The ZIP family zinc transporters support the virulence of Cryptococcus neoformans

Do, E.,Hu, G.,Caza, M. l.,Kronstad, J. W.,Jung, W. H. Informa Healthcare 2016 Medical mycology Vol.54 No.6

<P>Zinc is an essential element in living organisms and a cofactor for various metalloproteins. To disseminate and survive, a pathogenic microbe must obtain zinc from the host, which is an environment with extremely limited zinc availability. In this study, we investigated the roles of the ZIP family zinc transporters Zip1 and Zip2 in the human pathogenic fungus Cryptococcus neoformans. Zip1 and Zip2 are homologous to Zrt1 and Zrt2 of the model fungus, Saccharomyces cerevisiae, respectively. We found that the expression of ZIP1 was regulated by the zinc concentration in the environment. Furthermore, the mutant lacking ZIP1 displayed a severe growth defect under zinc-limited conditions, while the mutant lacking ZIP2 displayed normal growth. Inductively coupled plasma-atomic emission spectroscopy analysis showed that the absence of Zip1 expression significantly reduced total cellular zinc levels relative to that in the wild type, while overexpression of Zip1 was associated with increased cellular zinc levels. These findings suggested that Zip1 plays roles in zinc uptake in C. neoformans. We also constructed a Zip1-FLAG fusion protein and found, by immunofluorescence, not only that the protein was localized to the periphery implying it is a membrane transporter, but also that the protein was N-glycosylated. Furthermore, the mutant lacking ZIP1 showed attenuated virulence in a murine inhalation model of cryptococcosis and reduced survival within murine macrophages. Overall, our data suggest that Zip1 plays essential roles in zinc transport and the virulence of C. neoformans.</P>

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.

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.

The lysine biosynthetic enzyme Lys4 influences iron metabolism, mitochondrial function and virulence in Cryptococcus neoformans

Do, E.,Park, M.,Hu, G.,Caza, M.,Kronstad, J.W.,Jung, W.H. Academic Press 2016 Biochemical and biophysical research communication Vol.477 No.4

The lysine biosynthesis pathway via α-aminoadipate in fungi is considered an attractive target for antifungal drugs due to its absence in mammalian hosts. The iron-sulfur cluster-containing enzyme homoaconitase converts homocitrate to homoisocitrate in the lysine biosynthetic pathway, and is encoded by LYS4 in the model yeast Saccharomyces cerevisiae. In this study, we identified the ortholog of LYS4 in the human fungal pathogen, Cryptococcus neoformans, and found that LYS4 expression is regulated by iron levels and by the iron-related transcription factors Hap3 and HapX. Deletion of the LYS4 gene resulted in lysine auxotrophy suggesting that Lys4 is essential for lysine biosynthesis. Our study also revealed that lysine uptake was mediated by two amino acid permeases, Aap2 and Aap3, and influenced by nitrogen catabolite repression (NCR). Furthermore, the lys4 mutant showed increased sensitivity to oxidative stress, agents that challenge cell wall/membrane integrity, and azole antifungal drugs. We showed that these phenotypes were due in part to impaired mitochondrial function as a result of LYS4 deletion, which we propose disrupts iron homeostasis in the organelle. The combination of defects are consistent with our observation that the lys4 mutant was attenuated virulence in a mouse inhalation model of cryptococcosis.

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>