Bacterial Culture
C. acnes KPA171202 was grown on Brucella agar plates (Condalab) from glycerol and incubated for 3 days, 37ºC anaerobically using GasPak EZ anaerobe pouch system (BD). When grown in liquid, C. acnes KPA171202 was inoculated to an OD600 of 0,1 and grown in Brain-Heart Infusion broth (BHI) (Sigma) at 37ºC,110 rpm, anaerobically using AnaeroGen system (Thermo Scientific), until reaching the desired optical density. E. coli DH5a (NZYTech) was used for routinely cloning and E. coli dam-dcm- (New England Biolabs) to create a C. acnes methylase proficient shuttle strain. E. coli strains were grown in LB medium either supplemented with Ampicillin 50 µg/mL or Kanamycin 50 µg/mL and grown at 37ºC at 225 rpm.
Cloning of plasmids
Plasmids for homologous recombination were cloned as previously described 35. In short, homology arms were amplified from genomic DNA including restriction sites for NcoI, SpeI and Acc65I. Homology arms were digested with Acc65I and ligated and finally cloned into the pGEM-T-easy vector by restriction-enzyme-mediated cloning. In a second step the gene of interest was cloned between the homology arms in a second step. Replicative plasmids are based on the P. freudenreichii vector pBRESP36A 45 and were optimized by Lood et al. for protein production in C. acnes 46. Selected secretion peptides and LCN2 genes were cloned downstream of the p1340 promoter using NheI and SpeI restriction sites.
Minicircle production
Minicircle strains ZYCY10P3S2T 47 (System Biosciences), JMC2 and JMC3 48 were used to produce minicircles being methylated or lacking endogenous methylation. Gene flanking homology arms were cloned into the parental plasmid pMC BESPx MCS1 (Systems Biosciences) and transformed into ZYCY10P3S2T (ZYCY) (Systems Biosciences), JMC2 or JMC3 minicircle Production Strains. MC production was performed according to the manufacturer’s protocol.
Creating a MIIIB proficient dam- dcm- hdsMS- E. coli strain
To create an E. coli strain that produces plasmids with the C. acnes methylation pattern, lambda-Red recombineering was used as described by Datsenko & Wanner 49. Briefly, a recipient dam-/dcm- strain (NEB) was first transformed with pKD46 plasmid 49 and selected with 100 µg/ml ampicillin at 30ºC. Transformants were grown at 30ºC and induced with 0.4% arabinose for 1h before transformation with the linear DNA fragment to be recombined into the chromosome (see below). Transformants were selected at 37ºC with 25 µg/ml kanamycin, and successful recombineering was verified by colony PCR and Sanger sequencing. The temperature-dependent loss of pKD46 was confirmed through the absence of growth in Amp 100 µg/ml plates.
The linear DNA fragment to be recombined into the E. coli chromosome was prepared as follows. First, the MIIIB methylase gene was amplified from the genome of C. acnes KPA171202, and it was cloned under Bba_J23100 promoter and Bba_B0034 RBS in a pJET1.2 vector, together with the Tn5 neomycin phosphotransferase (KanR) gene amplified from pKD13 49. The region containing the MIIIB methylase and the KanR genes was then amplified with primers PP-19 and PP20, which provide 50 bp homology regions to the E. coli genome. In particular, primers were designed so that recombination occurs in the hsdMS locus, thereby inactivating the hsdMS methylation system upon insertion of the C. acnes methylase gene. To ensure that only the linear PCR product (and not the template plasmid) was transformed, the template plasmid was digested with XhoI and AatII for 1h at 37ºC prior to the PCR, and then the PCR product was treated with DpnI for 1h at 37ºC and the correct band was purified from a 1% agarose gel. The template-free, purified PCR product was transformed into dam-/dcm- cells carrying pKD46 as described above.
