Survival strategies of yeast and filamentous fungi against the antifungal protein AFP

Abstract

The activities of signaling pathways are critical for fungi to survive antifungal attack and to maintain cell integrity. However, little is known about how fungi respond to antifungals, particularly if these interact with multiple cellular targets. The antifungal protein AFP is a very potent inhibitor of chitin synthesis and membrane integrity in filamentous fungi and has so far not been reported to interfere with the viability of yeast strains. With the hypothesis that the susceptibility of fungi toward AFP is not merely dependent on the presence of an AFP-specific target at the cell surface but relies also on the cell's capacity to counteract AFP, we used a genetic approach to decipher defense strategies of the naturally AFP-resistant strain Saccharomyces cerevisiae. The screening of selected strains from the yeast genomic deletion collection for AFP-sensitive phenotypes revealed that a concerted action of calcium signaling, TOR signaling, cAMP-protein kinase A signaling, and cell wall integrity signaling is likely to safeguard S. cerevisiae against AFP. Our studies uncovered that the yeast cell wall gets fortified with chitin to defend against AFP and that this response is largely dependent on calcium/Crz1p signaling. Most importantly, we observed that stimulation of chitin synthesis is characteristic for AFP-resistant fungi but not for AFP-sensitive fungi, suggesting that this response is a successful strategy to protect against AFP. We finally propose the adoption of the damage-response framework of microbial pathogenesis for the interactions of antimicrobial proteins and microorganisms in order to comprehensively understand the outcome of an antifungal attack.

FIGURE 1.

AFP susceptibilities of the S. cerevisiae wild-type strain BY4741 and selected deletion mutants when cultivated in YPD medium in the presence of 400 μg/ml AFP. Growth is expressed as percentages compared with the negative control, which consisted of the strains cultivated in the absence of AFP. Error bars, S.D. for triplicate experiments.

FIGURE 2.

S. cerevisiae cell morphology and membrane integrity in the absence and presence of AFP. Cells were treated as described under “Experimental Procedures.” A, differential interference contrast micrographs of WT BY4741 and mutant strains. Bars, 10 μm. B, AFP-mediated membrane-permeabilizing effect on mutant and wild-type strains of S. cerevisiae using the SYTOX Green uptake assay. This dye can only enter plasma membranes when compromised, and previous work has shown that the degree of AFP activity can be correlated with the extent of fluorescence (13, 14).

FIGURE 3.

Cell wall remodeling of S. cerevisiae in response to AFP. Amount of chitin (A) and β-1,3-glucan (D) in mutants of S. cerevisiae in the absence and presence of 150 μg/ml AFP. The data are given relative to the chitin/glucan amount determined in the WT strain BY4741 in the absence of AFP (set as 100%). Error bars, S.D. for quadruple experiments. B, microscopic images of WT and chs1Δ strains stained with CFW. Pictures were taken using fixed exposure time (100 ms). The increase in CFW fluorescence intensity in the presence of AFP (400 μg/ml) reflects enhanced chitin levels at cell walls. Bar, 10 μm. C, plate sensitivity assay using 50 μg/ml CFW. Equivalent numbers of cells were serially diluted, and aliquots were spotted on YPD medium containing or lacking CFW. Plates were photographed after 3 days of incubation at 28 °C.

FIGURE 4.

Calcium-dependent cell wall remodeling in S. cerevisiae. A, BY4741 cells harvested from a logarithmically grown culture were treated with different amounts of AFP (0, 150, and 400 μg/ml) for 1 h, after which total RNA was isolated and analyzed. Transcript levels were quantified by densitometry using the 18 S rRNA signal for calibration. All values are expressed relative to the respective untreated control. Data from a representative experiment are shown. B and C, amount of chitin in S. cerevisiae strains in the absence or presence of 150 μg/ml AFP. The data are given relative to the chitin amount determined in the WT strain BY4741 in the absence of AFP (set as 100%). Error bars, S.D. for triplicate experiments. D, AFP sensitivity assay of BY4741 (using 150 μg/ml AFP) in the presence of 50 nm BAPTA or 10 nm FK506. These concentrations of BAPTA and FK506 were chosen because they are the maximum concentrations that do not inhibit growth of BY4741 (data not shown). The data are given relative to the growth of the WT strain BY4741 in the absence of AFP (set as 100%). Error bars, S.D. for quadruple experiments.

