Caffeine and Other Methylxanthines as Interceptors of Food-Borne Aromatic Mutagens: Inhibition of Trp-P‑1 and Trp-P‑2 Mutagenic Activity

Caffeine is one of the most important biologically active food components. In this article, we demonstrate that caffeine and other methylxanthines significantly reduce the mutagenic activity of two food-derived heterocyclic aromatic amines, Trp-P-1 and Trp-P-2 in the <i>Salmonella typhimurium</i> TA98 strain. Moreover, protection against Trp-P-1-induced mutagenicity was independent of liver S9 enzymatic fraction, suggesting that mechanisms other than modulation of mutagen bioactivation can contribute to the observed protective effects. UV–vis spectroscopy and computational studies revealed that methylxanthines intercept Trp-P-1 and Trp-P-2 in noncovalent molecular complexes, with association constants (<i>K</i>_AC) in the 10² M^–1 range. Enthalpy values (Δ<i>H</i> about −30 kJ·mol^–1) of mutagen–methylxanthine heterocomplexation obtained microcalorimetrically correspond to stacking (π–π) interactions. Finally, we demonstrated that the biological activity of Trp-P-1 and Trp-P-2 is strictly dependent on the presence of the mutagen in a free (unbound with methylxanthine) form, suggesting that mutagen sequestration in stacking heterocomplexes with methylxanthines can decrease its bioavailability and diminish its biological effects

Data_Sheet_1_Molecular characterization of the PhiKo endolysin from Thermus thermophilus HB27 bacteriophage phiKo and its cryptic lytic peptide RAP-29.docx

IntroductionIn the era of increasing bacterial resistance to antibiotics, new bactericidal substances are sought, and lysins derived from extremophilic organisms have the undoubted advantage of being stable under harsh environmental conditions. The PhiKo endolysin is derived from the phiKo bacteriophage infecting Gram-negative extremophilic bacterium Thermus thermophilus HB27. This enzyme shows similarity to two previously investigated thermostable type-2 amidases, the Ts2631 and Ph2119 from Thermus scotoductus bacteriophages, that revealed high lytic activity not only against thermophiles but also against Gram-negative mesophilic bacteria. Therefore, antibacterial potential of the PhiKo endolysin was investigated in the study presented here.MethodsEnzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3′-Dipropylthiacarbocyanine iodide (DiSC3(5)).Results and discussionThe PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7–7.1 log units and reaching the minimum inhibitory concentration values in the range of 2–31 μM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously.</p