Transformation of C. acnes
C. acnes KPA171202 was grown to an OD600 of 0.4, 0.7, 1.0 and 1.8. Cells were spun down at 1700 x g for 10 min at 4ºC and washed with equal volume of EPB (272 mM sucrose, sterile filtered) followed by a second spin under the same conditions. Cells were resuspended in 1 mL EPB and washed another 5 times, 9400 x g, 1 min, 4ºC. Then the pellet was resuspended in residual liquid. Competent cells were diluted 1:4 in EPB to a final volume of 50 ul and 500–8000 ng of DNA were added to each sample. Cells were transferred to a pre-cooled 0.1 cm Electroporation cuvette (BioRad) and electroporated 1.5 kV, 400 Ω and 25 uF. Cells were recovered in 100 µl BHI media and plated on a Brucella agar plate. After 24 h anaerobic incubation at 37ºC, cells were resuspended in 1 mL of BHI to remove remaining non incorporated DNA, spun down 5 min at 1700 x g and resuspended in 200 ul BHI. 50 µl of resuspended cells were then plated on 4x Brucella agar plates supplemented with 10 µg/mL erythromycin per sample. Transformants were obtained following anaerobic incubation at 37ºC for 7 days.
To improve electroporation efficiency different pre-treatments were tested. For glycine of penicillinG pre-treatment, cells were grown in presence or absence of 1.25% of glycine or 10 ug/mL PenicillinG, 4 h prior to electroporation. For osmotic stabilization 0.4 M of sucrose was added also 4 h prior.
Thymidine kinase knock-in functional testing
The tdk knock-in was created as previously described in Sörensen et al. 2010 35 targeting the thymidine kinase locus and cloning the pGEM-T-easy plasmid with homologous arms to the region. For simplicity the antibiotic resistance cassette, coding for erythromycin (ermE) was cloned together with the LCN2 gene between the homology arms to replace the tdk gene with an ermE cassette. To evaluate the functionality of the tdk dual selection marker 41, we tested the knock-in in sensitivity to FUDR (50 ug/mL) and erythromycin (10 ug/mL) and compared it to the wild type. Therefore, we plated C. acnes KPA171202 wild type bacteria and knock-in on Brucella agar plates either containing ery (10 ug/mL), FUDR (50 ug/mL) or neither of the components. Only the knock-in strain could grow on both erythromycin and FUDR-containing plates, while the wild type was sensitive to both.
TCA precipitation and protein purification
Recombinant C. acnes strains were inoculated in 25 mL BHI media supplemented with 10 ug/mL erythromycin to a starting OD600 of 0.1 and grown for 48 h at 37ºC, anaerobically, 110 rpm. Cells were spun down and the supernatant and pellet were treated separately. The supernatant was used for protein purification or trichloroacetic acid (TCA) precipitation while the pellet was subjected to lysis by a FastPrep FP120. Protein was precipitated as previously described 50 and deoxycholate (DOC)-trichloroacetic acid precipitation was followed.
For protein purification, cell-free supernatants were prepared by centrifugation and directly loaded onto a 5-mL HisTrap FF column (Cytiva). After washing the column with binding buffer A (50 mM Tris-HCl pH7.4, 500mM NaCl, 5mM Imidazole, 10% Glycerol), protein was eluted with elution buffer (Buffer A with 300mM Imidazole). The fractions containing NGAL protein were pooled and dialyzed with a storage buffer (50mM TRIS-HCl pH7.4, 500 mM NaCl and 10% Glycerol). Pooled fraction was analyzed by Western blotting with anti 6xHIS Tag antibody. Protein concentration was quantified by Qubit Protein Assay Kit (Thermo Fisher Scientific).
Western blot
NuPAGE LDS sample buffer (4x) containing β-mercaptoethanol was added to samples to a final concentration of 1x. Proteins were denatured for 10 min at 95ºC and loaded on a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and run for 1,5 h, 120 V in 1x MOPS SDS running buffer (Invitrogen). Proteins were transferred to an activated PVDF membrane (Brand) in NuPAGE 1x transfer buffer supplemented with methanol (10%) for 1 h, 20 V. Proteins were blocked with 4% milk TBST (1x) for 1 h at RT and then incubated o/n at 4 degrees with a mouse anti-histidine primary antibody (1:800) (MCA1396; BioRad). Membrane was washed three times with 1xTBST buffer and incubated with secondary anti-mouse IgGk BP-HRP (1:1000) (sc-516102; Santa Cruz Biotechnology) for 1 h RT. After three washes in TBST (1x), the membrane was developed using Pierce™ ECL Western Blotting Substrate (Thermo Fisher). This reaction emits light at 428 nm and this light signal was captured by a digital imager (BioRad).
