scholarly journals Role of Ferroxidases in Iron Uptake and Virulence of Cryptococcus neoformans

2009 ◽  
Vol 8 (10) ◽  
pp. 1511-1520 ◽  
Author(s):  
Won Hee Jung ◽  
Guanggan Hu ◽  
Wayne Kuo ◽  
James W. Kronstad
Keyword(s):  
Cryptococcus Neoformans ◽  
Membrane Trafficking ◽  
Iron Uptake ◽  
Defense Mechanism ◽  
Fluorescent Protein ◽  
Antifungal Drugs ◽  
Iron Limitation ◽  
Ergosterol Biosynthesis ◽  
Uptake System ◽  
Intracellular Iron

ABSTRACT 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.

scholarly journals Iron Acquisition in Mycobacterium avium subsp. paratuberculosis

2015 ◽  
Vol 198 (5) ◽  
pp. 857-866 ◽  
Author(s):  
Joyce Wang ◽  
Jalal Moolji ◽  
Alex Dufort ◽  
Alfredo Staffa ◽  
Pilar Domenech ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Iron Uptake ◽  
Genomic Island ◽  
Iron Acquisition ◽  
Laboratory Data ◽  
Uptake System ◽  
Intracellular Iron ◽  
Content Type ◽  
Iron Uptake System ◽  
Environmental Bacterium

ABSTRACTMycobacterium aviumsubsp.paratuberculosisis a host-adapted pathogen that evolved from the environmental bacteriumM. aviumsubsp.hominissuisthrough gene loss and gene acquisition. Growth ofM. aviumsubsp.paratuberculosisin the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system inM. aviumsubsp.paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed thatM. aviumsubsp.paratuberculosisis impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, inM. aviumsubsp.paratuberculosisgenes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on aM. aviumsubsp.paratuberculosis-specific genomic island, LSPP15. We obtained a transposon (Tn) mutant with a disruption in the LSPP15 geneMAP3776cfor targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775ctoMAP3772c[MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressingMAP3775-2cin wild-typeM. aviumsubsp.paratuberculosis. These data indicate that the horizontally acquired LSPP15 genes contribute to iron acquisition byM. aviumsubsp.paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen.IMPORTANCEMany microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception isMycobacterium aviumsubsp.paratuberculosis, a fastidious, slow-growing animal pathogen whose growth needs to be supported by exogenous mycobacterial siderophore (mycobactin) in the laboratory. Data presented here demonstrate that, compared to other closely relatedM. aviumsubspecies, mycobactin production and iron uptake are different inM. aviumsubsp.paratuberculosis, and these phenotypes may be caused by numerous deletions in its mycobactin biosynthesis pathway. Using a genomic approach, supplemented by targeted genetic and biochemical studies, we identified that LSPP15, a horizontally acquired genomic island, may encode an alternative iron uptake system. These findings shed light on the potential physiological consequence of horizontal gene transfer inM. aviumsubsp.paratuberculosisevolution.

scholarly journals The Sec1/Munc18 (SM) protein Vps45 is involved in iron uptake, mitochondrial function and virulence in the pathogenic fungusCryptococcus neoformans.

2018 ◽  
Author(s):  
Mélissa Caza ◽  
Guanggan Hu ◽  
Eric David Neilson ◽  
Minsu Cho ◽  
Won Hee Jung ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mitochondrial Function ◽  
Iron Uptake ◽  
Pathogenic Fungus ◽  
Antifungal Drugs ◽  
Uptake System ◽  
Poor Growth ◽  
Enhanced Sensitivity ◽  
Electron Transport Complexes ◽  
Sm Protein

ABSTRACTThe 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, avps45deletion 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 avps45mutant showed enhanced sensitivity to inhibitors of electron transport complexes as well as changes in mitochondrial membrane potential. Consistent with mitochondrial function, thevps45mutant was impaired in calcium homeostasis. To assess the relevance of these defects for virulence, we examined cell surface properties of thevps45mutant and found increased sensitivity to agents that challenge cell wall integrity and 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.

scholarly journals A defect in iron uptake enhances the susceptibility of Cryptococcus neoformans to azole antifungal drugs

