Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat
- Beijing Key Laboratory of Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, China
- National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, China
- Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology, China Agricultural University, China
- Tianjin Academy of Agricultural Sciences, China
- Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest Agriculture and Forest University, China
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, China
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, China
Figures
Figure 1 with 2 supplements
Construction of fibroblast cell line expressing Toll-like receptor 2-4 (TLR2-4).
(A) Structure of fusion protein TLR2-4. Total RNA was extracted from goat peripheral blood and was reverse- transcribed into cDNA. TLR2 extracellular domain and TLR4 transmembrane and intracellular …
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Figure 1—source data 1
The original blots of Toll-like receptor 2-4 (TLR2-4) (TLR2-4 blot was repeated twice) and wild-type (WT) fibroblasts.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig1-data1-v1.zip
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Figure 1—source data 2
The original blots.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig1-data2-v1.zip
Figure 1—figure supplement 1
Establishment and identification of the Toll-like receptor 2-4 (TLR2-4) fibroblast cell line.
Establishment of the TLR2-4 fibroblast cell line. (A) Gene editing efficiency was evaluated by the T7E1 cleavage assay. Fibroblasts were individually transfected with the eight small guide RNA …
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Figure 1—figure supplement 1—source data 1
The original gels of Figure 1—figure supplement 1A.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
The original gels of Figure 1—figure supplement 1C.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig1-figsupp1-data2-v1.zip
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Figure 1—figure supplement 1—source data 3
The original gels of Figure 1—figure supplement 1C.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig1-figsupp1-data3-v1.zip
Figure 1—figure supplement 2
Heatmap and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed genes (DEGs) between Toll-like receptor 2-4 (TLR2-4) and wild-type (WT) fibroblasts after S. aureus infection (n=3 biologically independent samples).
The effect of TLR2-4 on global transcriptome in killed S. aureus-infected fibroblasts. Both TLR2-4 and WT fibroblasts with knockdown endogenous TLR2 were stimulated with heat-killed S. aureus for 10 …
Figure 2 with 1 supplement
Generation of clone goat expressing Toll-like receptor 2-4 (TLR2-4).
(A) The clone goat expressing TLR2-4 was prepared by somatic cell nuclear transfer. (B) Schematic representation of the site-specific integration of primers S-M and T-R. (C) Genomic DNA of …
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Figure 2—source data 1
The original gels of Figure 2C.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig2-data1-v1.zip
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Figure 2—source data 2
The original gels of Figure 2E.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig2-data2-v1.zip
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Figure 2—source data 3
The original gels of Figure 2F.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig2-data3-v1.zip
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Figure 2—source data 4
The original blots of Figure 2G.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig2-data4-v1.zip
Figure 2—figure supplement 1
Identification of the insertion site and sequences of Toll-like receptor 2-4 (TLR2-4) in macrophages.
And the ligand recognition function of TLR2-4 receptor in macrophages was identified. The effect of TLR2-4 on the phagocytose of S. aureus in macrophages. (A) The PCR products of 3795 bp were …
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Figure 2—figure supplement 1—source data 1
The original blots of Figure 2—figure supplement 1H.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig2-figsupp1-data1-v1.zip
Figure 3
Toll-like receptor 2-4 (TLR2-4) promoted autophagy-dependent elimination of Staphylococcus aureus in macrophages.
(A) S. aureus burden in macrophages was estimated by the mean fluorescence intensity (MFI) of macrophages infected with fluorescein isothiocyanate (FITC)-labeled S. aureus using flow cytometry …
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Figure 3—source data 1
The original blots of Figure 3F.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig3-data1-v1.zip
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Figure 3—source data 2
The original blots of Figure 3G.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig3-data2-v1.zip
Figure 4 with 1 supplement
Identification of autophagy-related genes involved in clearance of Staphylococcus aureus in transgenic macrophages.
Macrophages from the Toll-like receptor 2-4 (TLR2-4) and two wild-type goats (WT1, WT2) were infected with S. aureus for 1 hr (multiplicity of infection [MOI] = 10), and then after washing three …
Figure 4—figure supplement 1
The principal component analysis (PCA) between two wild-type (WT) macrophages and the heatmap of the differentially expressed genes (DEGs) between TLR2-4 and WT macrophages after S. aureus infection.
