Gelatinases

Total DNA from enterococcal strains was isolated from overnight cultures grown in BHI, and 500 ng was spotted onto Hybond-N⁺ nylon membranes (Amersham). DNA was fixed by UV crosslinking with 70,000 µJ/cm². Membranes were washed in 2×SSC buffer [27] and blotted dry. Hybridization was carried out using the DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche Diagnostics), per manufacturer's instructions. PCR products used for probes included amplified internal fragments of a putative bile acid hydrolase, cbh; capsule locus cpsF; cytolysin locus cylB; biofilm-related protein encoding esp; gelatinase, gelE; putative stress regulator, gls-24-like (EF0117); putative glycosyl hydrolase (EF0077); putative nuclease (EF0031); S. pneumoniae psaA Mn transporter homolog (EF0095); bifunctional aminoglycoside inactivating gene aac6′-aph2″; and the chloramphenicol acetyltransferase gene, cat. Each was amplified using primers listed Table S1. To sample genomes for the presence of portions of the E. faecalis pathogenicity island (PAI), genes from across the pathogenicity island were selected as shown in Fig S1; sampled regions of the PAI did not include the 5′ most region that contained high homology to plasmid pAM373, due to the extrachromosomal and highly variable nature of this DNA in isolates. Genes blaZ and ermB were detected by PCR using primer pairs ermB-1/ermB-2 and blaZ-1/blaZ-2, respectively. β-lactamase activity was confirmed by colorimetric assay. Due to the large diversity of tetracycline resistance determinants, genotyping for tetracycline resistance was performed only by PCR for the most common resistance determinants, tetL, and tetM[29] (link).

Gelatin zymography was performed to assess MMP-9 and -2 activity following treatment of THP-1 cells with DTG, CAB, BIC, or DOX. This assay was used as preliminary confirmation of the inhibition of MMPs by individual INSTIs. Due to the nature of this assay, only the gelatinases, MMP-2 and -9, could be assessed. Cells were plated at a density of 1 × 10⁶ in 12 well plates and treated with phorbol-12-myristate-13-acetate (PMA) for 24 h. This was done to promote cell differentiation to stimulate MMP secretion. Following PMA treatment, cells were treated with DTG, CAB, BIC, or DOX at concentrations of 25, 50, 75, or 100 µM or control vehicle for 24 h. In our previous study, no DTG-induced cytotoxicity was recorded in PMA-stimulated THP-1 cells up to 100 µM (Bade et al., 2021 (link)). Thus, for comparative assessments among different INSTIs (DTG, BIC, and CAB) and DOX (positive control) drug concentrations of up to 100 µM were utilized. Each of the experimental tests were performed in triplicate. Following treatment, media was collected and centrifuged at 15,000 x g for 10 min at 4°C. Supernatant was collected and stored at −80°C for further analysis. For gelatin zymography, 3 µg of protein from cell medium was loaded in a 10% SDS-polyacrylamide gel containing 0.1% gelatin. Gels were ran at 55 V until the loading dye passed through its bottom. The gel was then removed and washed with water for 15 min, then incubated with renaturation buffer [2.5% (v/v) Triton X-100 in Milli-Q water] for 90 min at room temperature. The used renaturation buffer was replaced with fresh buffer every 30 min. Renaturation buffer was then replaced with developing buffer (50 mM Tris–HCl, pH 7.5, 5 mM CaCl2, 0.2 M NaCl, and 0.02% Brij-35) and the gel was incubated at 37°C in a shaker (Innova 42, New Brunswick Scientifc, Edison, NJ) for 48 h. After 48 h, the gel was washed with water for 15 min and then stained using 0.2% Coomassie Brilliant Blue R-250 (BIO-RAD, Hercules, CA) for 1 h. After staining, the gel was washed with water for 15 min before washing with destaining solution (30% methanol, 10% acetic acid, 60% water) for 45 min. The gel was then washed with water for 20 min to remove any destaining solution. Finally, the stained gel was imaged using the iBright 750 Imaging System (Invitrogen, Carlsbad, CA). ImageJ software was used to quantitate band density recorded as a measure of relative MMP activity.

Enzymatic activities from cells and cell-free culture supernatant of B25 were determined using API-ZYM^® (bioMérieux, Marcy l'Etoile, France), according to the manufacturer's instructions. The supernatant was obtained from a B25 fermentation in the following conditions: VEG medium (10 g/L yeast extract, 10 g/L glucose, 0.1 g/L KH₂PO₄, 0.1 g/L K₂HPO₄ and 0.2 g/L MgSO₄ × 7H₂O), 30 °C and 24 h. After fermentation, the culture was centrifuged at 5000 g for 10 min (Hitachi CR22N) and the supernatant was filtered by passing through a 0.22 µm pore size polyethersulfone (PES) filter to obtain the cell-free culture supernatant. To confirm that the supernatants were free of bacterial cells, aliquots of 100 µL were plated in NA and the absence of growth was confirmed after incubation at 28 °C for 2 days.
In addition, the presence of chitinase, protease, lipase, cellulase, and gelatinase activity were analyzed using classic methods. Briefly, 10 µL of overnight grown culture were spot plated on chitin agar medium [31] (link), casein agar medium [32] (link), tween 80 agar medium [33] (link) and microcrystalline cellulose (MC)-agar medium [34] (link). Plates were incubated for seven days at 28 °C. For observation, the cellulase detection plates were stained using 1 % Congo red dye and, after 15 min, the stained plates were rinsed with 1 M NaCl solution. In all cases, the appearance of a clear halo around colonies indicates enzymatic degradation. On the other hand, the qualitative assay for gelatinase activity was determined by using the nutrient gelatin plate method and nutrient gelatin stab method, as described [35] .

Fresh brain tissues were immersed in RIPA lysis buffer (P0013K, Beyotime, China) at a ratio of 1:4 (w: v) and centrifuged at 4°C (12,000 rpm, 15 min) after homogenization. The protein concentration was determined by a BCA concentration determination kit (P0010, Beyotime, China). The supernatant and loading buffer (P0016N, Beyotime, China) were mixed at a ratio of 4:1. A total of 120 μg of total protein was loaded into an 8% SDS‒PAGE gel containing 10% gelatin. After electrophoresis at 80 V in ice water for 3 h, each gel was washed in 2.5% Triton X-100 for 30 min 3 times. Then, gels were treated with incubation buffer (50 mM Tris-HCL, 0.2 M NaCl, 5 mM CaCl2, 1 µM ZnCl2, 0.02% Brij-35, pH 7.5) for 48 h at 37°C on a shaking table. Gels were stained with 0.5% Coomassie brilliant blue R250 (ST1123, Beyotime, China) for 3 h and then dyed with 30% methanol containing 10% acetic acid until the emergence of an appropriate color discrepancy. The clear bands on the zymogram represented gelatinase activity.