Bacteria strains and growth mediums
E. coli BL21 (DE3) was used for recombinant protein expression. Strains used for antimicrobial activity assays were carbapenem-resistant K. pneumoniae (KPC) and extended-spectrum beta-lactamase producing Enterococcus spp. CTX-M-14 (EC) (kindly provided by Dr. Lourdes Migura-Garcia, IRTA). E. coli strains were grown in Luria–Bertani (LB) medium and KPC and EC were grown in Brain–Heart Infusion (BHI) broth (Scharlau).
Genetic construct design
From N-terminal to C-terminal, the gene for the JAMF1 construct consisted of the sequences encoding Jun257-318 (Uniprot entry P05412), human α-defensin-5 (HD5) (Uniprot entry Q01523), gelsolin188-196 (Uniprot entry P06396), group-XIIA secretory phospholipase A2 (sPLA2) (Uniprot entry Q9BZM1) and Fos118-210 (Uniprot entry P01100)13. The gene encoding for the JAMF1.2 construct is identical to JAMF1 except for the human α -defensin-5, where we used HD563-94 instead. The sequence encoding for the JAMF2 construct is identical to JAMF1.2 but the sPLA2 domain was changed to sPLA2 23-189. A linker sequence (SGGGSGGS) was used between each of the domains. For HD5-GFP, HD5 63-94 was fused to GFP23 using a linker sequence (GGSSRSS). In all constructs an H6-Tag was placed at the C-terminal for protein purification. The fusion constructs were codon-optimized by GeneArt (Lifetechnologies) and cloned into pET22b (AmpR) vector (Novagene).
Antimicrobial protein production
All constructs were produced recombinantly in E. coli BL21 (DE3) using the expression vector pET22b. Cultures (1–2 L) were grown at 37 °C and 250 rpm in LB broth with ampicillin at 100 μg/ml. Protein expression was induced by 1 mM isopropyl-β-d-thiogalactoside (IPTG) at an OD600 = 0.4–0.6. Cultures were grown 3 h post-induction. The whole volume of each protein production was centrifuged at 6000× g and the pellet was resuspended in 120 ml of PBS 1 × with protease inhibitors (cOmplete EDTA-free, Roche). Samples of 30 ml were sonicated for 5 min at 10% amplitude (0.5 s ON/OFF cycles) for 4 rounds, resting for 5 min in ice between rounds.
Protein solubilization and purification
Only HD5-GFP was obtained directly from the soluble fraction. After sonication, HD5-GFP was centrifuged 45 min at 15,000× g at 4 °C and the supernatant was recovered for Immobilized Metal Affinity Chromatography (IMAC) purification, as explained below. For all other constructs, protein pellets were recovered after sonication and centrifugation (45 min at 15,000× g at 4 °C) and washed twice with distilled water and then weighted. After that, pellets were solubilized under non-denaturing conditions for 40 h at room temperature (RT) under agitation with 40 ml/g of pellet of solubilization buffer (0.2% w/v N-lauroylsarcosine, 40 mM Tris and protease inhibitors), as previously described24. After the incubation, the supernatant was recovered through centrifugation at 15,000× g for 45 min at 4 °C for purification. Imidazole (20 mM) and NaCl (500 mM) were added to equilibrate the solubilized samples with the binding buffer composition to prepare the sample for IMAC purification. IMAC purification was performed in an ÄKTA start protein purification system (GE Healthcare) using 1 ml HisTrap HP columns (GE Healthcare). Both the binding (20 mM Tris pH = 8, 500 mM NaCl, 20 mM imidazole) and the elution buffer (20 mM Tris pH = 8, 500 mM NaCl, 500 mM imidazole) contained 0.2% N-lauroyl sarcosine. The buffer of the selected fraction was changed to 10 mM KPi (K/PO4 buffer, pH 7.4) with a HiTrap desalting column (GE Healthcare). Protein integrity and quantity were analyzed by SDS electrophoresis (TGX™ FastCast™, Bio-Rad), followed by western blotting with a monoclonal anti-His antibody (1:1,000, His-probe, Santa Cruz). Quantification was performed by interpolation to a standard curve of soluble T22-GFP25.
