Biphasic zinc compartmentalisation in a human fungal pathogen

Abstract

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.

Eukaryotic transposable elements as “cargo carriers”: the forging of metal resistance in the fungusPaecilomyces variotii

“…In S. cerevisiae and Cryptococcus neoformans Zrc1 is localized to the vacuolar membrane and leads to the accumulation of zinc in the vacuole 40,41 . However, in Candida albicans and Hebeloma cylindrosporum the Zrc1 homologs appear to be localized to the endoplasmic reticulum and leads to zinc storage in so called “Zincosomes” 42,43 . The presence of P. variotii ZrcA in this membrane suggests a possible role in exporting zinc ions out of the cell rather than in involvement in intracellular zinc storage as in the case of Zrc1 in S. cerevisiae, C. albicans and H. cylindrosporum .…”

TheAspergillus fumigatustranscription factor AceA is involved not only in Cu but also in Zn detoxification through regulating transporters CrpA and ZrcA

“…Zrc1, an AfZrcA homologue, has been recently demonstrated to involve in Zn tolerance in the human fungal pathogens C. albicans and C. neoformans (Cho, Hu, Caza, Horianopoulos, Kronstad & Jung, ; Crawford et al, ). Here, Cu‐ and Zn‐responsive AfzrcA implies its potential involvement in AfAceA‐mediated Cu and Zn metabolism; however, the AfZrcA deletion mutant displayed WT‐like Cu resistance, and overexpression of AfZrcA was unable to rescue the high Cu growth defects associated with either AfaceA or AfcrpA deletion, suggesting that AfZrcA lacks an AfCrpA‐like function of responding to high Cu conditions.…”

Memory in Fungal Pathogens Promotes Immune Evasion, Colonisation, and Infection

“…Therefore, C. albicans induces iron and zinc scavenging mechanisms, just as it initiates hyphal growth and tissue invasion. Tissue invasion coincides with the imposition of nutritional immunity by the host as it attempts to limit fungal colonisation by limiting micronutrient availability [38,54]. Therefore, this appears to represent an excellent example of adaptive prediction, where the pathogen is essentially anticipating micronutrient deprivation before it invades micronutrient limiting domains [55,56] (Figure 2).…”

“…These mechanisms include the ability to bind complement regulatory proteins on the fungal cell surface [91,92] or to degrade complement proteins [93,94], thereby inhibiting the complement cascade. They include the development of growth forms that are recalcitrant to phagocytosis, such as C. neoformans Titan cells [44,95], C. immitis spherules, A. fumigatus germination and C. albicans hyphae, switching phenotypes and Goliath cells [96][97][98][99]. Even after phagocytosis, fungal pathogens are able to evade killing by perturbing phagolysosomal maturation and fungal killing [100][101][102], by escaping from macrophages by non-lytic vomocytosis [103,104], or by destroying macrophages via pyroptosis and other killing mechanisms [105][106][107][108].…”