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.

Secondary growth is a common post-harvest problem when pre-infected crops are attacked by filamentous fungi during storage or processing. Several antifungal approaches are thus pursued based on chemical, physical, or bio-control treatments; however, many of these methods are inefficient, affect product quality, or cause severe side effects on the environment. A protein that can potentially overcome these limitations is the antifungal protein AFP, an abundantly secreted peptide of the filamentous fungus Aspergillus giganteus. This protein specifically and at low concentrations disturbs the integrity of fungal cell walls and plasma membranes but does not interfere with the viability of other pro- and eukaryotic systems. We thus studied in this work the applicability of AFP to efficiently prevent secondary growth of filamentous fungi on food stuff and chose, as a case study, the malting process where naturally infested raw barley is often to be used as starting material. Malting was performed under lab scale conditions as well as in a pilot plant, and AFP was applied at different steps during the process. AFP appeared to be very efficient against the main fungal contaminants, mainly belonging to the genus Fusarium. Fungal growth was completely blocked after the addition of AFP, a result that was not observed for traditional disinfectants such as ozone, hydrogen peroxide, and chlorine dioxide. We furthermore detected reduced levels of the mycotoxin deoxynivalenol after AFP treatment, further supporting the fungicidal activity of the protein. As AFP treatments did not compromise any properties and qualities of the final products malt and wort, we consider the protein as an excellent biological alternative to combat secondary growth of filamentous fungi on food stuff.

tolerance of microorganisms to abiotic stress is enabled by regulatory mechanisms that coordinate the expression and activity of resistance genes. Alkalinity and high salt concentrations are major environmental physicochemical stresses. Here, we analyzed the roles of sodium-extrusion family (ENA) transporters EnaA, EnaB and EnaC in the response to these stress conditions in the filamentous fungus Aspergillus nidulans. While enac has a minor role, enaB is a key element for tolerance to na + and Li + toxicity. Adaptation to alkaline pH requires the concerted action of enaB with enaA. Accordingly, expression of enaA and enaB was induced by na + , Li + and pH 8. These expression patterns are altered in a sltAΔ background and completely inhibited in a mutant expressing non-functional pacc protein (palH72). However, a constitutively active PacC form was not sufficient to restore maximum enaA expression. in agreement with their predicted role as membrane Atpases, enaA localized to the plasma membrane while enaB accumulated at structures resembling the endoplasmic reticulum. Overall, results suggest different PacC-and SltA-dependent roles for EnaB in pH and salt homeostasis, acting in coordination with EnaA at pH 8 but independently under salt stress. Ion homeostasis is an essential biochemical process for cell life influencing a wide range of cellular functions from osmotic regulation to enzyme activity. In addition, certain ions, such as sodium, calcium, and lithium become toxic at a high cytoplasmic concentration 1. Therefore, microorganisms have developed specific transport mechanisms to maintain an adequate intra and extracellular distribution of certain ions. Examples are the maintenance of low cytoplasmic Na + /K + ratio and the extremely low cytoplasmic concentrations of Li + (< 10-7 M) and Ca 2+ (aprox. 10-7 M) 2-4. Modulation of cytoplasmic cation contents is mainly achieved by active transmembrane transporters that facilitate efflux and influx of ions through the plasma membrane or by an efficient storage of ions in intracellular compartments such as vacuoles 5. In addition, the activity of plasma-membrane transporters generates an electrochemical gradient across the membrane which stores energy that can be used by other transport mechanisms acting as antiporters and/or symporters. In this respect, the transmembrane electrochemical proton gradient has a major influence on the transmembrane potential and on the functionality of ion transport across the plasma membrane in both directions 6. But, most importantly, proton concentration determines the pH value and is the basis of a major abiotic stress/stimulus for the cell 7. Filamentous fungi are able to grow over a wide pH range with almost constant intracellular pH and transmembrane potential of approximately − 200 mV 8,9. A change in the extracellular pH, either towards acidification or to alkalinization, is an important stimulus to a cell or organism. This might cause alterations in the electrochemical gradient and its maintenance, with direct impact on the b...

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The afp gene encoding the antifungal protein (AFP) of Aspergillus giganteus has a prototypical alkaline gene expression pattern, which suggests that the gene might be under the control of the ambient pH-dependent zinc-finger transcription factor PacC. This notion is corroborated by the presence in the upstream region of afp of two putative PacC binding sites, afpP1 and afpP2, which are specifically recognised by the PacC protein of A. nidulans in vitro. However, in this report we provide several lines of evidence to show that pH-dependent up-regulation of afp is not mediated by transcriptional activation through PacC. (1) The temporal expression pattern of the A. giganteus pacC gene does not parallel the accumulation of the afp mRNA during cultivation. (2) Inactivation of afpP1 and afpP2 did not reduce promoter activity under alkaline conditions, as determined from the relative wild-type and mutant afp::lacZ reporter activities in A. nidulans. (3) Reporter activities in acidity- and alkalinity-mimicking mutant strains are inconsistent with a positive role for PacC in afp expression. (4) In A. giganteus, the pH-dependent increase in afp mRNA and AFP levels can be completely prevented by the calcineurin inhibitor FK506, suggesting that the calcineurin signalling pathway might control the in vivo activation of the afp promoter by alkaline pH.

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