Immunomodulatory activity in colonic epithelial cells
Human adenocarcinoma colonic epithelial cells (HT29 or CaCo-2) were maintained in Dulbecco’s Modified Eagle’s media (DMEM; Gibson, Life Technologies) containing 4.5 g/L glucose with 10% (v/v) fetal bovine serum (FBS; Benchmark Gemini Bio-Products), 1 mM sodium pyruvate (Gibco, Life Technologies), and 1% penicillin (100 U/ml)/streptomycin (100 μg/ml; HyClone Thermo, Fischer Scientific) in a humidified environment with 5% CO2 and at 37 °C. Cells were seeded in 24-well plates (Greiner, Bio-One, Monroe), cultured to 80–90% confluency and stimulated for up to 24 h in culture medium without FBS or antibiotics. CaCo-2 cultures were stimulated with recombinant JAMF2 at 1 μM and using KPi buffer as a negative control. Supernatants were collected after 24 h and simultaneous detection of multiple cytokines was performed using MILLIPLEX MAP KIT Human High Sensitivity T Cell Magnetic Bead Panel by Luminex technology. Similarly, HT29 cells were stimulated (or not) with recombinant HD5-GFP, JAMF1 or JAMF1.2 or HD5 (PeptaNova, commercial synthetic peptide, positive control) at 0.1 and 1 μM in presence or absence of LPS, 1 μg/ml for up to 24 h in culture medium without FBS or antibiotics. The dose of LPS was determined in a preliminary study using different concentrations (0.1–2 μg/ml), and in agreement with the literature26. Supernatants were collected from cells and levels of IL-8 determined using a DuoSet ELISA (DY208, R&D Systems).
Antibacterial activity
Bacterial cell viability was determined with a BacTiter-Glo™ Microbial Cell Viability assay (Promega). Briefly, bacterial cells were grown O/N at 37 °C and 250 rpm and then diluted 1:100 in 10 mM KPi buffer. After that, 150 μL from the KPi diluted cells were centrifuged in 1 ml tubes at 6200× g at 4 °C for 15 min. The supernatant was removed, and the pelleted cells were resuspended with 150 μL of either KPi buffer (negative control) or 150 μL of 1 and 3 μM of the synthetic HD5 peptide or the protein constructs (HD5-GFP, JAMF1, JAMF1.2 or JAMF2). After 0.5 h, 2 h or 5 h incubation at 37 °C in a 96-well plate, 100 μL were taken and mixed with 100 μL of the BacTiter-Glo™ reagent for 5 min. Luminescence was measured in a microplate luminometer (LUMIstar®, BMG LABTECH). The measured luminescence values (arbitrary units) were normalized to the control (KPi buffer treatment; equivalent to 100% bacterial survival). HD5 (PeptaNova) served as a control.
Scanning electron microscope (SEM)
To evaluate the mode of action of JAMF multidomain construct a SEM technique was selected. Shortly, an O/N culture of KPC was diluted 100-folds in a sterile KPi 10 mM buffer. A total of 400 μL were centrifuged (6,200× g, 15 min, 4 °C). The supernatant was removed and the bacterial pellet was resuspended in 400 μL of each treatment (KPi as a negative control and JAMF2 at 5 µM). Samples were deposited on circular coverslips and incubated in sterile 24-well plates for 5 min at 37 °C without agitation. Next, the supernatant was carefully removed. Coverslips were fixed with 2.5% glutaraldehyde in PB 0.1 M for 2 h at 4 °C, washed with PB 0.1 M, post-fixed with osmium tetroxide-potassium ferrocyanide for 2 h, washed with miliQ water, dehydrated in graded ethanol series (50, 70, 90, 96, 100%) and desiccated with HMDS. Samples were micrographed by field emission scanning electron microscopy (FESEM) Zeiss Merlin (Zeiss) running at 1 kV.
Enzymatic assay
A fluorometric assay kit was used to measure sPLA2 activity (Cayman), using a sPLA2 substrate consisting of 1,2-dithio analog of diheptanoyl phosphatidylcholine. Upon enzymatic hydrolysis of the thio ester bond at the sn-2 position, free thiols were detected using 5,5’-dithio-bis-(2-nitrobenzonic acid) (DTNB) at 412 nm. Absorbance measurements were taken each minute during 15 min. The reaction rate (µmol/min/ml) was determined for constructs JAMF1, JAMF1.2 and JAMF2 from the absorbance change per minute of the linear portion of the curve, using the DTNB extinction coefficient. At least 8 time points were used for the calculations.
Statistical analysis
Results are expressed as means with error bars representing standard errors of the mean (SEM). Data were obtained in triplicates and normality was assessed using a Shapiro–Wilk test. All comparisons were performed using either a two-sided unpaired Student’s t-test (asterisks) or one-way analysis of variance (ANOVA) with a post hoc Tukey HSD test for multiple group comparisons (letters). A P value < 0.05 was considered statistically significant. Analysis was conducted in R (R Core Team, 2019) with RStudio software (RStudio, Inc.), and figures were produced using the package ggplot2 (Wickham, 2016)27,28,29.
