Vacuolar zinc transporter Zrc1 is required for detoxification of excess intracellular zinc in the human fungal pathogen Cryptococcus neoformans

조민수,Guanggan Hu,Mélissa Caza,Linda C. Horianopoulos,James W. Kronstad,정원희 한국미생물학회 2018 The journal of microbiology Vol.56 No.1

Zinc is an important transition metal in all living organisms and is required for numerous biological processes. However, excess zinc can also be toxic to cells and cause cellular stress. In the model fungus Saccharomyces cerevisiae, a vacuolar zinc transporter, Zrc1, plays important roles in the storage and detoxification of excess intracellular zinc to protect the cell. In this study, we identified an ortholog of the S. cerevisiae ZRC1 gene in the human fungal pathogen Cryptococcus neoformans. Zrc1 was localized in the vacuolar membrane in C. neoformans, and a mutant lacking ZRC1 showed significant growth defects under high-zinc conditions. These results suggested a role for Zrc1 in zinc detoxification. However, contrary to our expectation, the expression of Zrc1 was induced in cells grown in zinc-limited conditions and decreased upon the addition of zinc. These expression patterns were similar to those of Zip1, the high-affinity zinc transporter in the plasma membrane of C. neoformans. Furthermore, we used the zrc1 mutant in a murine model of cryptococcosis to examine whether a mammalian host could inhibit the survival of C. neoformans using zinc toxicity. We found that the mutant showed no difference in virulence compared with the wildtype strain. This result suggests that Zrc1-mediated zinc detoxification is not required for the virulence of C. neoformans, and imply that zinc toxicity may not be an important aspect of the host immune response to the fungus.

Involvement of Mrs3/4 in Mitochondrial Iron Transport and Metabolism in Cryptococcus neoformans

최유정,도은수,Hu Guanggan,Caza Mélissa,Linda C. Horianopoulos,Kronstad James W.,정원희 한국미생물·생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.8

Mitochondria play a vital role in iron uptake and metabolism in pathogenic fungi, and also influence virulence and drug tolerance. However, the regulation of iron transport within the mitochondria of Cryptococcus neoformans, a causative agent of fungal meningoencephalitis in immunocompromised individuals, remains largely uncharacterized. In this study, we identified and functionally characterized Mrs3/4, a homolog of the Saccharomyces cerevisiae mitochondrial iron transporter, in C. neoformans var. grubii. A strain expressing an Mrs3/4-GFP fusion protein was generated, and the mitochondrial localization of the fusion protein was confirmed. Moreover, a mutant lacking the MRS3/4 gene was constructed; this mutant displayed significantly reduced mitochondrial iron and cellular heme accumulation. In addition, impaired mitochondrial iron-sulfur cluster metabolism and altered expression of genes required for iron uptake at the plasma membrane were observed in the mrs3/4 mutant, suggesting that Mrs3/4 is involved in iron import and metabolism in the mitochondria of C. neoformans. Using a murine model of cryptococcosis, we demonstrated that an mrs3/4 mutant is defective in survival and virulence. Taken together, our study suggests that Mrs3/4 is responsible for iron import in mitochondria and reveals a link between mitochondrial iron metabolism and the virulence of C. neoformans.

Defects in Phosphate Acquisition and Storage Influence Virulence of <i>Cryptococcus neoformans</i>

Kretschmer, Matthias,Reiner, Ethan,Hu, Guanggan,Tam, Nicola,Oliveira, Debora L.,Caza, Melissa,Yeon, Ju Hun,Kim, Jeongmi,Kastrup, Christian J.,Jung, Won Hee,Kronstad, James W. American Society for Microbiology 2014 Infection and immunity Vol.82 No.7

