Our pursuit for effective antifungal agents targeting fungal cell wall components: where are we?

Invasive mycotic infections present unacceptably high mortality rates in humans. These infections are initiated by the fungal cell wall, which mediates host-fungi interactions. The cell wall is associated with the physiology of fungi and is involved in essential processes in the entire cell functionality. Components of the fungal cell wall are synthesised and modified in the cell wall space by the activities of cell wall proteins through a range of signalling pathways that have been described uniquely in many fungi, therefore making them suitable drug targets. The echinocandin class of cell-wall-active drugs blocks cell wall ß-1,3-glucan biosynthesis through inhibiting the catalytic subunit of the synthetic protein complex. Resistance to echinocandins can occur through the acquisition of single nucleotide polymorphisms and/or through activation of cell wall signalling pathways resulting in an altered cell wall proteome and elevated chitin content in the cell wall. Countering the cell wall remodelling process will enhance the effectiveness of ß-1,3-glucan-active antifungal agents. Cell surface proteins are also important antifungal targets that can be used to develop rapid and robust diagnostics and more effective therapeutics. The cell wall remains a crucial target in fungi that needs to be harnessed to combat mycotic infections.

Nutritional immunity: targeting fungal zinc homeostasis.

Transition metals, such as Zn2+, are essential dietary constituents of all biological life, including mammalian hosts and the pathogens that infect them. Therefore, to thrive and cause infection, pathogens must successfully assimilate these elements from the host milieu. Consequently, mammalian immunity has evolved to actively restrict and/or pool metals to toxic concentrations in an effort to attenuate microbial pathogenicity - a process termed nutritional immunity. Despite host-induced Zn2+ nutritional immunity, pathogens such as Candida albicans, are still capable of causing disease and thus must be equipped with robust Zn2+ sensory, uptake and detoxification machinery. This review will discuss the strategies employed by mammalian hosts to limit Zn2+ during infection, and the subsequent fungal interventions that counteract Zn2+ nutritional immunity.

Biphasic zinc compartmentalisation in a human fungal pathogen.

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.

Zinc Limitation Induces a Hyper-Adherent Goliath Phenotype in Candida albicans.

Pathogenic microorganisms often face acute micronutrient limitation during infection due to the action of host-mediated nutritional immunity. The human fungal pathogen Candida albicans is polymorphic and its morphological plasticity is one of its most widely recognized pathogenicity attributes. Here we investigated the effect of zinc, iron, manganese, and copper limitation on C. albicans morphology. Restriction of zinc specifically resulted in the formation of enlarged, spherical yeasts, a phenotype which we term Goliath cells. This cellular response to zinc restriction was conserved in C. albicans, C. dubliniensis and C. tropicalis, but not in C. parapsilosis, C. lusitaniae or Debaryomyces hansenii, suggesting that it may have emerged in the last common ancestor of these related pathogenic species. Cell wall analysis revealed proportionally more chitin exposure on the Goliath cell surface. Importantly, these cells were hyper-adherent, suggesting a possible role in pathogenicity. Interestingly, the zincophore-encoding gene PRA1 was expressed by Goliath cells in zinc limited media and lack of Pra1 inhibited both cellular enlargement and adhesion. Goliath cells represent a further layer of Candida phenotypic plasticity.