Addition. The presence of Trp in Staphylococcus culturesonly partially restored their

Addition. The Tenofovir alafenamide supplier presence of Trp in Staphylococcus culturesonly partially restored their growth. However, MSSA could grow in Trp-depleted medium if Phe was present, while CNS growth was only restored by the presence of both amino acids. Thus, these three strains were variably auxotrophic for Phe and Trp. We next explored the role of the three catabolites of the enzymatic reaction (Figure 2B, C, D). Phenylpyruvate was weakly toxic at high concentrations (10 mM). In contrast, H2O2 totally inhibited the growth of the four MedChemExpress GR79236 bacterial strains at 1 mM, despite their endogenous catalase production. This effect was abolished by the addition of glutathione in the culture media. Ammonium (NH4+) is considered as a source of nitrogen for bacterial growth. However, addition of the basic compound ammonia (NH3) to the E. coli and staphylococci cultures displayed a toxic effect at doses 5 mM, which was reverted by buffering the media with HEPES. We verified if combining all three IL4I1 catabolites could amplify their individual effect. Isomolar amounts of phenylpyruvate, H2O2 and NH3 were added to a culture medium lacking HEPES but containing minimal amounts of glutathione, Trp and Phe (Figure 3). Under these conditions, the growth of E.coli and MSSA was sensitive to doses 10 times lower than when H2O2 was used alone. A similar effect was obtained by mixing H2O2 and NH3 only (Figure S3). Moreover, the inhibition of bacterial growth was lost in a HEPES-buffered medium containing the three catabolites (Figure S4), suggesting that NH3 potentiates the toxic effect of H2O2 via basification of the medium. This was confirmed using NaOH as basificating agent. Indeed, E coli was sensitive to ten times less H2O2 when the pH was increased from 7.5 to 8.5, an increase that can also be obtained with 5 mM NH3 and that is not toxic by itself (Figure S5). In summary, both amino acid depletion and the presence of phenylpyruvate, H2O2 or NH3 affected, to variable degrees, bacterial growth (Table 1). Moreover, the presence of equimolar quantities of H2O2 and NH3 from the IL4I1 reaction could have a cumulative effect, indicating that their simultaneous production by the enzyme may amplify its antibacterial properties. In order to confirm these putative mechanisms of IL4I1 action, we added Phe, Trp, glutathione, and HEPES, either separately or in combination, to bacterial cultures grown in conditioned media containing or not IL4I1. While IL4I1 was still toxic for the bacteria after the addition of a large excess (0.5 mg/ml) of Phe orFigure 1. IL4I1 inhibitory growth effect. (A) IL4I1 activity in conditioned medium from THP1 and THP1-IL4I1 cells (0.56106 cells/ml cultured in DMEM/F12+1 FCS, 48 hours at 37uC). Results are expressed as 23977191 pmol H2O2 produced in the presence of phenylalanine per hour per ml of conditioned medium. (B) Bacteria were diluted in conditioned medium (CM) from THP1 containing different ratios of IL4I1-conditioned medium. After 24 hours, bacterial growth was monitored at an OD of 595 nm. Data are given as mean 6 SEM from five independent experiments performed in duplicate. Mean OD in 100 CM THP1 was 0.227 for E. coli, 0.266 for B2599, 0.450 for MSSA and 0.516 CNS, respectively. These OD were considered as 100 bacterial growth and the OD measured in the other culture conditions were calculated as percentages with respect to this control. Statistical analyses were performed using the Mann-Whitney test in comparison to 100 CM THP1. ***p,0.001 for all the b.Addition. The presence of Trp in Staphylococcus culturesonly partially restored their growth. However, MSSA could grow in Trp-depleted medium if Phe was present, while CNS growth was only restored by the presence of both amino acids. Thus, these three strains were variably auxotrophic for Phe and Trp. We next explored the role of the three catabolites of the enzymatic reaction (Figure 2B, C, D). Phenylpyruvate was weakly toxic at high concentrations (10 mM). In contrast, H2O2 totally inhibited the growth of the four bacterial strains at 1 mM, despite their endogenous catalase production. This effect was abolished by the addition of glutathione in the culture media. Ammonium (NH4+) is considered as a source of nitrogen for bacterial growth. However, addition of the basic compound ammonia (NH3) to the E. coli and staphylococci cultures displayed a toxic effect at doses 5 mM, which was reverted by buffering the media with HEPES. We verified if combining all three IL4I1 catabolites could amplify their individual effect. Isomolar amounts of phenylpyruvate, H2O2 and NH3 were added to a culture medium lacking HEPES but containing minimal amounts of glutathione, Trp and Phe (Figure 3). Under these conditions, the growth of E.coli and MSSA was sensitive to doses 10 times lower than when H2O2 was used alone. A similar effect was obtained by mixing H2O2 and NH3 only (Figure S3). Moreover, the inhibition of bacterial growth was lost in a HEPES-buffered medium containing the three catabolites (Figure S4), suggesting that NH3 potentiates the toxic effect of H2O2 via basification of the medium. This was confirmed using NaOH as basificating agent. Indeed, E coli was sensitive to ten times less H2O2 when the pH was increased from 7.5 to 8.5, an increase that can also be obtained with 5 mM NH3 and that is not toxic by itself (Figure S5). In summary, both amino acid depletion and the presence of phenylpyruvate, H2O2 or NH3 affected, to variable degrees, bacterial growth (Table 1). Moreover, the presence of equimolar quantities of H2O2 and NH3 from the IL4I1 reaction could have a cumulative effect, indicating that their simultaneous production by the enzyme may amplify its antibacterial properties. In order to confirm these putative mechanisms of IL4I1 action, we added Phe, Trp, glutathione, and HEPES, either separately or in combination, to bacterial cultures grown in conditioned media containing or not IL4I1. While IL4I1 was still toxic for the bacteria after the addition of a large excess (0.5 mg/ml) of Phe orFigure 1. IL4I1 inhibitory growth effect. (A) IL4I1 activity in conditioned medium from THP1 and THP1-IL4I1 cells (0.56106 cells/ml cultured in DMEM/F12+1 FCS, 48 hours at 37uC). Results are expressed as 23977191 pmol H2O2 produced in the presence of phenylalanine per hour per ml of conditioned medium. (B) Bacteria were diluted in conditioned medium (CM) from THP1 containing different ratios of IL4I1-conditioned medium. After 24 hours, bacterial growth was monitored at an OD of 595 nm. Data are given as mean 6 SEM from five independent experiments performed in duplicate. Mean OD in 100 CM THP1 was 0.227 for E. coli, 0.266 for B2599, 0.450 for MSSA and 0.516 CNS, respectively. These OD were considered as 100 bacterial growth and the OD measured in the other culture conditions were calculated as percentages with respect to this control. Statistical analyses were performed using the Mann-Whitney test in comparison to 100 CM THP1. ***p,0.001 for all the b.