Genetic and Physiologic Characterization of Ferric/Cupric Reductase Constitutive Mutants ofCryptococcus neoformans

We have identified a cell surface ferric reductase activity in the fission yeast Schizosaccharomyces pombe. A mutant strain deficient in this activity was also deficient in ferric iron uptake, while ferrous iron uptake was not impaired. Therefore, reduction is a required step in cellular ferric iron acquisition. We have cloned frp1+, the wild-type allele of the mutant gene. frp1+ mRNA levels were repressed by iron addition to the growth medium. Fusion of 138 nucleotides of frp1+ promoter sequences to a reporter gene, the bacterial chloramphenicol acetyltransferase gene, conferred iron-dependent regulation upon the latter when introduced into S. pombe. The predicted amino acid sequence of the frp1+ gene exhibits hydrophobic regions compatible with transmembrane domains. It shows similarity to the Saccharomyces cerevisiae FRE1 gene product and the gp91-phox protein, a component of the human NADPH phagocyte oxidoreductase that is deficient in X-linked chronic granulomatous disease.

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The fission yeast ferric reductase gene frp1+ is required for ferric iron uptake and encodes a protein that is homologous to the gp91-phox subunit of the human NADPH phagocyte oxidoreductase.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4342 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4342-4350 ◽

Cited By ~ 84

Author(s):

D G Roman ◽

A Dancis ◽

G J Anderson ◽

R D Klausner

Keyword(s):

Fission Yeast ◽

Iron Uptake ◽

Ferric Iron ◽

Reductase Activity ◽

Iron Acquisition ◽

Protein A ◽

Mutant Gene ◽

Predicted Amino Acid Sequence ◽

Mrna Levels ◽

Ferric Reductase

We have identified a cell surface ferric reductase activity in the fission yeast Schizosaccharomyces pombe. A mutant strain deficient in this activity was also deficient in ferric iron uptake, while ferrous iron uptake was not impaired. Therefore, reduction is a required step in cellular ferric iron acquisition. We have cloned frp1+, the wild-type allele of the mutant gene. frp1+ mRNA levels were repressed by iron addition to the growth medium. Fusion of 138 nucleotides of frp1+ promoter sequences to a reporter gene, the bacterial chloramphenicol acetyltransferase gene, conferred iron-dependent regulation upon the latter when introduced into S. pombe. The predicted amino acid sequence of the frp1+ gene exhibits hydrophobic regions compatible with transmembrane domains. It shows similarity to the Saccharomyces cerevisiae FRE1 gene product and the gp91-phox protein, a component of the human NADPH phagocyte oxidoreductase that is deficient in X-linked chronic granulomatous disease.

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

Infection and Immunity ◽

10.1128/iai.01357-13 ◽

2013 ◽

Vol 82 (2) ◽

pp. 839-850 ◽

Cited By ~ 42

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.

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Genetic evidence that ferric reductase is required for iron uptake in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2294 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2294-2301 ◽

Cited By ~ 219

Author(s):

A Dancis ◽

R D Klausner ◽

A G Hinnebusch ◽

J G Barriocanal

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Iron Uptake ◽

Reductase Activity ◽

Gene Transcript ◽

Single Mutation ◽

Uptake System ◽

Ferric Reductase ◽

Wild Type ◽

Iron Starvation

The requirement for a reduction step in cellular iron uptake has been postulated, and the existence of plasma membrane ferric reductase activity has been described in both procaryotic and eucaryotic cells. In the yeast Saccharomyces cerevisiae, there is an externally directed reductase activity that is regulated by the concentration of iron in the growth medium; maximal activity is induced by iron starvation. We report here the isolation of a mutant of S. cerevisiae lacking the reductase activity. This mutant is deficient in the uptake of ferric iron and is extremely sensitive to iron deprivation. Genetic analysis of the mutant demonstrates that the reductase and ferric uptake deficiencies are due to a single mutation that we designate fre1-1. Both phenotypes cosegregate in meiosis, corevert with a frequency of 10(-7), and are complemented by a 3.5-kilobase fragment of genomic DNA from wild-type S. cerevisiae. This fragment contains FRE1, the wild-type allele of the mutant gene. The level of the gene transcript is regulated by iron in the same was as the reductase activity. The ferrous ion product of the reductase must traverse the plasma membrane. A high-affinity (Km = 5 microM) ferrous uptake system is present in both wild-type and mutant cells. Thus, iron uptake in S. cerevisiae is mediated by two plasma membrane components, a reductase and a ferrous transport system.

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High stability ferric chelates result in decreased iron uptake by the green alga Chlorella kessleri owing to decreased ferric reductase activity and chelation of ferrous iron

Botany ◽

10.1139/b09-063 ◽

2009 ◽

Vol 87 (10) ◽

pp. 922-931 ◽

Cited By ~ 9

Author(s):

Harold G. Weger ◽

Jackie Lam ◽

Nikki L. Wirtz ◽

Crystal N. Walker ◽

Ron G. Treble

Keyword(s):

Stability Constant ◽

Green Alga ◽

Iron Uptake ◽

High Stability ◽

Reductase Activity ◽

Iron Acquisition ◽

Free Form ◽

Ferric Reductase ◽

Chlorella Kessleri ◽

Green Alga Chlorella

Cells of the green alga Chlorella kessleri Fott et Nováková use a reductive mechanism for iron acquisition. Iron-limited cells acquired iron more rapidly from a chelator with a lower stability constant for Fe3+ (hydroxyethylethylenediaminetriacetic acid (HEDTA)) than from a chelator with a higher stability constant (N,N′-di[2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED)). Furthermore, iron uptake rates decreased with increasing chelator concentrations at constant iron concentration. The negative effects of elevated HBED levels on iron uptake could be partly alleviated by the addition of Ga3+, which suggests that iron-free chelator has a negative effect on iron acquisition by competing for Fe2+ with the ferrous transport system. Furthermore, ferric reductase activity progressively decreased with increasing concentrations of both chelators (in the iron-free form). This effect was not alleviated by Ga3+ addition and was apparently caused by the direct inhibition of the reductase. Overall, we conclude that chelators with high stability constants for Fe3+ decrease iron acquisition rates by Strategy I organisms via three separate mechanisms.

