Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat

  1. Mengyao Wang
  2. Yu Qi
  3. Yutao Cao
  4. Xiaosheng Zhang
  5. Yongsheng Wang
  6. Qingyou Liu
  7. Jinlong Zhang
  8. Guangbin Zhou
  9. Yue Ai
  10. Shao Wei
  11. Linli Wang
  12. Guoshi Liu
  13. Zhengxing Lian
  14. Hongbing Han  Is a corresponding author
  1. Beijing Key Laboratory of Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, China
  2. National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, China
  3. 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
  4. Tianjin Academy of Agricultural Sciences, China
  5. Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest Agriculture and Forest University, China
  6. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, China
  7. Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, China
7 figures, 1 table and 3 additional files

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 …

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
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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Capra hircus)TLR2GenBankNC_030824.1
Gene (Capra hircus)TLR4GenBankNC_030815.1
Strain, strain background (Staphylococcus aureus)ATCC29213GenBankU77328Bacteria cells
Transfected constructPX458 (pSpCas9(BB)–2A-GFP)AddgeneRRID: Addgene_48138PMID:24157548
Transfected constructpRosa26-promoterAddgeneRRID: Addgene_21710PMID:9108056
Transfected construct (Capra hircus)siRNA to endogenous TLR2GenePharmaSense: 5’-GCACUUCAACCCUCCCUUUTT-3’Antisense: 5’-AAAGGGAGGGUUGAAGUGCTT-3’
Sequence-based reagentsiRNA: nontargetin controlGenePharmaSilencer Select
Antibodynti-MyC (Mouse monoclonal)ProteintechCat# 60003–2-lg
RRID: AB_2883088
IF (1:100),
WB (1:1000)
AntibodyAnti-NF-kB p65 antibody
(Rabbit polyclonal)
AbcamCat# ab16502
RRID: AB_443394
WB (1:1000)
AntibodyAnti-LMNB2
(Rabbit polyclonal)
BeyotimeCat#: AF0219WB (1:1000)
AntibodyPhospho-p44/42 MAPK (Rabbit polyclonal)CSTCat#: 9,101
RRID: AB_331646
WB (1:1000)
AntibodyPhospho-JNK1/2/3-T183/T183/T221 (Rabbit monoclonal)ABclonalCat#: AP0631
RRID: AB_2771232
WB (1:1000)
AntibodyTubulin (Mouse monoclonal)BeyotimeCat#: AT819WB (1:1000)
AntibodyAnti-LC3 (Rabbit polyclonal)ProteintechCat#: 14600–1-AP
RRID: AB_2137737
WB (1:1000)
AntibodyAnti-SQSTM1/p62 (Rabbit polyclonal)AffinityCat#: AF5384
RRID: AB_2837869
WB (1:1000)
AntibodyPhospho-p38 MAPK-T180 (Rabbit polyclonal)ABclonalCat#: AP0238
RRID: AB_2771307
WB (1:1000)
AntibodyAnti-GAPDH (Rabbit polyclonal)Sangon BiotechCat#: D110016
RRID: AB_2904600
WB (1:1000)
AntibodyAnti-TFEB (Rabbit polyclonal)ProteintechCat#: 13372–1-AP
RRID: AB_2199611
WB (1:1000)
AntibodyAnti-Optineurin (Rabbit polyclonal)AffinityCat#: DF6655
RRID: AB_2838617
WB (1:1000)
AntibodyPhospho-pan-AKT1/2/3 (Rabbit polyclonal)AffinityCat#: AF0908
RRID: AB_2834079
WB (1:1000)
AntibodyPhospho-FOXO1A (Rabbit polyclonal)AffinityCat#: AF3416
RRID: AB_2834858
WB (1:1000)
AntibodyAcetyl-FOXO1A (Rabbit polyclonal)AffinityCat#: AF2305
RRID: AB_2845319
WB (1:1000)
AntibodyAnti-PRKACA (Rabbit polyclonal)ABclonalCat#: A0798
RRID: AB_2757400
WB (1:1000)
AntibodyPhospho-IKB alpha (Rabbit monoclonal)CSTCat#: 2,859
RRID: AB_561111
WB (1:1000)
AntibodyPhospho-IRF3-S386 (Rabbit polyclonal)ABclonalCat#: AP0857
RRID: AB_2771209
WB (1:1000)
AntibodyPeroxidase-Conjugated Goat anti-Rabbit IgGZSGB-BIOCat#: ZB-2301
RRID: AB_2747412
WB (1:5000)
AntibodyPeroxidase-Conjugated Goat anti-Mouse IgGZSGB-BIOCat#: ZB-2305
RRID: AB_2747415
WB (1:5000)
AntibodyAlexa Fluor 594 goat anti-mouse IgGThermoFisher ScientificCat#: A11005
RRID: AB_2534073
IF (1 μg/ml)
Commercial assay or kitNuclear Extraction Kits for CellsInvent BiotechnologiesCat#: SC-003
Chemical compound, drugDynasoreMCECat#: HY-1530450 μM
Chemical compound, drugSP600125MCECat#: HY-12041200 μM
Chemical compound, drugPD98059MCECat#: HY-12028200 μM
Chemical compound, drugTakinibMCECat#: HY-103490200 μM
Chemical compound, drugAmlexanoxMCECat#: HY-B0713100 μM
SoftwareImageJhttp://imagej.nih.gov/ij/RRID: SCR_003070
SoftwareFlowjohttps://www.flowjo.com/solutions/flowjoRRID: SCR_008520
SoftwareOriginhttp://www.originlab.com/index.aspx?go=PRODUCTS/OriginRRID: SCR_014212
OtherDAPI stainSolarbioCat#: C006510 μg/ml

Additional files

Supplementary file 1

Quantification and statistical analysis.

https://cdn.elifesciences.org/articles/78044/elife-78044-supp1-v1.docx
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
Transparent reporting form
https://cdn.elifesciences.org/articles/78044/elife-78044-transrepform1-v1.docx

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