2012 ◽  
Vol 49 (11) ◽  
pp. 955-966 ◽  
Author(s):  
Jeongmi Kim ◽  
Yong-Joon Cho ◽  
Eunsoo Do ◽  
Jaehyuk Choi ◽  
Guanggan Hu ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Iron Uptake ◽  
Antifungal Drugs

scholarly journals Physical and functional interaction of FgFtr1–FgFet1 and FgFtr2–FgFet2 is required for iron uptake in Fusarium graminearum

2007 ◽  
Vol 408 (1) ◽  
pp. 97-104 ◽  
Author(s):  
Yong-Sung Park ◽  
Ji-Hyun Kim ◽  
Jin-Hwa Cho ◽  
Hyo-Ihl Chang ◽  
Seung-Wook Kim ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusarium Graminearum ◽  
Iron Uptake ◽  
Fluorescent Protein ◽  
Amino Acid Level ◽  
Normal Function ◽  
Vacuolar Membrane ◽  
Uptake System ◽  
Genes Encoding ◽  
Green Fluorescent

FgFtr1 and FgFtr2 are putative iron permeases, and FgFet1 and FgFet2 are putative ferroxidases of Fusarium graminearum. They have high homologies with iron permease ScFtr1 and ferroxidase ScFet3 of Saccharomyces cerevisiae at the amino acid level. The genes encoding iron permease and ferroxidase were localized to the same chromosome in the manner of FgFtr1/FgFet1 and FgFtr2/FgFet2. The GFP (green fluorescent protein)-fused versions of FgFtr1 and FgFtr2 showed normal functions when compared with FgFtr1 and FgFtr2 in an S. cerevisiae system, and the cellular localizations of FgFtr1 and FgFtr2 in S. cerevisiae depended on the expression of their putative ferroxidase partners FgFet1 and FgFet2 respectively. Although FgFtr1 was found on the plasma membrane when FgFet1 and FgFtr1 were co-transformed in S. cerevisiae, most of the FgFtr1 was found in the endoplasmic reticulum compartment when co-expressed with FgFet2. Furthermore, FgFtr2 was found on the vacuolar membrane when FgFet2 was co-expressed. From the two-hybrid analysis, we confirmed the interaction of FgFtr1 and FgFet1, and the same result was found between FgFtr2 and FgFet2. Iron-uptake activity also depended on the existence of the respective partner. Finally, the FgFtr1 and FgFtr2 were found on the plasma and vacuolar membrane respectively, in F. graminearum. Taken together, these results strongly suggest that FgFtr1 and FgFtr2 from F. graminearum encode the iron permeases of the plasma membrane and vacuolar membrane respectively, and require their specific ferroxidases to carry out normal function. Furthermore, the present study suggests that the reductive iron-uptake system is conserved from yeast to filamentous fungi.

scholarly journals A Genomewide Screen in Schizosaccharomyces pombe for Genes Affecting the Sensitivity of Antifungal Drugs That Target Ergosterol Biosynthesis

2012 ◽  
Vol 56 (4) ◽  
pp. 1949-1959 ◽  
Author(s):  
Yue Fang ◽  
Lingling Hu ◽  
Xin Zhou ◽  
Wurentuya Jaiseng ◽  
Ben Zhang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Schizosaccharomyces Pombe ◽  
Membrane Trafficking ◽  
Cellular Response ◽  
Acid Metabolism ◽  
Antifungal Drugs ◽  
Ergosterol Biosynthesis ◽  
Nucleic Acid Metabolism ◽  
Regulation Of Transcription ◽  
Functional Categories

ABSTRACTWe performed a genomewide screen for altered sensitivity to antifungal drugs, including clotrimazole and terbinafine, that target ergosterol biosynthesis using aSchizosaccharomyces pombegene deletion library consisting of 3,004 nonessential haploid deletion mutants. We identified 109 mutants that were hypersensitive and 11 mutants that were resistant to these antifungals. Proteins whose absence rendered cells sensitive to these antifungals were classified into various functional categories, including ergosterol biosynthesis, membrane trafficking, histone acetylation and deacetylation, ubiquitination, signal transduction, ribosome biosynthesis and assembly, regulation of transcription and translation, cell wall organization and biogenesis, mitochondrion function, amino acid metabolism, nucleic acid metabolism, lipid metabolism, meiosis, and other functions. Also, proteins whose absence rendered cells resistant to these antifungals were classified into functional categories including mitochondrion function, ubiquitination, membrane trafficking, cell polarity, chromatin remodeling, and some unknown functions. Furthermore, the 109 sensitive mutants were tested for sensitivity to micafungin, another antifungal drug that inhibits (1,3)-β-d-glucan synthase, and 57 hypersensitive mutants were identified, suggesting that these mutants were defective in cell wall integrity. Altogether, our findings in fission yeast have shed light on molecular pathways associated with the cellular response to ergosterol biosynthesis inhibitors and may provide useful information for developing strategies aimed at sensitizing cells to these drugs.

