Vivaflow 200

In order to obtain a sufficient quantity of OMVs to detect a metabolomic signal, 2 l of RM (three culture flasks grown independently) and DM (three culture flasks grown independently) were inoculated with overnight culture of B. thetaiotaomicron at an initial OD of 0.0005 and 0.05, respectively. The OMV extraction was performed as previously described (Stentz et al., 2014 (link)) with slight modifications. After 16 h (OD approximately 4.0), the cell cultures were rapidly cooled in a manually shaken ice bath. The cultures were then centrifuged at 5000 × g for 15 min at 4°C, and the supernatants filtered through a 0.22-μm Steritop filtration unit (Millipore, Billerica, MA, United States) to remove debris and cells. The sterility of the filtrate containing the vesicles was confirmed by plating onto BHI–hemin agar. OMVs in the 2 l filtrates were concentrated by molecular weight (100 kDa MWCO, Vivaflow 200, Sartorius) down to 2 ml, diluted by addition of 1 l of ice-cold PBS, pH 7.4, and the suspensions were filtered and concentrated again, down to 9 ml. The 9 ml retentate was ultracentrifuged [150,000 × g for 2 h at 4°C in a Ti70 rotor (Beckman Instruments)]. The supernatant was completely removed using a vacuum pump and a needle and the OMV pellets were snap frozen in liquid nitrogen and stored at -80°C before extraction.

Saccharomyces cerevisiae strain FGY217 (MATα, ura3-52, lys2_201, and pep4) was used to express and secrete proteins. Yeast was transformed with the Lili-Mip plasmid using the lithium acetate method [19 (link)]. Transformed yeast colonies were inoculated in synthetic media lacking uracil with 2% dextrose and grown overnight at 30°C and 220 rpm. The Optical Density (OD) at 600 nm was measured, and the culture was diluted with fresh YPD media to an OD of 0.2/ml. This was again grown until the OD reached 0.6–0.7, induced with 2% galactose, and incubated for 24 h at 30°C and 220 rpm. The supernatant was clarified by centrifugation at 4000 ×g for 10 min. The clarified media was concentrated using a Sartorius Vivaflow200, with a 10kDa molecular weight cutoff filter to a final volume of 500 ml. The media was buffer exchanged into 50 mM sodium acetate at pH 5.0 and 10 mM NaCl while concentrating the media. The buffer-exchanged media were purified using cation exchange chromatography on an SP FF column (Cytiva). The Lili-Mip protein eluted between 100 mM and 200 mM NaCl (on a gradient that extended to 1.0M NaCl). The protein fractions were concentrated using a 10 kDa filter in an Amicon spin filtration system. The concentrated protein was purified using the size exclusion chromatography column S75 (Prep Grade—Cytiva) in 50 mM Sodium acetate pH 5.0 and 50 mM NaCl buffer.

Bacteria from −80 °C stocks were cultured on TSA-1 for 24–72 h at 25 °C, resuspended in TSB-1 at an OD_600nm of 0.5–0.6, inoculated 1:100 in 100 mL of TSB-1, and grown for an additional 24 h at 25 °C with shaking (160 rpm). This culture was then used to inoculate 6 × 800 mL of TSB-1 (in 2 L-Erlenmeyer flasks) at 1:100 dilution, followed by incubation at 25 °C with shaking until an OD_600nm of 0.9–1.0 was reached. The culture was then centrifuged at 3993× g for 30 min at 4 °C (Beckman Avanti J-26 XP, rotor JLA-8.1000), the pellet was discarded, and the supernatant was centrifuged again. The supernatant of this centrifugation was then filtered through a 0.22 µm pore-size filter to remove any remaining bacterial cells. Cell-free supernatant (~4.5 L) was concentrated 45-fold by tangential filtration (Vivaflow 200, 100,000 MWCO PES, VF20H4, Sartorius, Goettingen, Germany); dialyzed against 20 mM Tris pH 8.0, 150 mM NaCl, using the same system; and ultracentrifuged ON at 175,000× g, 4 °C (Beckman Optima XE 100, rotor SW 32 TI; Ultra-Clear Tubes, 344058, Beckman Coulter, Brea, CA, USA). The supernatant was discarded and the pellet (crude OMVs) was resuspended in 1.2 mL 0.9% NaCl, aliquoted, frozen in liquid nitrogen, and stored at −80 °C. The sterility of the crude OMV preparations was confirmed by plating aliquots on TSA-1.