RNA sequencing of S. aureus infected TLR2-4 and WT macrophages. Macrophages were infected with S. aureus for 1 hr (multiplicity of infection [MOI] = 10). Cells were washed three times using PBS, and …
Figure 5 with 1 supplement
Toll-like receptor (TLR2-4) enhanced Staphylococcus aureus-induced autophagy via activating JNK/ERK signaling.
(A) The change in phosphorylation of JNK, ERK, and p38 in S. aureus-infected macrophages. Macrophages were pretreated with DMSO (carrier), Takinib (200 μM), or Amlexanox (100 μM) for 12 hr, then …
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Figure 5—source data 1
The original blots of Figure 5A, B and D.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig5-data1-v1.zip
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Figure 5—source data 2
The original blots of Figure 5A,B,D.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig5-data2-v1.zip
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Figure 5—source data 3
The original blots of Figure 5A,B,D.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig5-data3-v1.zip
Figure 5—figure supplement 1
The phosphorylation of IRF3 was detected by Western blotting in Staphylococcus aureus-infected Toll-like receptor 2-4 (TLR2-4) macrophages.
Signaling pathways involved in S. aureus-infected TLR2-4 macrophages. (A) The protein level of MyD88 in macrophages was detected by Western blot. Tubulin was used to normalize the data of each …
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Figure 5—figure supplement 1—source data 1
The original blots of Figure 5—figure supplement 1A.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
The original blots of Figure 5—figure supplement 1B.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig5-figsupp1-data2-v1.zip
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Figure 5—figure supplement 1—source data 3
The original blots of Figure 5—figure supplement 1D.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig5-figsupp1-data3-v1.zip
Figure 6
Toll-like receptor 2-4 (TLR2-4) induced autophagy flux through the cyclic adenosine phosphate (cAMP) pathway in Staphylococcus aureus-infected macrophages.
(A) TLR2-4 promoted the expression of ATG5 and ATG12 in macrophages after S. aureus infection. The mRNA relative expression of ATG5 and ATG12 was monitored by real-time reverse transcription PCR …
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Figure 6—source data 1
The original blots of Figure 6D, E and G.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig6-data1-v1.zip
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Figure 6—source data 2
The original blots of Figure 6D,E,G.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig6-data2-v1.zip
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Figure 6—source data 3
The original blots of Figure 6D,E,G.
- https://cdn.elifesciences.org/articles/78044/elife-78044-fig6-data3-v1.zip
Figure 7
A proposed model for Toll-like receptor 2-4 (TLR2-4) enhancing autophagy-dependent clearance of Staphylococcus aureus.
Upon S. aureus stimulation, TLR2-4 on the surface of macrophages augmented LC3-promoted phagophore formation to enhance xenophagy through the MyD88-dependent TAK1-JNK/ERK signaling. Internalized …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Capra hircus) | TLR2 | GenBank | NC_030824.1 | |
| Gene (Capra hircus) | TLR4 | GenBank | NC_030815.1 | |
| Strain, strain background (Staphylococcus aureus) | ATCC29213 | GenBank | U77328 | Bacteria cells |
| Transfected construct | PX458 (pSpCas9(BB)–2A-GFP) | Addgene | RRID: Addgene_48138 | PMID:24157548 |
| Transfected construct | pRosa26-promoter | Addgene | RRID: Addgene_21710 | PMID:9108056 |
| Transfected construct (Capra hircus) | siRNA to endogenous TLR2 | GenePharma | Sense: 5’-GCACUUCAACCCUCCCUUUTT-3’ | Antisense: 5’-AAAGGGAGGGUUGAAGUGCTT-3’ |
