Cytosolic Acidification Is the First Transduction Signal of Lactoferrin-induced Regulated Cell Death Pathway

Abstract

In yeast, we reported the critical role of K⁺-efflux for the progress of the regulated cell death (RCD) induced by human lactoferrin (hLf), an antimicrobial protein of the innate immune system that blocks Pma1p H⁺-ATPase. In the present study, the K⁺ channel Tok1p was identified as the K⁺ channel-mediating K⁺-efflux, as indicated by the protective effect of extracellular K⁺ (30 mM), K⁺-channel blockers, and the greater hLf-resistance of TOK1-disrupted strains. K⁺-depletion was necessary but not sufficient to induce RCD as inferred from the effects of valinomycin, NH₄Cl or nigericin which released a percentage of K⁺ similar to that released by lactoferrin without affecting cell viability. Cytosolic pH of hLf-treated cells decreased transiently (0.3 pH units) and its inhibition prevented the RCD process, indicating that cytosolic acidification was a necessary and sufficient triggering signal. The blocking effect of lactoferrin on Pma1p H⁺-ATPase caused a transitory decrease of cytosolic pH, and the subsequent membrane depolarization activated the voltage-gated K⁺ channel, Tok1p, allowing an electrogenic K⁺-efflux. These ionic events, cytosolic accumulation of H⁺ followed by K⁺-efflux, constituted the initiating signals of this mitochondria-mediated cell death. These findings suggest, for the first time, the existence of an ionic signaling pathway in RCD.

Keywords: Candida albicans; apoptosis-like; cell signaling pathway; cytosolic acidification; lactoferrin; potassium efflux; regulated cell death.

Figure 1

Influence of extracellular factors on lactoferrin-induced regulated cell death. C. albicans cells (10⁵ cells/mL) were incubated with 5 μM lactoferrin (hLf) for 90 min at 37 °C in the presence of (A) different extracellular K⁺ concentrations in Tris buffer (circles) or potassium phosphate buffer (PPB, squares); (B) different ionic strength calculated in the presence of KCl (solid circles) and LiCl (open circles) solutions; or (C) equivalent osmolarity values of KCl (circles) or sorbitol (triangles) solutions. The cell viability was determined by a plate-count method. (D) Effect of extracellular K⁺ on the interaction of lactoferrin with the H⁺-ATPase. The H⁺-extrusion mediated by the plasma membrane Pma1p H⁺-ATPase of C. albicans cells suspended in 30 mM KCl (squares) or 50 mM KCl (circles) was determined in the presence (red lines) or in the absence (black lines, control) of lactoferrin by monitoring glucose-induced acidification of the external medium. The results are the means ± SD from duplicates of at least three independent experiments. In Figure 1D, only the mean data (n = 3) are shown, and the bars representing standard errors (coefficient of variation of <10%) are omitted for clarity.

Figure 2

Identification of K⁺-channel involved in the lactoferrin-induced RCD process. (A) Viability of yeast cells (10⁵ cells/mL) suspended in Tris buffer and treated with 5 μM lactoferrin (hLf, red column) or pre-incubated for 15 min at 37 °C with the K⁺-channel inhibitors tetraethylammonium (10 mM TEA; gray column) or Ba²⁺ (10 µM BaCl₂; white column) before the addition of 5 μM hLf. (B) Potassium efflux measured from non-treated cells (NT) assayed under the above conditions (black column; negative control), treated with hLf alone (red column) or pre-incubated with 10 mM TEA (gray column) or 10 µM BaCl₂ (white column) before the addition of lactoferrin. Nystatin (100 μg/mL) was used as a positive control of K⁺ released by permeabilized cells (dashed column). (C) Antifungal activity of three concentrations of hLf on C. albicans TOK1-disrupted strains. The strains (10⁵ cells/mL) were incubated for 90 min at 37 °C with 1.25 μM (white column), 2.5 μM (gray column), or 5 μM (black column) of lactoferrin. All the percentages of viability and K⁺ released correspond to cells suspended in Tris buffer and incubated with hLf for 90 min at 37 °C. The cell viability was determined by a plate-count method. The results are the means ± SD from duplicates of at least three independent experiments. Statistical significance was assessed by Student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Effect of different K⁺-depletion inducers on cell viability. (A) Antifungal activity of lactoferrin and valinomycin. The cells (10⁵ cells/mL) were incubated in Tris buffer for 90 min at 37 °C with 5 µM lactoferrin (hLf, red column) or 50 µg/mL valinomycin (Val; green column) and cell viability was calculated at 24 h and 48 h. (B) Time-course of K⁺ release induced by lactoferrin and valinomycin. The K⁺ released was measured at different times after the addition of 20 µM lactoferrin (red line), 50 µg/mL valinomycin (green line), or 100 µg/mL nystatin (black line, positive control) to cell suspensions (10⁷ cells/mL). (C) Effect of NH₄Cl on viability of lactoferrin-treated cells. The time-kill curve of cells (10⁵ cells/mL), pre-incubated in the presence of 10 mM NH₄Cl and treated with (blue line) or without 5 µM hLf (purple line) to determine cell viability. Lactoferrin-treated cells in the absence of NH₄Cl (red line) were used as control. (D) Time-course of K⁺-release in the presence of NH₄Cl. Cell suspensions were treated with (blue line) or without 20 µM hLf (purple line) in the presence of 10 mM NH₄Cl. The K⁺ release was determined at different times of the incubation period performed at 37 °C. Nystatin (100 µg/mL) was used as positive control in the measurements of released K⁺. Each value shown is the mean ± SD from duplicates of at least three independent experiments.

