PapRIV, a BV-2 microglial cell activating quorum sensing peptide

Quorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteria to communicate with their peers in a cell-density dependent manner. These peptides do not only act as interbacterial communication signals, but can also have effects on the host. Compelling evidence demonstrates the presence of a gut-brain axis and more specifically, the role of the gut microbiota in microglial functioning. The aim of this study is to investigate microglial activating properties of a selected QSP (PapRIV) which is produced by Bacillus cereus species. PapRIV showed in vitro activating properties of BV-2 microglia cells and was able to cross the in vitro Caco-2 cell model and reach the brain. In vivo peptide presence was also demonstrated in mouse plasma. The peptide caused induction of IL-6, TNFα and ROS expression and increased the fraction of ameboid BV-2 microglia cells in an NF-κB dependent manner. Different metabolites were identified in serum, of which the main metabolite still remained active. PapRIV is thus able to cross the gastro-intestinal tract and the blood–brain barrier and shows in vitro activating properties in BV-2 microglia cells, hereby indicating a potential role of this quorum sensing peptide in gut-brain interaction.

Once the synthesis of the peptide sequence was completed, the Fmoc group was removed and the peptidyl resin was treated with trifluoroacetic acid (TFA)/H2O/triisopropylsilane (TIS) (95:2.5:2.5) for 2 h. Following TFA evaporation and diethyl ether extraction, the peptide was dissolved in H2O and analyzed by HPLC. Finally, peptides were purified using a CombiFlash instrument, lyophilized, analyzed by HPLC and characterized by mass spectrometry.
Once the synthesis of the peptide sequence was completed, the Fmoc group was removed and the peptidyl resin was treated with TFA/H2O/TIS (95:2.5:2.5) for 2 h. Following TFA evaporation and diethyl ether extraction, the peptide was dissolved in H2O and analyzed by HPLC. Finally, peptides were purified using a CombiFlash instrument, lyophilized, analyzed by HPLC and characterized by mass spectrometry.

Synthesis of the scrambled peptide DEHSFLP
This peptide was synthesized manually by the solid-phase method following standard Fmoc chemistry as described for peptide SDLPFEA. After the coupling of the second amino acid, Fmoc-Leu-OH, the Fmoc group removal was achieved with tetrabutylammonium fluoride (TBAF)/ethanol/DMF (20 nM:2%:10 mL, 1 × 5 min) followed by washing the resin with DMF (6 × 10 sec) and CH2Cl2 (2 × 10 sec). The completion of the coupling was checked with the ninhydrin test [1]. Peptide elongation, cleavage and purification were then performed as described for peptide SDLPFEA.

Supplementary method S2: UPLC-MS parameters Caco-2 assay
Aliquots of the acceptor compartment are analyzed using the following validated UPLC-MS/MS method.

Preparation of Krebs-Henseleit buffer (pH 7.4) (KH-buffer)
The powdered medium was dissolved in 900 mL water while stirring. To this solution, 0.3790 g CaCl2×2H2O and 2.098 g NaHCO3 was subsequently added while stirring. NaOH or HCl was used to adjust to pH 7.4. This solution was then further diluted to 1000 mL using ultrapure water.
Preparation of tissue homogenates -The liver (n = 1), kidneys (n = 6), brains (n = 3), colons (n = 4) and faeces (0.196 g) of ICR-CD-1 mice were collected. -After cleaning and rinsing the tissues using ice-cold KH buffer, the tissues were cut in little pieces and transferred into a 50 mL tube to which 36 mL ice-cold KH buffer was added. -The tissues were then homogenized with at tissue homogenizer.
-After the larger particles were allowed to settle for about 30 minutes at 5°C, approximately 25 mL of the middle layer was transferred into a 50 mL Falcon tube using a plastic pipette. -After shaking the homogenates, the homogenates were dispensed into 2 mL Eppendorf tubes, and stored at -35°C until use. -The protein content of the homogenates were determined using the Pierce Modified Lowry Protein Assay method. -Just before use, the homogenates were diluted to a protein concentration of 0.6 mg/mL (i.e. 300 µg / 500 µL) with KH buffer.
Sample preparation -Into a 1.5 ml Protein LoBind tube, 900 µl of serum/tissue extract (500 µl of homogenate/serum and 400 µl of KH buffer, pH 7.4) was transferred. -After temperature-equilibration at 37°C for 15 min while shaking at 750 rpm, 100 µl of peptide solution (0.1 mg/mL) was added followed by continued incubation at 37°C (while shaking), with 6×100 µl aliquots taken after 0, 5, 10, 30 and 60 minutes. -The aliquots were immediately transferred into 500 µl Protein LoBind Eppendorf tubes containing 100 µl of 1% V/V trifluoroacetic acid solution in water, heated for 5 minutes at 95°C, and cooled for 30 minutes in an ice-bath. -The samples were then centrifuged at 16,000 g for 5 minutes at 5°C. -The supernatants was then analyzed by UPLC-UV/MS.

Supplementary method S4: UPLC-UV/MS parameters ex vivo metabolization
The analytical parameters of the UPLC-UV/MS method for the ex vivo metabolization are given in Table 3. The gradient program for the peptide is given in Table 4.