FIGURE 5.

Crz1p-dependent chitin response in S. cerevisiae. A, growth of BY4741 and derived mutants in the presence of 400 μg/ml AFP. Growth is expressed as percentages compared with the negative control, which consisted of the strains cultivated in the absence of AFP. Error bars, S.D. for triplicate experiments. B, amount of chitin in the chs1Δcrz1Δ double mutant (strain JPSc1.4) when cultivated in the absence or presence of 150 μg/ml AFP. As a control, a chs1Δ strain was used in which the URA3 selection marker, used to delete CRZ1 (see “Experimental Procedures”), was integrated heterologously into the genome of the chs1Δ strain (strain JPSc1.6). We used this strain as a reference to avoid artifacts due to nonmatching auxotrophies between mutant and reference strain (66). The data are given relative to the chitin amount determined in the WT strain BY4741 in the absence of AFP (set as 100%). Mean values of a duplicate experiment are given.

FIGURE 6.

Effects of stimulated calcium signaling on AFP susceptibility of A. niger. 10⁷ spores/ml of the WT strain N402 were allowed to germinate in YPD medium for 5 h, after which 100 mm of CaCl₂ were added (as a negative control, H₂O was added). After 3 or 6 h of further cultivation, germlings were washed twice with YPD and thereafter incubated in YPD supplemented without or with 10 μg/ml AFP. A, growth was assessed by measuring A₆₀₀ after 24 h and is expressed as percentages compared with the growth of N402 in the absence of AFP (set as 100%). Error bars, S.D. for triplicate experiments. B, Northern analysis of chsD gene expression after 6 h of calcium priming. 5 μg of total RNA from calcium treated and non-treated samples were hybridized with a chsD probe. Methylene blue-stained 18 S RNA confirmed equal loading.

FIGURE 7.

Cytotoxic activities of AMPs and the microbial response. A, the damage-response framework of microbial pathogenesis is reflected by a parabolic curve (–55). When translating this concept to the AMP-microorganism interaction, the y axis denotes microbial damage, which is defined as a perturbation of cell homeostasis. The microbial damage is a function of the microbial response, which, in terms of quality and quantity, can be considered as weak, appropriate, or (too) strong. Both a too weak and a too excessive response will damage or kill the microorganism. B, the csmB and the chsV mutation are protective for A. niger and F. oxysporum, respectively, whereby the latter makes F. oxysporum insensitive against AFP. With a question mark we denote the possible scenario in which a signaling pathway meant to protect the microorganisms causes self-damage when deregulated (e.g. increased cytoplasmic calcium concentrations might induce apoptosis). C, the curve in C is more flattened compared with B to indicate that S. cerevisiae is in general less susceptible to AFP than filamentous fungi. Plotted are different S. cerevisiae deletion mutants, which we observed to enhance or diminish AFP-induced damage based on their chitin response. Most importantly, the damage-response framework also opens the possibilities to include the paradoxical effect of improved growth in the presence of AFP, which we have observed for three S. cerevisiae mutants (Table 1, group F).

FIGURE 8.

Proteins and signaling pathways of S. cerevisiae involved in AFP counteraction. Proteins that are of major importance for the defense against AFP are indicated in boldface type, and proteins that contribute to a lesser extent are given in italics. Proteins that were not included in the screening assay (e.g. because of essential cell functions) are indicated with a question mark. The interconnection of proteins into signaling networks is based on Refs. , , and 36).