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Genetic evidence that ferric reductase is required for iron uptake in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2294-2301.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2294-2301

Author(s):

A Dancis ◽

R D Klausner ◽

A G Hinnebusch ◽

J G Barriocanal

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Iron Uptake ◽

Reductase Activity ◽

Gene Transcript ◽

Single Mutation ◽

Uptake System ◽

Ferric Reductase ◽

Wild Type ◽

Iron Starvation

The requirement for a reduction step in cellular iron uptake has been postulated, and the existence of plasma membrane ferric reductase activity has been described in both procaryotic and eucaryotic cells. In the yeast Saccharomyces cerevisiae, there is an externally directed reductase activity that is regulated by the concentration of iron in the growth medium; maximal activity is induced by iron starvation. We report here the isolation of a mutant of S. cerevisiae lacking the reductase activity. This mutant is deficient in the uptake of ferric iron and is extremely sensitive to iron deprivation. Genetic analysis of the mutant demonstrates that the reductase and ferric uptake deficiencies are due to a single mutation that we designate fre1-1. Both phenotypes cosegregate in meiosis, corevert with a frequency of 10(-7), and are complemented by a 3.5-kilobase fragment of genomic DNA from wild-type S. cerevisiae. This fragment contains FRE1, the wild-type allele of the mutant gene. The level of the gene transcript is regulated by iron in the same was as the reductase activity. The ferrous ion product of the reductase must traverse the plasma membrane. A high-affinity (Km = 5 microM) ferrous uptake system is present in both wild-type and mutant cells. Thus, iron uptake in S. cerevisiae is mediated by two plasma membrane components, a reductase and a ferrous transport system.

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Iron Acquisition in Mycobacterium avium subsp. paratuberculosis

Journal of Bacteriology ◽

10.1128/jb.00922-15 ◽

2015 ◽

Vol 198 (5) ◽

pp. 857-866 ◽

Cited By ~ 10

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.

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Identification and Characterization of a Streptococcus pyogenes Operon Involved in Binding of Hemoproteins and Acquisition of Iron

Infection and Immunity ◽

10.1128/iai.71.3.1042-1055.2003 ◽

2003 ◽

Vol 71 (3) ◽

pp. 1042-1055 ◽

Cited By ~ 101

Author(s):

Christopher S. Bates ◽

Griselle E. Montañez ◽

Charles R. Woods ◽

Rebecca M. Vincent ◽

Zehava Eichenbaum

Keyword(s):

Hydrogen Peroxide ◽

Streptococcus Pyogenes ◽

Iron Acquisition ◽

Iron Complex ◽

Surface Proteins ◽

Leader Peptide ◽

Hemoglobin Binding ◽

Membrane Bound ◽

Identification And Characterization

ABSTRACT The hemolytic Streptococcus pyogenes can use a variety of heme compounds as an iron source. In this study, we investigate hemoprotein utilization by S. pyogenes. We demonstrate that surface proteins contribute to the binding of hemoproteins to S. pyogenes. We identify an ABC transporter from the iron complex family named sia for streptococcal iron acquisition, which consists of a lipoprotein (siaA), membrane permease (siaB), and ATPase (siaC). The sia transporter is part of a highly conserved, iron regulated, 10-gene operon. SiaA, which was localized to the cell membrane, could specifically bind hemoglobin. The operon's first gene encodes a novel bacterial protein that bound hemoglobin, myoglobin, heme-albumin, and hemoglobin-haptoglobin (but not apo-haptoglobin) and therefore was named Shr, for streptococcal hemoprotein receptor. PhoZ fusion and Western blot analysis showed that Shr has a leader peptide and is found in both membrane-bound and soluble forms. An M1 SF370 strain with a polar mutation in shr was more resistant to streptonigrin and hydrogen peroxide, suggesting decreased iron uptake. The addition of hemoglobin to the culture medium increased cell resistance to hydrogen peroxide in SF370 but not in the mutant, implying the sia operon may be involved in hemoglobin-dependent resistance to oxidative stress. The shr mutant demonstrated reduced hemoglobin binding, though cell growth in iron-depleted medium supplemented with hemoglobin, whole blood, or ferric citrate was not affected, suggesting additional systems are involved in hemoglobin utilization. SiaA and Shr are the first hemoprotein receptors identified in S. pyogenes; their possible role in iron capture is discussed.

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Transferrin-mediated iron acquisition by pathogenic Neisseria

Biochemical Society Transactions ◽

10.1042/bst0300705 ◽

2002 ◽

Vol 30 (4) ◽

pp. 705-707 ◽

Cited By ~ 6

Author(s):

R. W. Evans ◽

J. S. Oakhill

Keyword(s):

Bacterial Cell ◽

Iron Uptake ◽

Binding Proteins ◽

Current Knowledge ◽

Iron Acquisition ◽

Direct Interaction ◽

Acquisition System ◽

Uptake System ◽

Short Account ◽

Transferrin Binding Proteins

The pathogenic Neisseria have a siderophore-independent iron-uptake system reliant on a direct interaction between the bacterial cell and transferrin. In the meningococcus this uptake system is dependent on two surface-exposed transferrin-binding proteins. This short account will review our current knowledge of the transferrin-mediated iron-acquisition system of pathogenic Neisseria.

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