Unexpected Link between Iron and Drug Resistance of Candida spp.: Iron Depletion Enhances Membrane Fluidity and Drug Diffusion, Leading to Drug-Susceptible
Cells

ABSTRACT Inthis study, we show that iron depletion in Candida albicans with bathophenanthrolene disulfonic acid and ferrozine as chelators enhanced its sensitivity to several drugs, including the most common antifungal, fluconazole (FLC). Several other species of Candida also displayed increased sensitivity to FLC because of iron restriction. Iron uptake mutations, namely,Δ ftr1 and Δftr2, as well as the copper transporter mutation Δccc2, which affects high-affinity iron uptake in Candida, produced increased sensitivity to FLC compared to that of the wild type. The effect of iron depletion on drug sensitivity appeared to be independent of the efflux pump proteins Cdr1p and Cdr2p. We found that iron deprivation led to lowering of membrane ergosterol by 15 to 30%. Subsequently, fluorescence polarization measurements also revealed that iron-restricted Candida cells displayed a 29 to 40% increase in membrane fluidity, resulting in enhanced passive diffusion of the drugs. Northern blot assays revealed that the ERG11 gene was considerably down regulated in iron-deprived cells, which might account for the lowered ergosterol content. Our results show a close relationship between cellular iron and drug susceptibilities of C. albicans. Considering that multidrug resistance is a manifestation of multifactorial phenomena, the influence of cellular iron on the drug susceptibilities of Candida suggests iron as yet another novel determinant of multidrug resistance.

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Expression Patterns in Reductive Iron Assimilation and Functional Consequences during Phagocytosis of Lichtheimia corymbifera, an Emerging Cause of Mucormycosis

Journal of Fungi ◽

10.3390/jof7040272 ◽

2021 ◽

Vol 7 (4) ◽

pp. 272

Author(s):

Felicia Adelina Stanford ◽

Nina Matthias ◽

Zoltán Cseresnyés ◽

Marc Thilo Figge ◽

Mohamed I. Abdelwahab Hassan ◽

...

Keyword(s):

Gene Expression ◽

Iron Uptake ◽

Intracellular Transport ◽

Pathogenic Fungus ◽

Expression Patterns ◽

Iron Depletion ◽

Iron Assimilation ◽

Human Pathogenic Fungus ◽

Hyphal Formation ◽

Yeast Mutants

Iron is an essential micronutrient for most organisms and fungi are no exception. Iron uptake by fungi is facilitated by receptor-mediated internalization of siderophores, heme and reductive iron assimilation (RIA). The RIA employs three protein groups: (i) the ferric reductases (Fre5 proteins), (ii) the multicopper ferroxidases (Fet3) and (iii) the high-affinity iron permeases (Ftr1). Phenotyping under different iron concentrations revealed detrimental effects on spore swelling and hyphal formation under iron depletion, but yeast-like morphology under iron excess. Since access to iron is limited during pathogenesis, pathogens are placed under stress due to nutrient limitations. To combat this, gene duplication and differential gene expression of key iron uptake genes are utilized to acquire iron against the deleterious effects of iron depletion. In the genome of the human pathogenic fungus L. corymbifera, three, four and three copies were identified for FRE5, FTR1 and FET3 genes, respectively. As in other fungi, FET3 and FTR1 are syntenic and co-expressed in L. corymbifera. Expression of FRE5, FTR1 and FET3 genes is highly up-regulated during iron limitation (Fe-), but lower during iron excess (Fe+). Fe- dependent upregulation of gene expression takes place in LcFRE5 II and III, LcFTR1 I and II, as well as LcFET3 I and II suggesting a functional role in pathogenesis. The syntenic LcFTR1 I–LcFET3 I gene pair is co-expressed during germination, whereas LcFTR1 II- LcFET3 II is co-expressed during hyphal proliferation. LcFTR1 I, II and IV were overexpressed in Saccharomyces cerevisiae to represent high and moderate expression of intracellular transport of Fe3+, respectively. Challenge of macrophages with the yeast mutants revealed no obvious role for LcFTR1 I, but possible functions of LcFTR1 II and IVs in recognition by macrophages. RIA expression pattern was used for a new model of interaction between L. corymbifera and macrophages.

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The TonB system in Aeromonas hydrophila NJ-35 is essential for MacA2B2 efflux pump-mediated macrolide resistance

Veterinary Research ◽

10.1186/s13567-021-00934-w ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Yuhao Dong ◽

Qing Li ◽

Jinzhu Geng ◽

Qing Cao ◽

Dan Zhao ◽

...

