Metals are essential for life, and they play a central role in the struggle between infecting microbes and their hosts. In fact, an important aspect of microbial pathogenesis is the ‘nutritional immunity’, in which metals are actively restricted (or, in an extended definition of the term, locally enriched) by the host to hinder microbial growth and virulence. Consequently, fungi have evolved often complex regulatory networks, uptake and detoxification systems for essential metals such as iron, zinc, copper, nickel and manganese. These systems often differ fundamentally from their bacterial counterparts, but even within the fungal pathogens we can find common and unique solutions to maintain metal homeostasis. Thus, we here compare the common and species-specific mechanisms used for different metals among different fungal species—focusing on important human pathogens such as Candida albicans, Aspergillus fumigatus or Cryptococcus neoformans, but also looking at model fungi such as Saccharomyces cerevisiae or A. nidulans as well-studied examples for the underlying principles. These direct comparisons of our current knowledge reveal that we have a good understanding how model fungal pathogens take up iron or zinc, but that much is still to learn about other metals and specific adaptations of individual species—not the least to exploit this knowledge for new antifungal strategies.

Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH-dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2Δ growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrt1/Zrt2-cellular import, followed by Zrc1-dependent intracellular compartmentalisation.

Iron availability is a central factor in infections, since iron is a critical micronutrient for all living organisms. The host employs both iron limitation and toxicity strategies to control microbial growth, and successful pathogens are able to tightly coordinate iron homeostasis in response to changing iron levels. As a commensal and opportunistic pathogen, Candida albicans copes with both iron deficiency and excess via the precise regulation of iron acquisition, consumption and storage. The C. albicans transcription factor Hap43 is known to be required for the iron starvation response, while specific domains of its ortholog, HapX, in Aspergillus fumigatus, were recently shown to regulate iron uptake and consumptions genes under both low and high iron levels. Therefore, we investigated the contribution of C. albicans Hap43 domains in response to changing iron levels. We found the C-terminus of Hap43 to be essential for the activation of iron uptake genes during iron starvation, whereas, in contrast to A. fumigatus, Hap43 was not required in mediating adaptation to iron resistance. These data indicate that the generally conserved metal acquisition systems in fungal pathogens can show individual adaptations to the host environment.

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