<P>Nutrient acquisition and sensing are critical aspects of microbial pathogenesis. Previous transcriptional profiling indicated that the fungal pathogen <I>Cryptococcus neoformans</I>, which causes meningoencephalitis in immunocompromised individuals, encounters phosphate limitation during proliferation in phagocytic cells. We therefore tested the hypothesis that phosphate acquisition and polyphosphate metabolism are important for cryptococcal virulence. Deletion of the high-affinity uptake system interfered with growth on low-phosphate medium, perturbed the formation of virulence factors (capsule and melanin), reduced survival in macrophages, and attenuated virulence in a mouse model of cryptococcosis. Additionally, analysis of nutrient sensing functions for <I>C. neoformans</I> revealed regulatory connections between phosphate acquisition and storage and the iron regulator Cir1, cyclic AMP (cAMP)-dependent protein kinase A (PKA), and the calcium-calmodulin-activated protein phosphatase calcineurin. Deletion of the <I>VTC4</I> gene encoding a polyphosphate polymerase blocked the ability of <I>C. neoformans</I> to produce polyphosphate. The <I>vtc4</I> mutant behaved like the wild-type strain in interactions with macrophages and in the mouse infection model. However, the fungal load in the lungs was significantly increased in mice infected with <I>vtc4</I> deletion mutants. In addition, the mutant was impaired in the ability to trigger blood coagulation <I>in vitro</I>, a trait associated with polyphosphate. Overall, this study reveals that phosphate uptake in <I>C. neoformans</I> is critical for virulence and that its regulation is integrated with key signaling pathways for nutrient sensing.</P>

Role of Ferroxidases in Iron Uptake and Virulence of Cryptococcus neoformans

Jung, Won Hee,Hu, Guanggan,Kuo, Wayne,Kronstad, James W. American Society for Microbiology 2009 EUKARYOTIC CELL Vol.8 No.10

<B>ABSTRACT</B><P>Iron acquisition is a critical aspect of the virulence of many pathogenic microbes, and iron limitation is an important defense mechanism for mammalian hosts. We are examining mechanisms of iron regulation and acquisition in the fungal pathogen <I>Cryptococcus neoformans</I>, and here, we characterize the roles of the ferroxidases Cfo1 and Cfo2. Cfo1 is required for the reductive iron uptake system that mediates the utilization of transferrin, an important iron source for <I>C. neoformans</I> during infection. The virulence of a <I>cfo1</I> mutant was attenuated in a mouse model of cryptococcosis, and the mutant also displayed increased sensitivities to the antifungal drugs fluconazole and amphotericin B. Wild-type levels of drug sensitivity were restored by the addition of exogenous heme, which suggested that reduced levels of intracellular iron may curtail heme levels and interfere with ergosterol biosynthesis. We constructed green fluorescent protein (GFP) fusion proteins and found elevated expression of Cfo1-GFP upon iron limitation, as well as localization of the fusion to the plasma membrane. Trafficking to this location was disrupted by a defect in the catalytic subunit of cyclic AMP-dependent protein kinase. This result is consistent with findings from studies indicating an influence of the kinase on the expression of protein-trafficking functions in <I>C. neoformans</I>.</P>

Expanding fungal pathogenesis: Cryptococcus breaks out of the opportunistic box

Kronstad, James W.,Attarian, Rodgoun,Cadieux, Brigitte,Choi, Jaehyuk,D'Souza, Cletus A.,Griffiths, Emma J.,Geddes, Jennifer M. H.,Hu, Guanggan,Jung, Won Hee,Kretschmer, Matthias,Saikia, Sanjay,Wang, J Nature Publishing Group, a division of Macmillan P 2011 Nature reviews. Microbiology Vol.9 No.3

Cryptococcus neoformans is generally considered to be an opportunistic fungal pathogen because of its tendency to infect immunocompromised individuals, particularly those infected with HIV. However, this view has been challenged by the recent discovery of specialized interactions between the fungus and its mammalian hosts, and by the emergence of the related species Cryptococcus gattii as a primary pathogen of immunocompetent populations. In this Review, we highlight features of cryptococcal pathogens that reveal their adaptation to the mammalian environment. These features include not only remarkably sophisticated interactions with phagocytic cells to promote intracellular survival, dissemination to the central nervous system and escape, but also surprising morphological and genomic adaptations such as the formation of polyploid giant cells in the lung.