To investigate cation adaptation and homoeostasis in Aspergillus nidulans, two transcription-factor-encoding genes have been characterized. The A. nidulans orthologue crzA of the Saccharomyces cerevisiae CRZ1 gene, encoding a transcription factor mediating gene regulation by Ca(2+), has been identified and deleted. The crzA deletion phenotype includes extreme sensitivity to alkaline pH, Ca(2+) toxicity and aberrant morphology connected with alterations of cell-wall-related phenotypes such as reduced expression of a chitin synthase gene, chsB. A fully functional C-terminally GFP (green fluorescent protein)-tagged form of the CrzA protein is apparently excluded from nuclei in the absence of added Ca(2+), but rapidly accumulates in nuclei upon exposure to Ca(2+). In addition, the previously identified sltA gene, which has no identifiable homologues in yeasts, was deleted, and the resulting phenotype includes considerably enhanced toxicity by a number of cations other than Ca(2+) and also by alkaline pH. Reduced expression of a homologue of the S. cerevisiae P-type ATPase Na(+) pump gene ENA1 might partly explain the cation sensitivity of sltA-null strains. Up-regulation of the homologue of the S. cerevisiae vacuolar Ca(2+)/H(+) exchanger gene VCX1 might explain the lack of Ca(2+) toxicity to null-sltA mutants, whereas down-regulation of this gene might be responsible for Ca(2+) toxicity to crzA-null mutants. Both crzA and sltA encode DNA-binding proteins, and the latter exerts both positive and negative gene regulation.

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 AFPsensitive 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.

In Aspergillus nidulans a combination of null mutations in halA, encoding a protein kinase, and sltA, encoding a zinc-finger transcription factor having no yeast homologues, results in an elevated calcium requirement (‘calcium auxotrophy’) without impairing net calcium uptake. sltA− (±halA−) mutations result in hypertrophy of the vacuolar system. In halA−sltA− (and sltA−) strains, transcript levels for pmcA and pmcB, encoding vacuolar Ca2+-ATPase homologues, are highly elevated, suggesting a regulatory relationship between vacuolar membrane area and certain vacuolar membrane ATPase levels. Deletion of both pmcA and pmcB strongly suppresses the ‘calcium auxotrophy’. Therefore the ‘calcium auxotrophy’ possibly results from excessive vacuolar calcium sequestration, causing cytosolic calcium deprivation. Null mutations in nhaA, homologous to Saccharomyces cerevisiaeNHA1, encoding a plasma membrane Na+/H+ antiporter effluxing Na+ and K+, and a non-null mutation in trkB, homologous to S. cerevisiaeTRK1, encoding a plasma membrane high affinity K+ transporter, also suppress the calcium auxotrophy.