Luria bertani medium

PCL (M_w 80,000) was obtained from Sigma-Aldrich (Shanghai, China). SF was extracted from Bombyx mori silkworms (Second Silk Company, Zhejiang, China) by our group. AMX (>98%) and MWCNTs were purchased from Adamas (Emeryville, CA, USA) and Aladdin Industrial Company (China), respectively. The carboxylated MWCNTs with lengths of 10–30 μm had inner and outer diameters of approximately 5 and 20 nm, respectively. Hexafluoroisopropanol (HFIP), EDC, and NHS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Escherichia coli was purchased from Shanghai Fuzhong Biotechnology Development Co., Ltd. (Shanghai, China). Luria–Bertani (LB) medium was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). L929 cells were obtained from the Institute of Biochemistry and Cell Biology (The Chinese Academy of Sciences, Shanghai, China). Dulbecco’s modified Eagle’s medium, fetal bovine serum, and glutaraldehyde were purchased from Shanghai Limin Industrial Co., Ltd. (Shanghai, China). Mouse polyclonal anti-CD11b and anti-CD68 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX). Rabbit anti-collagen I antibody was purchased from Sigma-Aldrich (St. Louis, MO).

The APEC17 strain is a serotype O2 strain isolated from a case of avian colibacillosis in Anhui Province, China (Tu et al., 2016 (link)). All strains and plasmids are listed in Table 1. Bacterial strains were grown in Luria-Bertani (LB) medium (Sangon, Shanghai, China) at 28°C or 37°C. If required, antibiotics were added at the following concentrations: ampicillin (100 µg/mL) and chloramphenicol (30 µg/mL) (Sangon).Table 1

Strains and plasmids used in this study.

Table 1

Strains	Relevant genotype	Source
AE17	APEC wild-type strain	Laboratory stock
AE17ΔenvZ	AE17 envZ-deletion mutant strain	This study
AE17C-envZ	Cmr, AE17ΔenvZ with the complement plasmid pSTV28-envZ	This study
Plasmids
pKD46	Ampr, expresses λ red recombinase, temperature-sensitive	Takara
pKD3	Ampr Cmr, cat gene, confers resistance to chloramphenicol template plasmid	Takara
pCP20	Ampr Cmr, temperature-sensitive plasmid	Takara
pSTV28	Cmr, cloning vector with pACYC184 replication starting point	Takara
pSTV28-envZ	Cmr, pSTV28 with envZ gene	This study

Abbreviations: Cm^r, chloramphenicol-resistant; Amp^r, ampicillin-resistant; APEC, Avian pathogenic E. coli.

B.a (ATCC15703) was purchased from the American Type Culture Collection (ATCC, Rockville, Maryland, USA). The bacteria genome was extracted, and V4 of 16S ribosomal RNA sequencing was performed by Tsingke Biotechnology (Beijing, China), followed by comparing the sequence results with the strain sequence in the PubMed Nucleotide BLAST database (https://blast.ncbi.nlm.nih.gov) to confirm bacterial strain at the species level. The bacteria were cultured in anaerobic modified Reinforced Clostridium Medium (BD Difco, Sparks, MD, USA) under an atmosphere of 10% H₂, 10% CO₂, and 80% N₂ (AW500SG anaerobic workstations; ELECTROTEK, Keighley, West Yorkshire, United Kingdom) for 48 h. The non‐pathogenic commensal intestinal bacteria, Escherichia coli (E.coli) strain DH5a (Code No.9057, Takara, Dalian, Liaoning, China), which was used as a negative control, was cultured in Luria‐Bertani medium (Cat. #A507002, Sangon Biotech, Shanghai, China) at 37°C. When the optical density (OD) at 600nm of B.a reached 1.0, the cultures were centrifuged at 1000 × g for 5 min at 4°C and then washed twice with sterile anaerobic phosphate‐buffered saline (PBS), then resuspended at a final concentration of 1 × 10⁹ CFU/300 μL under strictly anaerobic conditions.