scholarly journals Role of Ferric Reductases in Iron Acquisition and Virulence in the Fungal Pathogen Cryptococcus neoformans

2013 ◽  
Vol 82 (2) ◽  
pp. 839-850 ◽  
Author(s):  
Sanjay Saikia ◽  
Debora Oliveira ◽  
Guanggan Hu ◽  
James Kronstad
Keyword(s):  
Cryptococcus Neoformans ◽  
Iron Acquisition ◽  
Antifungal Drugs ◽  
Growth Defect ◽  
Uptake System ◽  
Ferric Reductase ◽  
Pathogenic Yeast ◽  
Content Type ◽  
High Affinity ◽  
Cryptococcal Disease

ABSTRACTIron acquisition is critical for the ability of the pathogenic yeastCryptococcus neoformansto cause disease in vertebrate hosts. In particular, iron overload exacerbates cryptococcal disease in an animal model, defects in iron acquisition attenuate virulence, and iron availability influences the expression of major virulence factors.C. neoformansacquires iron by multiple mechanisms, including a ferroxidase-permease high-affinity system, siderophore uptake, and utilization of both heme and transferrin. In this study, we examined the expression of eight candidate ferric reductase genes and their contributions to iron acquisition as well as to ferric and cupric reductase activities. We found that loss of theFRE4gene resulted in a defect in production of the virulence factor melanin and increased susceptibility to azole antifungal drugs. In addition, theFRE2gene was important for growth on the iron sources heme and transferrin, which are relevant for proliferation in the host. Fre2 may participate with the ferroxidase Cfo1 of the high-affinity uptake system for growth on heme, because a mutant lacking both genes showed a more pronounced growth defect than thefre2single mutant. A role for Fre2 in iron acquisition is consistent with the attenuation of virulence observed for thefre2mutant. This mutant also was defective in accumulation in the brains of infected mice, a phenotype previously observed for mutants with defects in high-affinity iron uptake (e.g., thecfo1mutant). Overall, this study provides a more detailed view of the iron acquisition components required forC. neoformansto cause cryptococcosis.

scholarly journals A Flucytosine-Responsive Mbp1/Swi4-Like Protein, Mbs1, Plays Pleiotropic Roles in Antifungal Drug Resistance, Stress Response, and Virulence of Cryptococcus neoformans

2011 ◽  
Vol 11 (1) ◽  
pp. 53-67 ◽  
Author(s):  
Min-Hee Song ◽  
Jang-Won Lee ◽  
Min Su Kim ◽  
Ja-Kyung Yoon ◽  
Theodore C. White ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Stress Responses ◽  
Drug Susceptibility ◽  
Antifungal Drugs ◽  
Ergosterol Biosynthesis ◽  
Dependent Manner ◽  
Content Type ◽  
Antifungal Drug Resistance ◽  
Cellular Processes ◽  
Basidiomycetous Fungus

ABSTRACTCryptococcosis, caused by the basidiomycetous fungusCryptococcus neoformans, is responsible for more than 600,000 deaths annually in AIDS patients. Flucytosine is one of the most commonly used antifungal drugs for its treatment, but its resistance and regulatory mechanisms have never been investigated at the genome scale inC. neoformans. In the present study, we performed comparative transcriptome analysis by employing two-component system mutants (tco1Δ andtco2Δ) exhibiting opposing flucytosine susceptibility. As a result, a total of 177 flucytosine-responsive genes were identified, and many of them were found to be regulated by Tco1 or Tco2. Among these, we discovered an APSES-like transcription factor, Mbs1 (Mbp1- andSwi4-like protein 1). Expression analysis revealed thatMBS1was regulated in response to flucytosine in a Tco2/Hog1-dependent manner. Supporting this,C. neoformanswith the deletion ofMBS1exhibited increased susceptibility to flucytosine. Intriguingly, Mbs1 played pleiotropic roles in diverse cellular processes ofC. neoformans. Mbs1 positively regulated ergosterol biosynthesis and thereby affected polyene and azole drug susceptibility. Mbs1 was also involved in genotoxic and oxidative stress responses. Furthermore, Mbs1 promoted production of melanin and capsule and thereby was required for full virulence ofC. neoformans. In conclusion, Mbs1 is considered to be a novel antifungal therapeutic target for treatment of cryptococcosis.

