The Archetypal Gamma-Core Motif of Antimicrobial Cys-Rich Peptides Inhibits H+-ATPases in Target Pathogens

Abstract

Human lactoferrin (hLf) is an innate host defense protein that inhibits microbial H⁺-ATPases. This protein includes an ancestral structural motif (i.e., γ-core motif) intimately associated with the antimicrobial activity of many natural Cys-rich peptides. Peptides containing a complete γ-core motif from hLf or other phylogenetically diverse antimicrobial peptides (i.e., afnA, SolyC, PA1b, PvD₁, thanatin) showed microbicidal activity with similar features to those previously reported for hLf and defensins. Common mechanistic characteristics included (1) cell death independent of plasma membrane (PM) lysis, (2) loss of intracellular K⁺ (mediated by Tok1p K⁺ channels in yeast), (3) inhibition of microbicidal activity by high extracellular K⁺, (4) influence of cellular respiration on microbicidal activity, (5) involvement of mitochondrial ATP synthase in yeast cell death processes, and (6) increment of intracellular ATP. Similar features were also observed with the BM2 peptide, a fungal PM H⁺-ATPase inhibitor. Collectively, these findings suggest host defense peptides containing a homologous γ-core motif inhibit PM H⁺-ATPases. Based on this discovery, we propose that the γ-core motif is an archetypal effector involved in the inhibition of PM H⁺-ATPases across kingdoms of life and contributes to the in vitro microbicidal activity of Cys-rich antimicrobial peptides.

Keywords: ATPase inhibitor; H+-ATPase; antimicrobial mechanism of action; antimicrobial motif; antimicrobial peptide; gamma-core motif; host defense; innate immunity; lactoferrin.

Figure 1

Cell viability and membrane permeabilization assays. C. albicans or P. aeruginosa (10⁶ cells/mL) were incubated (90 min, 37 °C) with each peptide/protein at the indicated concentrations. Aliquots were plated on SDA or TSB, and colonies were counted after 24–48 h (C. albicans) or 24 h (P. aeruginosa). At each point, samples were stained with propidium iodide (PI) and intracellular PI incorporation, indicative of membrane permeabilization, was quantified by flow cytometry. The relative cell viability (blue lines) and cellular permeabilization (red lines) were calculated as the percentage of untreated cells. Human lactoferrin (hLf) and the peptide Lfpep were used as negative and positive controls of cellular permeabilization, respectively. The results are the means of at least three independent experiments, and error bars represent standard deviations (±SD).

Figure 2

Potassium efflux induced by hLf γ-core-containing peptides. (A,D) Kinetics of K⁺ efflux. C. albicans or P. aeruginosa (10⁷ cells/mL) were incubated with kaliocin-1, Kdp15, human lactoferrin (hLf), and Lfpep. hLf and Lfpep were used as negative and positive controls of the membrane permeabilization, respectively. The effect of antifungal BM2 peptide was evaluated. The extracellular K⁺ concentration was quantified at different times. (B,C,E) Effect of extracellular K⁺ and TEA⁺ on microbicidal activity. (B,E) microbicidal activity was assessed in Tris buffer in the absence or in the presence of 50 mM KCl or (C) 10 mM TEA⁺. Cell viability was determined by plating aliquots of the cell suspensions. The percentage of viable cells was determined relative to that for cells incubated without peptides (control). Results are the mean ± SD from duplicates of at least three independent determinations. * p < 0.05, ** p < 0.01, and *** p < 0.001 were used.

Figure 3

Effect of cellular respiration on the microbicidal activity of hLf γ-core-containing peptides. (A,C) Oxygen consumption was measured in C. albicans and P. aeruginosa cellular suspensions in the absence (control) or the presence of kaliocin-1 (Kal-1), Kdp15, or human lactoferrin (hLf), and piericidin A (positive control). (B,D) Effect of cellular respiration on the microbicidal activity of hLf γ-core-containing peptides. Cells (10⁶ cells/mL) were pre-incubated (15 min, 37 °C) with 32 μM piericidin A and incubated for 90 min at 37 °C with the peptides hLf or BM2. The cell viability was determined by means of the plate counting method. The results are the mean ± SD from duplicates of at least three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 were used.

Figure 4

Effect of inhibition of mitochondrial ATP synthase on the candidacidal activity of hLf γ-core-containing peptides. Candidacidal effect of IC₅₀ of kaliocin-1 (Kal-1) and Kdp15 and 0.25 μM BM2 on cells (10⁶ cells/mL) pre-incubated without or with 16 μg/mL oligomycin A. Cell viability was determined by plating aliquots of the cell suspensions and the percentage of viable cells was determined relative to that for cells incubated without peptides. Human lactoferrin (hLf) and Lfpep were used as positive and negative controls, respectively. Data are the mean ± SD from at least three experiments. ** p < 0.01, and *** p < 0.001 were used.

Figure 5

Effect of hLf γ-core-containing peptides on PM H⁺-ATPases. (A) Effect of hLf γ-core-containing peptides on glucose-dependent external acidification. C. albicans cells (10⁷ cells/mL) in 50 mM KCl were preincubated with the peptides (2 × IC₅₀) for 20 min. Glucose (final concentration of 2.5 mM) was then added to induce H⁺ efflux via Pma1p H⁺-ATPase, as indicated by the subsequent external acidification. For clarity, only one set of three independent experiments and values measured every 5 min are represented. (B) Effect of hLf γ-core-containing peptides on intracellular ATP (iATP) levels. Measurement of iATP of C. albicans cells (B) cells in the presence of kaliocin-1 (Kal-1) or Kdp15. The PM H⁺-ATPases inhibitors, human lactoferrin (hLf), and the peptide BM2 were used as positive controls. The total cellular iATP concentrations were determined after 90 min of treatment. The mean data ± SD are indicated as the percent change compared to that for the untreated control sample.

Figure 6

Effect of different inhibitors on microbicidal activity of phylogenetically diverse γ-core-containing peptides. (A,D) Microbicidal effect of different γ-core-containing peptides. Cell suspensions (10⁶ cells/mL) in Tris buffer were incubated for 90 min at 37 °C with the peptides and cell viability was determined by a plating counting method. (B,E) Effect of different inhibitors on microbicidal activity. (C) Effect of different γ-core-containing peptides on glucose-dependent external acidification by C. albicans. For clarity, only one set of three independent experiments and values measured every 5 min are represented. (F) Effects of different γ-core-containing peptides on membrane integrity. Bars represent the percentage of propidium iodide (PI)-positive cells incubated with the peptides. The results are the mean ± SD from duplicates of at least three independent experiments.