| Sequence-based reagent | siRNA: nontargetin control | GenePharma | Silencer Select | |
| Antibody | nti-MyC (Mouse monoclonal) | Proteintech | Cat# 60003–2-lg RRID: AB_2883088 | IF (1:100), WB (1:1000) |
| Antibody | Anti-NF-kB p65 antibody (Rabbit polyclonal) | Abcam | Cat# ab16502 RRID: AB_443394 | WB (1:1000) |
| Antibody | Anti-LMNB2 (Rabbit polyclonal) | Beyotime | Cat#: AF0219 | WB (1:1000) |
| Antibody | Phospho-p44/42 MAPK (Rabbit polyclonal) | CST | Cat#: 9,101 RRID: AB_331646 | WB (1:1000) |
| Antibody | Phospho-JNK1/2/3-T183/T183/T221 (Rabbit monoclonal) | ABclonal | Cat#: AP0631 RRID: AB_2771232 | WB (1:1000) |
| Antibody | Tubulin (Mouse monoclonal) | Beyotime | Cat#: AT819 | WB (1:1000) |
| Antibody | Anti-LC3 (Rabbit polyclonal) | Proteintech | Cat#: 14600–1-AP RRID: AB_2137737 | WB (1:1000) |
| Antibody | Anti-SQSTM1/p62 (Rabbit polyclonal) | Affinity | Cat#: AF5384 RRID: AB_2837869 | WB (1:1000) |
| Antibody | Phospho-p38 MAPK-T180 (Rabbit polyclonal) | ABclonal | Cat#: AP0238 RRID: AB_2771307 | WB (1:1000) |
| Antibody | Anti-GAPDH (Rabbit polyclonal) | Sangon Biotech | Cat#: D110016 RRID: AB_2904600 | WB (1:1000) |
| Antibody | Anti-TFEB (Rabbit polyclonal) | Proteintech | Cat#: 13372–1-AP RRID: AB_2199611 | WB (1:1000) |
| Antibody | Anti-Optineurin (Rabbit polyclonal) | Affinity | Cat#: DF6655 RRID: AB_2838617 | WB (1:1000) |
| Antibody | Phospho-pan-AKT1/2/3 (Rabbit polyclonal) | Affinity | Cat#: AF0908 RRID: AB_2834079 | WB (1:1000) |
| Antibody | Phospho-FOXO1A (Rabbit polyclonal) | Affinity | Cat#: AF3416 RRID: AB_2834858 | WB (1:1000) |
| Antibody | Acetyl-FOXO1A (Rabbit polyclonal) | Affinity | Cat#: AF2305 RRID: AB_2845319 | WB (1:1000) |
| Antibody | Anti-PRKACA (Rabbit polyclonal) | ABclonal | Cat#: A0798 RRID: AB_2757400 | WB (1:1000) |
| Antibody | Phospho-IKB alpha (Rabbit monoclonal) | CST | Cat#: 2,859 RRID: AB_561111 | WB (1:1000) |
| Antibody | Phospho-IRF3-S386 (Rabbit polyclonal) | ABclonal | Cat#: AP0857 RRID: AB_2771209 | WB (1:1000) |
| Antibody | Peroxidase-Conjugated Goat anti-Rabbit IgG | ZSGB-BIO | Cat#: ZB-2301 RRID: AB_2747412 | WB (1:5000) |
| Antibody | Peroxidase-Conjugated Goat anti-Mouse IgG | ZSGB-BIO | Cat#: ZB-2305 RRID: AB_2747415 | WB (1:5000) |
| Antibody | Alexa Fluor 594 goat anti-mouse IgG | ThermoFisher Scientific | Cat#: A11005 RRID: AB_2534073 | IF (1 μg/ml) |
| Commercial assay or kit | Nuclear Extraction Kits for Cells | Invent Biotechnologies | Cat#: SC-003 | |
| Chemical compound, drug | Dynasore | MCE | Cat#: HY-15304 | 50 μM |
| Chemical compound, drug | SP600125 | MCE | Cat#: HY-12041 | 200 μM |
| Chemical compound, drug | PD98059 | MCE | Cat#: HY-12028 | 200 μM |
| Chemical compound, drug | Takinib | MCE | Cat#: HY-103490 | 200 μM |
| Chemical compound, drug | Amlexanox | MCE | Cat#: HY-B0713 | 100 μM |
| Software | ImageJ | http://imagej.nih.gov/ij/ | RRID: SCR_003070 | |
| Software | Flowjo | https://www.flowjo.com/solutions/flowjo | RRID: SCR_008520 | |
| Software | Origin | http://www.originlab.com/index.aspx?go=PRODUCTS/Origin | RRID: SCR_014212 | |
| Other | DAPI stain | Solarbio | Cat#: C0065 | 10 μg/ml |
Additional files
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Supplementary file 1
Quantification and statistical analysis.
- https://cdn.elifesciences.org/articles/78044/elife-78044-supp1-v1.docx
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Supplementary file 2
The data for generation of clone goat and part primers used in this study.
(A) Table displaying the generation of clone goats by nuclear transfer. (B) Primers of crRNA-oligo. (C) Table displaying the sequences of primers in T7 endonuclease 1 (T7E1) assay. (D) All primers used for PCR. (E) Table displaying the primers for real-time reverse transcription PCR (qRT-PCR).
- https://cdn.elifesciences.org/articles/78044/elife-78044-supp2-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/78044/elife-78044-transrepform1-v1.docx