Sebocyte culture
Immortalized human sebocytes, SZ95 51 were seeded in a density of 2x10^5 cells/well and maintained in DMEM/F12 (Gibco) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS), 100 U/mL Pen/Strep, 1mM CaCl2 and 5 ng/ml human epidermal growth factor (EGF; Sigma-Aldrich). Cells were incubated at 37ºC in a humidified 5% CO2 incubator.
PCi-SEB_Cau (Phenocell) were seeded and maintained following manufacturer protocols in PhenoCULT-SEB basal medium supplemented with Supplement A (1/1000) for 3 days prior to exposure and starting the assays.
Sebum modulation assay
PCi-SEB_Cau human iPSC derived sebocytes (Phenocell) were seeded in a 24 well plate at a cell density of 25000 viable cells/cm2 in PhenoCULT-SEB basal medium supplemented with 1/1000 Supplement A. Cells were grown in a humidified incubator (37 ºC, 5% CO2) for 3 days before being exposed and starting the assay. Experiment was performed in triplicates being treated with either vehicle, 5 uM arachidonic acid (AA) (Sigma), 5 uM AA + 10 uM ISO (13-cis retinoic acid)(Sigma), 5 uM AA + 50 ng/mL rhNGAL E. coli, 5 uM AA + 50 ng/mL C. acnes pBR13,14 or 16 NGAL. Cells were exposed for 48 h and then cell lipid content stained with the fluorescent marker BODIPY 493/503 (Sigma Aldrich) following manufacturer's recommendations. The nucleus was stained with DAPI. Samples were analyzed by confocal fluorescence microscopy and fluorescence measured and normalized.
MTT cytotoxicity assay
Human SZ95 sebocytes were seeded in a 12-well plate to a density of 2x10^5 cells/well and incubated in a humidified atmosphere (37ºC, 5% CO2) until 80% confluency in DMEM/F12 (Gibco) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS), 100 U/mL Pen/Strep, 1mM CaCl2 and 5 ng/ml human epidermal growth factor (EGF; Sigma-Aldrich Co.). PCi-SEB_Cau (Phenocell) were seeds to 2x10^5 cells/well incubated in a humidified atmosphere (37ºC, 5% CO2) until 80% confluency in PhenoCULT-SEB basal media supplemented with 1/1000 Supplement A for 3 days prior to treatment. Then, SZ95 and PCi-SEB_Cau were incubated with purified NGAL protein in four replicates, produced in E. coli or in HEK293 (LC2-H5222, ACROBiosystems), with concentration 0, 100, 1000 ng/mL for 96 h and then subsequently incubated with fresh medium and 10% MTT (5 mg/mL in phosphate buffered saline) for 2 h at 37°C. Afterwards, the medium was removed carefully and 500 µL of dimethyl sulfoxide (DMSO) 99% purity was added to lysate the cells and dissolve the purple insoluble crystals of MTT. The cell lysate was transferred to a new 96-well plate and then the absorbance was read using a Microplate Autoreader at excitation/emission of 540/630 nm (Tecan). Absorbance values were considered directly proportional to cell viability.
Tunel assay
SZ95 and PCi-SEB_Cau sebocytes were cultured in 96-well plates as described above. Wells were rinsed with PBS and treated in triplicates with vehicle control, 50 ng/ml, 100 ng/ml, 1 µg/ml of NGAL (E.coli, HEK293 or C. acnes) protein for 24 hours. Samples were prepared by manufacturer’s instructions for In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics). DNase I treatment of positive and negative controls (without terminal transferase) were included as assay controls according to manufacturer’s instructions. Digital images from Operetta HCS fluorescence microscopy experiments were processed using the Harmony high content analysis version 4.9 software package (Perkin-Elmer). When performed, image processing was restricted to changes in brightness and/or contrast and was applied equally across the entire image and different images of the same experiment, including controls. For image analysis, at least three fields per biological replicate were randomly chosen. To measure staining intensity, the Harmony high content analysis software package was used with custom algorithms. The DAPI channel was used to detect nuclei. Cytoplasmic area was determined as a ring of constant size around each nucleus and was used to measure the mean fluorescence intensity of the staining of interest.