Figure 4

Effect of lactoferrin on cytosolic pH. (A) Transient cytosolic acidification induced by lactoferrin. Cytosolic pH of C. albicans-adapted pHluorin cells (black line, control), exposed to 5 µM lactoferrin (red line) or 25 µM diethylstilbestrol (DES, blue line), was determined at different times. These inhibitors of fungal Pma1p H⁺-ATPase were added to the cell suspensions at the indicated time (arrow). Cytosolic pH values were derived from fluorescence intensity measurements at different times over 15 min and calculated according to the calibration curve. (B) Calibration curve showing measured ratio of fluorescence intensity at 405 nm to intensity at 485 nm versus pH of pHluorin-expressing permeabilized cells equilibrated in buffers of increasing pH. Data are shown as means ± SD from duplicates of at least three independent experiments. (C) The normal activity of Pma1p H⁺-ATPase of cytoplasmic membrane (CM) generates a proton gradient (ΔpH) and an electrical (ΔΨ) gradient (negative inside) indicated by dashed lines. Cytosolic K⁺ is retained by the negative electrical charge. (D) The figure shows the sequence of linked events induced by lactoferrin (i.e., cytosolic acidification and K⁺-efflux). The blocking effect of lactoferrin on Pma1p H⁺-ATPase (1) causes a transient accumulation of protons in cytosol, detected as cytosolic acidic pH shift (2), and the subsequent loss of electrochemical gradient (3). As a consequence, the voltage-dependent channel Tok1p opens upon plasma membrane depolarization (4) allowing K⁺-efflux (5).

Figure 5

Proposed model of the RCD signaling pathway induced by lactoferrin. The scheme includes hypothetical steps (gray boxes) of this process based on our previously reported data [2,4,32,34]. In yeast cells, Pma1p H⁺-ATPase actively extrudes H⁺ out the cell and generates a proton gradient (ΔpH) and an electrical gradient (ΔΨ) which allow pH regulation and the flux of other ions and nutrients across the plasma membrane (PM). The here proposed cell death signaling pathway includes a sequence of ionic events in the cytosol as response to an extracellular signal, as follows: (1) Lactoferrin (hLf, first messenger) binds to the plasma membrane Pma1p H⁺-ATPase (receptor) blocking this proton pump [4]. (2) Protons are transitorily accumulated in the cytosol, an event detected as a cytosolic acidic pH shift (second messenger-like signal), inducing a ΔΨ-decrease. (3) This PM-depolarization opens the voltage-gated K⁺ channel Tok1p, allowing a passive and electrogenic K⁺-efflux [2,32,34]. (4) Hypothetically, both cytosolic K⁺ depletion and transient cytosolic acidification could favor in turn the loss of mitochondrial potassium, probably via a K⁺/H⁺ exchanger, and the simultaneous entry of H⁺ to the mitochondrial matrix, causing a loss of ΔΨ_m, as reported in [2]. The H⁺-influx to the matrix, via ATP synthase, is an essential event because inhibition of either electron transfer chain (ETC) or ATP synthase prevented the progress of this cell death pathway [2,4]. (5) The supposed perturbation of the mitochondrial homeostasis could induce further events previously visualized as phenotypic apoptotic markers [2]. Events marked with (?) are hypothetical steps that are being studied.