The antimicrobial activity of chemerin-derived peptide p4 requires oxidative conditions

Abstract

Figure 3. p4 exhibits rapid concentration-dependent lytic activity against E. coli. A, E. coli HB101 was incubated with the indicated concentrations of p4 for 2 h. Cell viability was analyzed by MDA assay. n ϭ 3, mean Ϯ S.D. B, E. coli HB101 was incubated with 100 M p4 or vehicle for the indicated times. Cell viability was analyzed by MDA assay, n ϭ 3; mean Ϯ S.D. C, human erythrocytes were incubated with 1% Triton X-100, the indicated concentration of p4, or vehicle for 2 h. Hemolysis of erythrocytes is shown relative to lysis caused by Triton X-100. n ϭ 3, mean Ϯ S.D. D, E. coli HB101 was incubated with 100 M p4 or vehicle for the indicated times. Bacterial morphology was assessed by TEM. E, E. coli HB101 was incubated with 100 M p4 for 5 min. Alterations in bacterial permeability were visualized by fluorescence imaging. Bacteria were treated with FITC-labeled p4 (green), stained with PI (red), and counterstained with Hoechst to visualize DNA (blue). Arrows point to accumulation of p4 at the cell surface. F, ␤-galexpressing E. coli JM83 was incubated with the indicated concentrations of p4 for 15 min. The ␤-gal activity present in supernatants of p4-treated bacteria is shown as a percentage of the vehicle-treated bacteria. n ϭ 3, mean Ϯ S.D. G, E. coli HB101 was treated with p4 for 45 min, followed by TEM. Arrows and asterisks indicate outer membrane perturbations and the discontinuous inner membrane, respectively. H, intracellular localization of p4 is shown by immunogold labeling. E. coli HB101 was treated with biotin-p4 or p4 as a control, fixed, and stained with mouse anti-biotin Abs, followed by anti-mouse Abs conjugated to gold particles. Arrowheads indicate gold particles. The enlarged image (i) demonstrates interaction of p4 with the cell membrane. ***, p Ͻ 0.001; **, p Ͻ 0.01; *, p Ͻ 0.05 by Kruskal-Wallis one-way ANOVA with post hoc Dunn's test. TEM and fluorescence microscopy images are from one experiment and are representative of at least three experiments.

Bacteria Modify Their Sensitivity to Chemerin-Derived Peptides by Hindering Peptide Association With the Cell Surface and Peptide Oxidation

“…Changes in cytoplasmic ATP levels in response to p4 were at least partly dependent on electron transport chain (ETC), since R. capsulatus deficient in one of its key ETC enzymes, cytochrome bc 1 (mutant MT-RBC1 KO), was less sensitive to sublethal and lethal p4 levels ( Figure 4B). These data further support our previous findings, that p4 restricts the growth of R. capsulatus in association with cytochrome bc 1 activity, which has an ability to facilitate the formation of antimicrobial p4 dimers via p4 oxidation (Godlewska et al, 2019).…”

“…The prophylactic potential of p4 against skin infection with a variety of microorganisms, including MRSA infection, and the therapeutic efficacy of p4 in treating experimental S. aureus skin infections [(Godlewska et al, 2019) and Table 2] could form the basis of potential new drugs for the prevention/treatment of skin infections, independent of the endogenous abundance of p4 in vivo. Here, we show that p4 is likely to target multiple bacterial components, including the cell wall, cell membrane, and chromosome.…”

“…Since P4 is not uniformly bactericidal, it is possible that bacteria may employ countermeasures to ameliorate the damaging effects of p4. We showed that an oxidative environment can strongly augment p4 antimicrobial activity by supporting the formation of disulfide bonds leading to potent antimicrobial p4 dimers compared to barely active p4 monomers (Godlewska et al, 2019). To test whether bacteria themselves can modulate antimicrobial activity of p4 by oxidizing the peptide, we employed highly p4-sensitive Gram negative bacteria, Rhodobacterium capsulatus (R. capsulatus) (MIC = 5 µM) (Godlewska et al, 2019).…”

Rola chemeryny i jej antybakteryjnych pochodnych w utrzymaniu funkcji barierowych nabłonka

“…Taka modyfikacja, znana pod pojęciem konserwatywnej substytucji aminokwasów gwarantuje zachowanie identycznych parametrów fizykochemicznych cząsteczki. Na podstawie tych danych można stwierdzić, że oba parametry, silny ładunek dodatni wraz z wysoką amfipatycznością peptydu są konieczne do zapewnienia pełnej aktywności antybakteryjnej p4 [23].…”

“…Spośród 20 aminokwasów składających się na peptyd p4, 13 reszt aminokwasowych pozostaje niezmiennych, natomiast w przypadku jednej reszty obserwuje się konserwatywne podstawienie aminokwasów. Co istotne, ta wspomniana konserwatywność dotyczy wielu reszt aminokwasowych istotnych z punktu widzenia aktywności antybakteryjnej peptydu [23].…”