Bacteria engraftment on murine skin
Bacteria (WT and KI) was grown in 20 mL to a OD600 of 1, spun down at 1700 x g, 10 min and washed 3 times with 0.5% of peptone/PBS and resuspended in 0.5% peptone/PBS to a final volume of 500 ul. Mice (C57BL/6J) were shaved on the back at an area of 2cm x 5cm. Wild type, knock out or vehicle were applied with a cotton swab on the shaved areas and repeated for consecutive 3 days. Every day before application a sample was taken with a moistened cotton swab and dissolved in 0.5 mL QuickExtract™ DNA Extraction Solution (Lucigen) and treated for 6 min at 65ºC followed by 2 min at 95ºC with 15 seconds vortexing between steps. Samples were taken every 24 h during the full assay period of 7 days. 5 µl DNA extract was used for amplification of the SLST region or 16S V3-V4 region linked to illumina adaptors using KAPA HiFi HotStart Readymix (Roche) (Initial denaturation for 5 min at 95°C followed by 35 cycles of 98°C for 20 s, 62°C for 25 s, and 72°C 30 s; and a final elongation for 1 min at 72°C).
SLST_illumina_fwd:
5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTGCTCGCAACTGCAAGCA-3’ and
SLST_illumina_rev:
5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCGGCTGGCAAATGAGGCAT-3’
16S_illumina_fwd:
5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’ and
16S_illumina_rev:
5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3’.
Junctions were PCR amplified using KAPA HiFi HotStart Readymix (Roche) (Initial denaturation for 3 min at 95°C followed by 25 cycles of 98°C for 20 s, 62°C for 15 s, and 72°C 30 s; and a final elongation for 1 min at 72°C) using primer pairs Junction1_ fwd and rev and Junction2_fwd and rev (see Supplementary Table 1).
PCR products were loaded in a 1% agarose gel and visualized by a transilluminator.
Skin biopsies
Bacteria was applied to shaved back of mice for 3 consecutive days. 24h after the last application mice were sacrificed and skin biopsies were taken. Samples for RNA extraction were stored in RNAlater solution while samples for cryosections were frozen embedded in OCD solution.
RNA extraction and qPCR of skin biopsies
Total RNA was extracted from skin (NucleoSpin RNA extraction kit, Macherey-Nagel) according to the manufacturer instructions. RNA was then used for cDNA synthesis (High-Capacity cDNA Reverse Transcription Kit, Thermo Fisher Scientific). Quantitative PCR amplification reactions were performed in the QuantStudio 7 Pro Real-Time PCR System (Applied Biosystems) using TaqMan and Sybr Green Gene Expression Master Mix. Values were normalized to Sdha. The following TaqMan assays (Thermo Fisher Scientific) were used to quantify mRNA expression of mouse Sdha (Mm01352366_m1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1) and Tnfα (Mm00443258_m1) and primers LCN_qPCR_fwd and rev for detection of human NGAL.
Gram staining of skin sections
Cryosections from frozen tissue embedded in OCT (Optimal Cutting Temperature (OCT) compound Tissue-Tek) were cut at 7um thickness with cryotome. Frozen skin sections were air-dried at room temperature for 15 minutes, fixed with 4% paraformaldehyde for 15 minutes and stained with Gram following standard procedures. Crystal violet was applied for 1 minute following water washing, then iodine for 1 minute then water wash, rapid decolorization was achieved with acetone and a final 1 minute incubation with Safranin with subsequent washes were applied. After drying the samples DPX (Sigma Aldrich) mounting media was applied. Conventional images were captured using a Zeiss apotome microscope.