scholarly journals YdiV regulates Escherichia coli ferric uptake by manipulating the DNA-binding ability of Fur in a SlyD-dependent manner

2020 ◽  
Vol 48 (17) ◽  
pp. 9571-9588
Author(s):  
Fengyu Zhang ◽  
Bingqing Li ◽  
Hongjie Dong ◽  
Min Chen ◽  
Shun Yao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Iron Deficiency ◽  
Iron Uptake ◽  
Iron Homeostasis ◽  
Bacterial Invasion ◽  
Host Immune System ◽  
Uptake System ◽  
Dependent Manner ◽  
Intracellular Iron ◽  
E Coli

Abstract Iron is essential for all bacteria. In most bacteria, intracellular iron homeostasis is tightly regulated by the ferric uptake regulator Fur. However, how Fur activates the iron-uptake system during iron deficiency is not fully elucidated. In this study, we found that YdiV, the flagella gene inhibitor, is involved in iron homeostasis in Escherichia coli. Iron deficiency triggers overexpression of YdiV. High levels of YdiV then transforms Fur into a novel form which does not bind DNA in a peptidyl-prolyl cis-trans isomerase SlyD dependent manner. Thus, the cooperation of YdiV, SlyD and Fur activates the gene expression of iron-uptake systems under conditions of iron deficiency. Bacterial invasion assays also demonstrated that both ydiV and slyD are necessary for the survival and growth of uropathogenic E. coli in bladder epithelial cells. This reveals a mechanism where YdiV not only represses flagella expression to make E. coli invisible to the host immune system, but it also promotes iron acquisition to help E. coli overcome host nutritional immunity.

Efficacy of Novel Schiff base Derivatives as Antifungal Compounds in Combination with Approved Drugs Against Candida Albicans

2019 ◽  
Vol 15 (6) ◽  
pp. 648-658 ◽  
Author(s):  
Manzoor Ahmad Malik ◽  
Shabir Ahmad Lone ◽  
Parveez Gull ◽  
Ovas Ahmad Dar ◽  
Mohmmad Younus Wani ◽  
...  
Keyword(s):  
Schiff Base ◽  
Candida Albicans ◽  
Antifungal Activity ◽  
Fungal Infections ◽  
Total Sterol ◽  
Antifungal Drugs ◽  
New Drugs ◽  
Ergosterol Biosynthesis ◽  
Dependent Manner ◽  
Schiff Base Derivatives

Background: The increasing incidence of fungal infections, especially caused by Candida albicans, and their increasing drug resistance has drastically increased in recent years. Therefore, not only new drugs but also alternative treatment strategies are promptly required. Methods: We previously reported on the synergistic interaction of some azole and non-azole compounds with fluconazole for combination antifungal therapy. In this study, we synthesized some non-azole Schiff-base derivatives and evaluated their antifungal activity profile alone and in combination with the most commonly used antifungal drugs- fluconazole (FLC) and amphotericin B (AmB) against four drug susceptible, three FLC resistant and three AmB resistant clinically isolated Candida albicans strains. To further analyze the mechanism of antifungal action of these compounds, we quantified total sterol contents in FLC-susceptible and resistant C. albicans isolates. Results: A pyrimidine ring-containing derivative SB5 showed the most potent antifungal activity against all the tested strains. After combining these compounds with FLC and AmB, 76% combinations were either synergistic or additive while as the rest of the combinations were indifferent. Interestingly, none of the combinations was antagonistic, either with FLC or AmB. Results interpreted from fractional inhibitory concentration index (FICI) and isobolograms revealed 4-10-fold reduction in MIC values for synergistic combinations. These compounds also inhibit ergosterol biosynthesis in a concentration-dependent manner, supported by the results from docking studies. Conclusion: The results of the studies conducted advocate the potential of these compounds as new antifungal drugs. However, further studies are required to understand the other mechanisms and in vivo efficacy and toxicity of these compounds.

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