Iron source preference and regulation of iron uptake in Cryptococcus neoformans.
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ABSTRACT:
SUBMITTER: Jung WH
PROVIDER: S-EPMC2242830 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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 Cryptococcus neoformans, 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 C. neoformans during infection. The virulence of a cfo1 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 C. neoformans.
| S-EPMC2756870 | biostudies-literature
Project description:Iron is an essential nutrient for virtually all organisms and acts as a cofactor for many key enzymes of major metabolic pathways. Furthermore, iron plays a critical role in pathogen-host interactions. In this study, we analyzed metabolomic changes associated with iron availability and the iron regulatory protein Cir1 in a human fungal pathogen Cryptococcus neoformans. Our metabolite analysis revealed that Cir1 influences the glycolytic pathway, ergosterol biosynthesis and inositol metabolism, which require numerous iron-dependent enzymes and play important roles in pathogenesis and antifungal sensitivity of the fungus. Moreover, we demonstrated that increased cellular iron content and altered gene expression in the cir1 mutant contributed to metabolite changes. Our study provides a new insight into iron regulation and the role of Cir1 in metabolome of C. neoformans.
| S-EPMC3402442 | biostudies-literature
Project description:The high-affinity reductive iron uptake system that includes a ferroxidase (Cfo1) and an iron permease (Cft1) is critical for the pathogenesis of Cryptococcus neoformans. In addition, a mutant lacking CFO1 or CFT1 not only has reduced iron uptake but also displays a markedly increased susceptibility to azole antifungal drugs. Altered antifungal susceptibility of the mutants was of particular interest because the iron uptake system has been proposed as an alternative target for antifungal treatment. In this study, we used transcriptome analysis to begin exploring the molecular mechanisms of altered antifungal susceptibility in a cfo1 mutant. The wild-type strain and the cfo1 mutant were cultured with or without the azole antifungal drug fluconazole and their transcriptomes were compared following sequencing with Illumina Genome Analyzer IIx (GAIIx) technology. As expected, treatment of both strains with fluconazole caused elevated expression of genes in the ergosterol biosynthetic pathway that includes the target enzyme Erg11. Additionally, genes differentially expressed in the cfo1 mutant were involved in iron uptake and homeostasis, mitochondrial functions and respiration. The cfo1 mutant also displayed phenotypes consistent with these changes including a reduced ratio of NAD(+)/NADH and down-regulation of Fe-S cluster synthesis. Moreover, combination treatment of the wild-type strain with fluconazole and the respiration inhibitor diphenyleneiodonium dramatically increased susceptibility to fluconazole. This result supports the hypothesis that down-regulation of genes required for respiration contributed to the altered fluconazole susceptibility of the cfo1 mutant. Overall, our data suggest that iron uptake and homeostasis play a key role in antifungal susceptibility and could be used as novel targets for combination treatment of cryptococcosis. Indeed, we found that iron chelation in combination with fluconazole treatment synergistically inhibited the growth of C. neoformans.
| S-EPMC4706552 | biostudies-literature
Project description:Iron overload is known to exacerbate many infectious diseases, and conversely, iron withholding is an important defense strategy for mammalian hosts. Iron is a critical cue for Cryptococcus neoformans because the fungus senses iron to regulate elaboration of the polysaccharide capsule that is the major virulence factor during infection. Excess iron exacerbates experimental cryptococcosis and the prevalence of this disease in Sub-Saharan Africa has been associated with nutritional and genetic aspects of iron loading in the background of the HIV/AIDS epidemic. We demonstrate that the iron-responsive transcription factor Cir1 in Cr. neoformans controls the regulon of genes for iron acquisition such that cir1 mutants are "blind" to changes in external iron levels. Cir1 also controls the known major virulence factors of the pathogen including the capsule, the formation of the anti-oxidant melanin in the cell wall, and the ability to grow at host body temperature. Thus, the fungus is remarkably tuned to perceive iron as part of the disease process, as confirmed by the avirulence of the cir1 mutant; this characteristic of the pathogen may provide opportunities for antifungal treatment.
| S-EPMC1637126 | biostudies-literature
Project description:Transcriptome profiling of wild type and cfo1 mutant with fluconazole treatment in Cryptococcus neoformans var. grubii H99 Purpose: The goals of this study are to compare cfo1 mutant transcriptome profiling (RNA-seq) to wild-type with or without fluconazole treatment in Cryptococcus neoformans var. grubii H99. Methods: mRNA profiles of wild-type and cfo1 mutant with or without fluconazole treatment were generated by RNA-Seq, using Illumina GAIIx. The sequence reads that passed quality filters were mapped to reference genome and the normalized RPKM values were calculated by CLC Genomics Workbench. Results: Compared to wild-type, a number of genes were differentially expressed in the cfo1 mutant, especially genes involved in iron homeostasis and transport, ergosterol biosynthesis, mitochondrial function and respiration. Conclusions: Our data suggested reduced expression of the genes in the respiratory chain is the main reason for altered antifungal sensitivity of the cfo1 mutant. The results of our study revealed that iron uptake plays a key role in fluconazole sensitivity of C. neoformans.