Keyword(s):

Aeromonas Hydrophila ◽

Efflux Pump ◽

Fold Increase ◽

Activity Level ◽

Macrolide Resistance ◽

Wild Type ◽

Iron Deprivation ◽

Pump Activity ◽

Tonb System ◽

Increased Sensitivity

AbstractThe TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a significantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no effect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efflux. Compared with the WT strain, deletion of macA2B2, one of two ATP-binding cassette (ABC) types of the macrolide efflux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA2B2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance afforded by the MacA2B2 pump was completely abrogated by tonB123 deletion. In addition, macA2B2 expression was not altered in the ΔtonB123 mutant, indicating that any influence of TonB on MacA2B2-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system significantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA2B2-mediated macrolide efflux.

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CBIO-10. REDUCED IRON EXPORT FUNCTIONS IN A CELL INTRINSIC MANNER TO DRIVE GLIOBLASTOMA GROWTH

Neuro-Oncology ◽

10.1093/neuonc/noaa215.070 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii17-ii17

Author(s):

Katie Troike ◽

Erin Mulkearns-Hubert ◽

Daniel Silver ◽

James Connor ◽

Justin Lathia

Keyword(s):

Tumor Cells ◽

Iron Uptake ◽

Iron Homeostasis ◽

Iron Status ◽

Tumor Grade ◽

Hfe Gene ◽

Iron Release ◽

Female Patients ◽

Cellular Iron

Abstract Glioblastoma (GBM), the most common primary malignant brain tumor in adults, is characterized by invasive growth and poor prognosis. Iron is a critical regulator of many cellular processes, and GBM tumor cells have been shown to modulate expression of iron-associated proteins to enhance iron uptake from the surrounding microenvironment, driving tumor initiation and growth. While iron uptake has been the central focus of previous investigations, additional mechanisms of iron regulation, such as compensatory iron efflux, have not been explored in the context of GBM. The hemochromatosis (HFE) gene encodes a transmembrane glycoprotein that aids in iron homeostasis by limiting cellular iron release, resulting in a sequestration phenotype. We find that HFE is upregulated in GBM tumors compared to non-tumor brain and that expression of HFE increases with tumor grade. Furthermore, HFE mRNA expression is associated with significantly reduced survival specifically in female patients with GBM. Based on these findings, we hypothesize that GBM tumor cells upregulate HFE expression to augment cellular iron loading and drive proliferation, ultimately leading to reduced survival of female patients. To test this hypothesis, we generated Hfe knockdown and overexpressing mouse glioma cell lines. We observed significant alterations in the expression of several iron handling genes with Hfe knockdown or overexpression, suggesting global disruption of iron homeostasis. Additionally, we show that knockdown of Hfe in these cells increases apoptosis and leads to a significant impairment of tumor growth in vivo. These findings support the hypothesis that Hfe is a critical regulator of cellular iron status and contributes to tumor aggression. Future work will include further exploration of the mechanisms that contribute to these phenotypes as well as interactions with the tumor microenvironment. Elucidating the mechanisms by which iron effulx contributes to GBM may inform the development of next-generation targeted therapies.

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Intermediate steps in cellular iron uptake from transferrin. Detection of a cytoplasmic pool of iron, free of transferrin.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)36621-9 ◽

1992 ◽

Vol 267 (30) ◽

pp. 21384-21389

Author(s):

D.R. Richardson ◽

E Baker

Keyword(s):

Iron Uptake ◽

Cellular Iron ◽

Cellular Iron Uptake

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Relationship between Down-Regulation of Copper-Related Genes and Decreased Ferroportin Protein Level in the Duodenum of Iron-Deficient Piglets

Nutrients ◽

10.3390/nu13010104 ◽

2020 ◽

Vol 13 (1) ◽

pp. 104

Author(s):

Aneta Jończy ◽

Rafał Mazgaj ◽

Rafał Radosław Starzyński ◽

Piotr Poznański ◽

Mateusz Szudzik ◽

...

Keyword(s):

Iron Absorption ◽

Underlying Mechanism ◽

Metabolic Effects ◽

Deficiency Anemia ◽

Copper Transporter ◽

Cellular Iron ◽

Iron Deficient ◽

Copper Distribution ◽

Iron Deposits ◽

Neonatal Iron Deficiency

In mammals, 2 × 1012 red blood cells (RBCs) are produced every day in the bone marrow to ensure a constant supply of iron to maintain effective erythropoiesis. Impaired iron absorption in the duodenum and inefficient iron reutilization from senescent RBCs by macrophages contribute to the development of anemia. Ferroportin (Fpn), the only known cellular iron exporter, as well as hephaestin (Heph) and ceruloplasmin, two copper-dependent ferroxidases involved in the above-mentioned processes, are key elements of the interaction between copper and iron metabolisms. Crosslinks between these metals have been known for many years, but metabolic effects of one on the other have not been elucidated to date. Neonatal iron deficiency anemia in piglets provides an interesting model for studying this interplay. In duodenal enterocytes of young anemic piglets, we identified iron deposits and demonstrated increased expression of ferritin with a concomitant decline in both Fpn and Heph expression. We postulated that the underlying mechanism involves changes in copper distribution within enterocytes as a result of decreased expression of the copper transporter—Atp7b. Obtained results strongly suggest that regulation of iron absorption within enterocytes is based on the interaction between proteins of copper and iron metabolisms and outcompetes systemic regulation.

