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.
Highlights
Est is the exploitation of antimicrobial peptides (AMPs),2 which are natural products of pro- and eukaryotic organisms and function as defense molecules to combat nutrient competitors, colonizers or invaders [3, 4]
This set of strains included mutants affected in cell wall and plasma membrane assembly and mutants disturbed in signaling processes, such as cell wall integrity (CWI) signaling, calcium signaling, high osmolarity glycerol signaling, protein kinase A (PKA) signaling, and TOR signaling, known to be important to fortify and preserve the yeast cell wall [17, 21, 36]
Because for several yeast systems it has been reported that an elevated cell wall chitin content very often correlates with hypersensitivity to the chitin antagonist Calcofluor white (CFW) (36 –38), we wished to determine whether there is a general relationship between chitin levels in S. cerevisiae and susceptibility to AFP
Summary
Est is the exploitation of antimicrobial peptides (AMPs), which are natural products of pro- and eukaryotic organisms and function as defense molecules to combat nutrient competitors, colonizers or invaders [3, 4]. Most of them are derived from precursors, are small in size (Ͻ100 amino acids with frequent use of glycine, alanine, lysine, and cysteine), display cationic and amphipathic properties, and resist proteolytic degradation due to stabilizing disulfide bonds [2, 4, 5] Their positive net charge attracts them electrostatically to negatively charged cell surfaces of microorganisms, where hydrophobic and/or receptor-based interactions allow them to bind, traverse, or permeabilize biological membranes [4, 7, 8]. Fungal Survival Strategies against AFP cell membrane integrity of AFP-sensitive filamentous fungi, it is able to bind to chitin and nucleic acids under in vitro conditions [13] This loss of bioactivity implies that the primary inhibitory effect of AFP is exerted at the cell surface, where a potential target(s) of AFP resides. By means of screening mutants of an AFP-resistant fungus for AFP-sensitive phenotypes, it may be possible to spot and analyze defense mechanisms that are essential to survive an AFP attack
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