2013-05-31 | GSE37875 | GEO
Project description:Transcriptome profiling of wild type and cfo1 mutant with fluconazole treatment in Cryptococcus neoformans var. grubii H99 Purpose: The goals of this study are to compare cfo1 mutant transcriptome profiling (RNA-seq) to wild-type with or without fluconazole treatment in Cryptococcus neoformans var. grubii H99. Methods: mRNA profiles of wild-type and cfo1 mutant with or without fluconazole treatment were generated by RNA-Seq, using Illumina GAIIx. The sequence reads that passed quality filters were mapped to reference genome and the normalized RPKM values were calculated by CLC Genomics Workbench. Results: Compared to wild-type, a number of genes were differentially expressed in the cfo1 mutant, especially genes involved in iron homeostasis and transport, ergosterol biosynthesis, mitochondrial function and respiration. Conclusions: Our data suggested reduced expression of the genes in the respiratory chain is the main reason for altered antifungal sensitivity of the cfo1 mutant. The results of our study revealed that iron uptake plays a key role in fluconazole sensitivity of C. neoformans. mRNA profiles of wild-type and cfo1 mutant with fluconazole treatment were generated by RNA-Seq, using Illumina GAIIx.
2013-05-31 | E-GEOD-37875 | biostudies-arrayexpress
Project description:The biological impact of alternative splicing is poorly understood in fungi, although recent studies have shown that these microorganisms are usually intron-rich. In this study, we re-annotated the genome of C. neoformans var. neoformans using RNA-Seq data. Comparison with C. neoformans var. grubii revealed that more than 99% of ORF-introns are in the same exact position in the two varieties whereas UTR-introns are much less evolutionary conserved. We also confirmed that alternative splicing is very common in C. neoformans, affecting nearly all expressed genes. We also observed specific regulation of alternative splicing by environmental cues in this yeast. However, alternative splicing does not appear to be an efficient method to diversify the C. neoformans proteome. Instead, our data suggest the existence of an intron retention-dependent mechanism of gene expression regulation that is not dependent on NMD. This regulatory process represents an additional layer of gene expression regulation in fungi and provides a mechanism to tune gene expression levels in response to any environmental modification.
| S-EPMC5006051 | biostudies-literature
Project description:A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase A (PKA) is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential targets for anti-cryptococcal therapy.
| S-EPMC1828699 | biostudies-literature
Project description:The battle for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection. The pathogenic fungus, Cryptococcus neoformans, employs multiple mechanisms to compete for iron during cryptococcosis, a disease primarily of immunocompromised hosts. In this study, we examined the role of endocytic trafficking in iron uptake by characterizing a mutant defective in the Sec1/Munc18 (SM) protein Vps45. This protein is known to regulate the machinery for vesicle trafficking and fusion via interactions with SNARE proteins. As expected, a vps45 deletion mutant was impaired in endocytosis and showed sensitivity to trafficking inhibitors. The mutant also showed poor growth on iron-limited media and a defect in transporting the Cfo1 ferroxidase of the high-affinity iron uptake system from the plasma membrane to the vacuole. Remarkably, we made the novel observation that Vps45 also contributes to mitochondrial function in that a Vps45-Gfp fusion protein associated with mitotracker, and a vps45 mutant showed enhanced sensitivity to inhibitors of electron transport complexes as well as changes in mitochondrial membrane potential. Consistent with mitochondrial function, the vps45 mutant was impaired in calcium homeostasis. To assess the relevance of these defects for virulence, we examined cell surface properties of the vps45 mutant and found increased sensitivity to agents that challenge cell wall integrity and to antifungal drugs. A change in cell wall properties was consistent with our observation of altered capsule polysaccharide attachment, and with attenuated virulence in a mouse model of cryptococcosis. Overall, our studies reveal a novel role for Vps45-mediated trafficking for iron uptake, mitochondrial function and virulence.
| S-EPMC6091972 | biostudies-literature
Project description:Cryptococcus neoformans is an opportunistic fungal pathogen that causes serious human disease in immunocompromised populations. Its polysaccharide capsule is a key virulence factor which is regulated in response to growth conditions, becoming enlarged in the context of infection. We used microarray analysis of cells stimulated to form capsule over a range of growth conditions to identify a transcriptional signature associated with capsule enlargement. The signature contains 880 genes, is enriched for genes encoding known capsule regulators, and includes many uncharacterized sequences. One uncharacterized sequence encodes a novel regulator of capsule and of fungal virulence. This factor is a homolog of the yeast protein Ada2, a member of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex that regulates transcription of stress response genes via histone acetylation. Consistent with this homology, the C. neoformans null mutant exhibits reduced histone H3 lysine 9 acetylation. It is also defective in response to a variety of stress conditions, demonstrating phenotypes that overlap with, but are not identical to, those of other fungi with altered SAGA complexes. The mutant also exhibits significant defects in sexual development and virulence. To establish the role of Ada2 in the broader network of capsule regulation we performed RNA-Seq on strains lacking either Ada2 or one of two other capsule regulators: Cir1 and Nrg1. Analysis of the results suggested that Ada2 functions downstream of both Cir1 and Nrg1 via components of the high osmolarity glycerol (HOG) pathway. To identify direct targets of Ada2, we performed ChIP-Seq analysis of histone acetylation in the Ada2 null mutant. These studies supported the role of Ada2 in the direct regulation of capsule and mating responses and suggested that it may also play a direct role in regulating capsule-independent antiphagocytic virulence factors. These results validate our experimental approach to dissecting capsule regulation and provide multiple targets for future investigation.
| S-EPMC3234223 | biostudies-literature