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Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00456-12 ◽

2012 ◽

Vol 56 (8) ◽

pp. 4450-4458 ◽

Cited By ~ 59

Author(s):

Mark Veleba ◽

Paul G. Higgins ◽

Gerardo Gonzalez ◽

Harald Seifert ◽

Thamarai Schneiders

Keyword(s):

Multidrug Resistance ◽

Klebsiella Pneumoniae ◽

Efflux Pump ◽

Content Type ◽

Resistance Phenotype ◽

Serratia Proteamaculans ◽

Virulence Potential ◽

Article Type ◽

Acrab Efflux Pump

ABSTRACTTranscriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes.Klebsiella pneumoniaeis a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription oframAis associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the availableKlebsiellagenome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded inK. pneumoniae,Enterobactersp. 638,Serratia proteamaculans568, andEnterobacter cloacae. We show that the overexpression ofrarAresults in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show thatrarA(MGH 78578 KPN_02968) and its neighboring efflux pump operonoqxAB(KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest thatrarAoverexpression upregulates theoqxABefflux pump. Additionally, it appears thatoqxR, encoding a GntR-type regulator adjacent to theoqxABoperon, is able to downregulate the expression of theoqxABefflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.

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Bifunctional Chelating Supramolecular Polymer and Its Application in Downregulation of Cellular Iron Uptake

Biomacromolecules ◽

10.1021/acs.biomac.7b00765 ◽

2017 ◽

Vol 18 (9) ◽

pp. 2830-2838 ◽

Cited By ~ 2

Author(s):

Fan Xu ◽

Jing Jing ◽

Xiuman Zhou ◽

Yanwu Zhang

Keyword(s):

Iron Uptake ◽

Supramolecular Polymer ◽

Cellular Iron ◽

Cellular Iron Uptake

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Evidence for a copper-dependent iron transport system in the marine, magnetotactic bacterium strain MV-1

Microbiology ◽

10.1099/mic.0.27233-0 ◽

2004 ◽

Vol 150 (9) ◽

pp. 2931-2945 ◽

Cited By ~ 59

Author(s):

Bradley L. Dubbels ◽

Alan A. DiSpirito ◽

John D. Morton ◽

Jeremy D. Semrau ◽

J. N. E. Neto ◽

...

Keyword(s):

Iron Content ◽

Iron Uptake ◽

Iron Transport ◽

Dna Analysis ◽

Iron Concentration ◽

Protein A ◽

Uptake System ◽

Iron Uptake System ◽

Cellular Iron ◽

Solid Media

Cells of the magnetotactic marine vibrio, strain MV-1, produce magnetite-containing magnetosomes when grown anaerobically or microaerobically. Stable, spontaneous, non-magnetotactic mutants were regularly observed when cells of MV-1 were cultured on solid media incubated under anaerobic or microaerobic conditions. Randomly amplified polymorphic DNA analysis showed that these mutants are not all genetically identical. Cellular iron content of one non-magnetotactic mutant strain, designated MV-1nm1, grown anaerobically, was ∼20- to 80-fold less than the iron content of wild-type (wt) MV-1 for the same iron concentrations, indicating that MV-1nm1 is deficient in some form of iron uptake. Comparative protein profiles of the two strains showed that MV-1nm1 did not produce several proteins produced by wt MV-1. To understand the potential roles of these proteins in iron transport better, one of these proteins was purified and characterized. This protein, a homodimer with an apparent subunit mass of about 19 kDa, was an iron-regulated, periplasmic protein (p19). Two potential ‘copper-handling’ motifs (MXM/MX2M) are present in the amino acid sequence of p19, and the native protein binds copper in a 1 : 1 ratio. The structural gene for p19, chpA (copper handling protein) and two other putative genes upstream of chpA were cloned and sequenced. These putative genes encode a protein similar to the iron permease, Ftr1, from the yeast Saccharomyces cerevisiae, and a ferredoxin-like protein of unknown function. A periplasmic, copper-containing, iron(II) oxidase was also purified from wt MV-1 and MV-1nm1. This enzyme, like p19, was regulated by media iron concentration and contained four copper atoms per molecule of enzyme. It is hypothesized that ChpA, the iron permease and the iron(II) oxidase might have analogous functions for the three components of the S. cerevisiae copper-dependent high-affinity iron uptake system (Ctr1, Ftr1 and Fet3, respectively), and that strain MV-1 may have a similar iron uptake system. However, iron(II) oxidase purified from both wt MV-1 and MV-1nm1 displayed comparable iron oxidase activities using O2 as the electron acceptor, indicating that ChpA does not supply the multi-copper iron(II) oxidase with copper.

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