TLR2-4 macrophages upon JNK or ERK1/2 inhibitor, indicating that TLR2-4 triggered JNK 262 and ERK signaling to enhance S. aureus removal of macrophages. 263 TLRs can migrate from cell membrane into endosomes of the cytoplasm to activate TANK 264 binding kinase (TBK1) signaling cascade (Akira and Takeda, 2004;Wei et al., 2019). The   284 Autophagy is mediated by conserved autophagy-related proteins at the different steps of the 285 autophagy process. Autophagosomes are formed through the concerted action of the ATGs 286 (Cadwell, 2016). Consequently, we monitored the expression of ATG5 and ATG12 in S. 287 aureus-infected macrophages. The relative mRNA expression of ATG5 and ATG12 in TLR2-288 4 macrophages with S. aureus infection was significantly higher than that in the WT  306 These data suggested that the PI3K-AKT-FoxO1signaling was barely involved in regulating S. 307 aureus-induced autophagy in TLR2-4 macrophages.

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The cyclic adenosine monophosphate (cAMP)/cAMP-dependent protein kinase A (PKA) 309 signaling enhanced the induction of autophagy (Zhou et al., 2017). Based on the results of 310 KEGG ( Figure 4C), we analyzed the activation of cAMP-PKA signaling in macrophages.

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The intracellular level of cAMP in TLR2-4 macrophages was significantly higher than that in 312 WT macrophages regardless as S. aureus infection ( Figure 6C). Simultaneously, the level of  et al., 2005;Ouchi et al., 2000), and NF-B suppresses the expression of ATG5 and 320 ATG12 (Lim et al., 2012a;Shu et al., 2020). The levels of IκB-α phosphorylation and p65 321 nuclear translocation in TLR2-4 macrophages were extremely reduced than that in WT 322 macrophages regardless as S. aureus infection (Figure 6D), while inhibitor H-89 rescued the 323 phosphorylation of IκB-α and nuclear translocation of p65 ( Figure 6D). These results 324 suggested that TLR2-4 inhibited the activity of NF-B via cAMP-PKA signaling. Upon S. 325 aureus stimulation, the mRNA relative expression of ATG5 and ATG12 in TLR2-4 326 macrophages was significantly increased by cAMP activator Forskolin, but was decreased by 327 inhibitor H-89 ( Figure 6F). These data indicated that cAMP-PKA-NF-B signaling enhanced 328 the expression of ATG5 and ATG12. Meantime, the conversion of LC3-I to LC3-II was 329 increased in TLR2-4 macrophages with S. aureus treatment by activator Forskolin, while was 330 decreased by inhibitor H-89 ( Figure 6G, Figure 6-source data 3). Taken together, TLR2-4 331 increased the levels of ATG5 and ATG12 via cAMP-PKA-NF-B signaling to enhance S.

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It is crucial to exploit a new approach that solve the emergence of S. aureus infections.

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Modulation of host innate immunity may be a potential strategy to eliminate pathogens. TLR2 340 plays an essential role in recognizing S. aureus and activating autophagy pathway during S. 341 aureus infections. However, we cannot regulate autophagy by overexpressing TLR2, as it is 342 required to form heterodimerizes with TLR6 or TLR1. TLR4 and myeloid differentiation 343 factor 2 complex binds to lipopolysaccharide triggers formation of the activated homodimer 344 (Park et al., 2009). It is an essential component in host resistance via downstream signaling 345 pathways initiating autophagy to degrade pathogens (Pahari et al., 2020;Xu et al., 2007).

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Meanwhile, TLR4 is involved in immune response in S. aureus-induced infection (Liu et al., 347 2013;Stenzel et al., 2008). The function of TLR2 and TLR4 dependent autophagy has been 348 shown to be crucial in the bactericidal activity of S. aureus in mouse macrophages (Shu et al., 2020). Therefore, to achieve the enhanced host resistance against S. aureus, a recombinant   The Staphylococcus aureus strain ATCC29213 were preserved in our laboratory. Bacteria 433 were grown in tryptic soy broth (TSB) liquid medium with shaking overnight at 37 °C.

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Cultures were diluted with TSB at a ratio of 1:100 and incubated with shaking at 37 °C until 435 the optical density at 600 nm was 1.5.
Healthy dairy goats of Lao shan and Nubian were selected. All animal experiments and 438 treatments were approved and supervised by the Animal Welfare Committee of China All goats were fasted 12 h before oocyte collection for facilitating surgery. The ovulation 548 response was verified by laparoscopy. Goats displayed corpora lutea were selected for oocyte 549 collection at 62 h after CIDR removal. The oocytes were collected by oviduct flushing with 550 20 ml warm PBS containing 0.3% bovine serum albumin (BSA). Collected oocytes were 551 transferred into the holding medium using an inverted microscope (LX71 Olympus, Japan).

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The oocytes with cumulus cells were incubated in M199 medium (Gibico, USA) containing    The lysosomal activity of macrophages was measured using a Lysosomal Intracellular Macrophages (3×10 5 /well) were seeded in the 6-well plates and incubate for 12 h in DMEM 636 medium supplemented with 10% FBS at 37 °C with 5% CO2, and the medium was replaced 637 with fresh medium containing S. aureus (MOI 10). Bafilomycin A1 was added in the negative 638 control group. Cells were incubated for 1, 2, 4, 8 h at 37 °C with 5% CO2, then washed three 639 times with PBS. Cells were treated with medium containing gentamicin (200 µg/ml) and Self-         (1-3). The original gels of Figure 2C, E, F.        for 4 h. The conversion of LC3-I to LC3-II were checked by western blotting. All data are means ± SD of at least 3 experimental repeats (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure supplement 5.
Inhibiting JNK or ERK1/2 increased the mRNA level of ATG5 and ATG12 in wide type macrophages. In order to explore the signal pathway regulating ATGs, the AKT-FoxO1 pathway in S. aureus infected TLR2-4 macrophages was evaluated by western blotting.       All data are means ± SD of at least 3 experimental repeats (*p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance).