Chemigenius 2 imaging system

The 1-D bacterial overlay procedure was adapted from Odenbreit et al. [45 (link)]. In brief, purified mucins (1μg/μL in PBS or 4 M guanidine chloride in PBS) were spotted on dry nitrocellulose membranes using a Bio-Dot SF (Biorad Hercules, CA, USA) which were saturated with PFBB (Protein Free Blocking Buffer) (Thermo-Scientific, Waltham, MA, USA) for 1 h. Bovine serumalbumin at 1 µg/µL was used as a negative control. Bacteria were labeled either with 2.5 µg/mL DAPI or 50 µM syto9 in PBS for 15 min or with FITC at 0.1 mg/mL (for Gram− bacteria) or 0.2 mg/mL (Gram+ bacteria) in carbonate buffer (NaCl 0.15 M, NaHCO₃ 0.1 M) for 1 h. Labeled bacteria were collected by centrifugation at 3000× g for 5 min, washed three times in PBS, suspended in 1 mL of blocking buffer and added to the membrane in blocking buffer. In the case of FITC, an additional step, involving 2 h of incubation in blocking solution before the final three washes, was included to reduce the background signal. After incubation for 1 h at room temperature in the dark, followed by three washes of membranes in PBS containing 0.5% Tween 20, the fluorescence of adherent bacteria was detected by a ChemiGenius 2 imaging system (Syngene, Frederick, MD, USA). Mucins were chemically desialylated for 1 h at 80 °C in a 0.05 M trifluoroacetic acid (TFA) solution.

Colorectal tissues from healthy individuals arised from patients with diverticulosis. The use of human tissues for this study was approved by the local hospital ethics committee and French Ministry of Higher Education and Research (DC-2008-242). All subjects gave written informed consent in accordance with the Declaration of Helsinki.
The procedure was adapted from that of Da Silva et al. (2015) (link) to evaluate the binding of S. thermophilus LMD-9 to human colonic mucin. TIL448 and TIL1230 were used as high-adhesive and low-adhesive controls, respectively (Le et al., 2013 (link)). In brief, mucin was scraped and purified from human biopsies and spotted (10 μg) on dry nitrocellulose membranes. PGM was used as control. Bacteria (10⁹ CFU/mL in phosphate-buffered saline) were stained with DAPI for 15 min at room temperature in the dark. Labeled bacteria were added to the membrane in blocking buffer. After incubation for 1 h at room temperature in the dark, the fluorescence of adherent bacteria was detected by a ChemiGenius 2 imaging system (Syngene).

Activity of the purified ClpP1/P2 complex was tested by performing a degradation assay with the E. coli ClpX (EcClpX) chaperone and its model substrate GFP-SsrA. The assay was performed in buffer D (20 mM Tris HCl pH 7.5, 25 mM NaCl, 150 mM KCl, 4 mM MgCl₂, 1 mM DTT) and in the presence of an ATP-regeneration system (Dougan et al., 2002 (link)). EcClpX (0.9 μM) was added in excess of ClpP1/P2 (0.3 μM). The assay was performed at 37 °C and samples were collected at different time points, which were treated with NuPAGE sample buffer and denatured at 75 °C for 10 min. Samples were separated on 12% Bis-Tris gels (Life Technologies) and visualized by staining with colloidal coomassie blue. The amount of GFP-SsrA at each time point was quantified using the ChemiGenius2 imaging system (Syngene) and associated software.