The antifungal activity of the Penicillium chrysogenum protein PAF disrupts calcium homeostasis in Neurospora crassa

Abstract

The antifungal protein PAF from Penicillium chrysogenum exhibits growth-inhibitory activity against a broad range of filamentous fungi. Evidence from this study suggests that disruption of Ca(2+) signaling/homeostasis plays an important role in the mechanistic basis of PAF as a growth inhibitor. Supplementation of the growth medium with high Ca(2+) concentrations counteracted PAF toxicity toward PAF-sensitive molds. By using a transgenic Neurospora crassa strain expressing codon-optimized aequorin, PAF was found to cause a significant increase in the resting level of cytosolic free Ca(2+) ([Ca(2+)](c)). The Ca(2+) signatures in response to stimulation by mechanical perturbation or hypo-osmotic shock were significantly changed in the presence of PAF. BAPTA [bis-(aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid], a Ca(2+) selective chelator, ameliorated the PAF toxicity in growth inhibition assays and counteracted PAF induced perturbation of Ca(2+) homeostasis. These results indicate that extracellular Ca(2+) was the major source of these PAF-induced effects. The L-type Ca(2+) channel blocker diltiazem disrupted Ca(2+) homeostasis in a similar manner to PAF. Diltiazem in combination with PAF acted additively in enhancing growth inhibition and accentuating the change in Ca(2+) signatures in response to external stimuli. Notably, both PAF and diltiazem increased the [Ca(2+)](c) resting level. However, experiments with an aequorin-expressing Deltacch-1 deletion strain of N. crassa indicated that the L-type Ca(2+) channel CCH-1 was not responsible for the observed PAF-induced elevation of the [Ca(2+)](c) resting level. This study is the first demonstration of the perturbation of fungal Ca(2+) homeostasis by an antifungal protein from a filamentous ascomycete and provides important new insights into the mode of action of PAF.

Fig. 1.

Growth of N. crassa wt after 24 h of incubation in Vogel's* medium containing 1 μg/ml or 10 μg/ml PAF and supplemented with increasing concentrations of CaCl₂. Percent values were calculated from percent changes in OD₆₂₀ values of PAF-treated N. crassa cells compared to untreated controls (set at 100%). Results are expressed as mean ± SD (n = 5).

Fig. 2.

Increase in resting [Ca²⁺]_c of 6-h-old N. crassa germlings treated with 10 μg/ml or 100 μg/ml PAF. Before protein application, the [Ca²⁺]_c resting level was determined for all wells by obtaining the average of three measurements of [Ca²⁺]_c over a 2-min period. PAF was then added, and measurements were taken every minute. Untreated samples served as controls. The SD (n = 6) was less than 10% of the values presented.

Fig. 3.

Viability staining of networks of fused N. crassa (6 h old) germlings with fluorescein diacetate and propidium iodide. (A) The germlings were treated for 12 min (this was the time of pretreatment used prior to mechanical stimulation in the experiments involving aequorin-based calcium measurement) with (100 μg/ml) PAF in the presence of fluorescein diacetate and propidium iodide. The fungal cells are all alive because they are fluorescing green (fluorescein diacetate detects living cells), and there is no red fluorescence evident (propidium iodide is taken up into the cell only when the plasma membrane has been permeabilized). (B) Control in which a network of germlings was pretreated with (70%) ethanol (EtOH) for 40 min and stained with fluorescein diacetate and propidium iodide. Ethanol permeabilizes the plasma membrane and kills the cells. All of the germlings fluoresce red with propidium iodide, and no green fluorescence is evident.

Fig. 4.

Effects of PAF on the [Ca²⁺]_c responses to mechanical perturbation (A) and hypo-osmotic shock (B). Six-hour-old N. crassa germlings were treated with 10 μg/ml or 100 μg/ml PAF for 60 min before stimulation by mechanical perturbation (addition of 100 μl of Vogel's medium) or hypo-osmotic shock (addition of 5% Vogel's medium). The [Ca²⁺]_c signature was monitored for 12 min. The SD (n = 6) was less than 10% of the values presented. The histogram in panel C shows in detail the effects of a 60-min pretreatment with 10 μg/ml and 100 μg/ml PAF on the amplitude of the [Ca²⁺]_c responses to mechanical perturbation and hypo-osmotic shock.

Fig. 5.

Effect of the extracellular chelator BAPTA on the [Ca²⁺]_c resting level (A) and hyphal growth in the presence or absence of PAF (B). (A) BAPTA (10 mM final concentration) was applied 10 min before treatment with 100 μg/ml PAF (▪) or after 10 min of PAF treatment (▾). Samples without supplements were used as controls (○). The SD (n = 6) was less than 10% of the values presented. (B) Conidia of N. crassa were incubated with 10 μg/ml PAF, 1 mM BAPTA, or both chemicals combined. Percent values were calculated from percent changes in OD₆₂₀ values of PAF-treated N. crassa compared with untreated controls (set as 100%). Results are expressed as means ± SDs (n = 5).

Fig. 6.

Effects of the L-type channel blocker diltiazem and PAF on the [Ca²⁺]_c resting level. (A) Time-dependent effects on the [Ca²⁺]_c resting level after the substances were added at time zero. (B) The effect of the extracellular chelator BAPTA on germlings pretreated for 15 min with PAF, diltiazem, or both substances combined. In each experiment, final concentrations of 100 μg/ml PAF, 0.5 mM diltiazem, and 10 mM BAPTA were used. Untreated germlings were used as controls. Values represent averages of six wells; the SD was less than 10%.