Yeast processing conditions were optimized, changing the initial volume of yeast medium (in the range 0.5–-5 L) and the concentration factor (100–-1000×) to obtain a sufficient amount of killer protein for all the subsequent evaluations. The yeast culture (5 L) obtained after 36 h incubation was centrifuged at 4,000 rpm for 10 min (T = 4 °C) to remove yeast cells. The supernatant was filtered using 0.22 μm sterile filtering flasks (Millipore, Billerica, MA, USA) and concentrated 100× using the Sartorius Vivaflow 200 ultrafiltration system equipped with a polyethersulfone membrane (cutoff: 10 kDa). The obtained volume was further reduced (10×) using concentrators tube (cutoff: 10 kDa) centrifuged at 6,000 rpm for 1 h at 4 °C.

Escherichia coli (ATCC 8739) was cultured at three different temperatures: 20°C, 27°C and 37°C in essential M9 microbial growth medium consisting in Na₂HPO₄ (6.8 g/L), KH₂PO₄ (3 g/L), NH₄Cl (1 g/L) and NaCl (0.5 g/L). To M9 medium, a solution containing D-glucose (4 g/L), MgSO₄ (241 mg/L) and CaCl₂⋅2H₂O (15 mg/L) was added and the resulting solution was adjusted to pH = 7. Bacteria were cultured overnight until the culture reached an OD₆₀₀ of approximately 1. Lactobacillus rhamnosus LGG (ATCC 53103) (Dicoflor 60^®, DICOFARM) was cultured overnight in a De Man, Rogosa and Sharpe medium (SigmaAldrich, MRS broth, 51 g/L) at 37°C in anaerobic conditions up to an OD₆₀₀ of approximately 2. Aliquots (5 ml) of the bacterial cultures (both E. coli and L. rhamnosus) were retained for further analysis and to be used as controls.
To isolate either OMVs or MVs, bacteria were removed from culture media by centrifugation (Beckman Avanti J-25 centrifuge, JA-10 rotor, 6000 × g, 15 min), and the supernatant was filtered through a 0.45 μm filter unit (Sartorius). The supernatant, which contains vesicles, was concentrated by ultrafiltration (Vivaflow 200, Sartorius) up to small volume (50 mL), then filtered through a 0.45 μm filter.

Water (Table 1) was filtered over 0.22 µm polycarbonate membrane filters (293 mm diameter; Nuclepore; Whatman) to remove cellular organisms. Subsequently, viruses in the filtrate were concentrated successively using a spiral-wound ultra-filtration device suitable for large volumes (PTHK Prep/Scale-TFF; Millipore; Atlantic Ocean: 30 kDa molecular weight cutoff [MWCO]; Mediterranean Sea: 100 kDa MWCO) followed by a smaller ultra-filtration device (Vivaflow 200; 30 kDa MWCO; PES; Sartorius Stedim Biotech) until a final volume of ∼50 mL was reached. The virus concentrates were flash-frozen in liquid nitrogen and stored at −80°C until processed. Subsequently, virus concentrates were thawed and further concentrated to a final volume of 200 µL using Amicon Ultra-15 centrifugal ultra-filtration devices (Ultracel; 30 kDa MWCO; Millipore) by repeatedly loading ∼15 mL of concentrate onto the filters until the entire volume of each concentrate was passed through one filter. DNA extraction was performed using a QIAmp MinElute Virus Spin Kit (Qiagen) following the manufacturer's instructions. Viral metagenomic data sets were obtained from at least 100 ng of DNA per sample by pyro-sequencing on 1/4 of a pico-titer plate using Roche-454 GS FLX Titanium technology performed at the Broad Institute of MIT and Harvard (Cambridge, USA).

The extracellular domain of murine toll-like receptor 2 (aa 1–587) was fused to the human IgG1-Fc protein and expressed in HEK293 cells [21 (link)]. Following the concentration of the 5-L cell supernatant with a Vivaflow 200 (Sartorius) ultrafiltration cassette (50 kDa molecular weight cut-off), the TLR2 fusion protein was purified by protein A affinity chromatography, and the purity was verified by SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie staining.