A novel hybrid iron regulation network combines features from pathogenic and nonpathogenic yeasts

Aft1 is the main positive regulator under iron starvation conditions in C. glabrata.

“Specifically, similarly to baker’s yeast, Aft1 is the main positive regulator under iron starvation conditions, while Cth2 degrades mRNAs encoding iron-requiring enzymes.”

A Sef1 ortholog is required for full growth under iron limitation conditions in C. glabrata.

“However, unlike the case with S. cerevisiae, a Sef1 ortholog is required for full growth under iron limitation conditions, making C. glabrata an evolutionary intermediate to SEF1-dependent fungal pathogens.”

Category 2 contained 31 mutants with general growth defects that were independent of iron supplementation.

“Category 2 contained another 31 mutants with general growth defects that were independent of iron supplementation (Fig. 1b), including deletions of known and iron-associated genes (YFH1, ATM1) conserved in most fungi such as C. albicans (28, 29) and S. cerevisiae (30, 31).”

• cites [28] Santos R, Buisson N, Knight SA, Dancis A, Camadro JM, Lesuisse E 2004 Candida albicans lacking the frataxin homologue: a relevant yeast model for studying the role of frataxin. Mol Microbiol 54: 507– 519. doi: 10.1111/j.1365-2958.2004.04281.x. 15469520 sourceNot checked yet

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• cites [29] Lan CY, Rodarte G, Murillo LA, Jones T, Davis RW, Dungan J, Newport G, Agabian N 2004 Regulatory networks affected by iron availability in Candida albicans. Mol Microbiol 53: 1451– 1469. doi: 10.1111/j.1365-2958.2004.04214.x. 15387822 sourceNot checked yet

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• cites [30] Adamec J, Rusnak F, Owen WG, Naylor S, Benson LM, Gacy AM, Isaya G 2000 Iron-dependent self-assembly of recombinant yeast frataxin: implications for Friedreich ataxia. Am J Hum Genet 67: 549– 562. doi: 10.1086/303056. 10930361 PMC1287515 sourceNot checked yet

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• cites [31] Ueta R, Fujiwara N, Iwai K, Yamaguchi-Iwai Y 2012 Iron-induced dissociation of the Aft1p transcriptional regulator from target gene promoters is an initial event in iron-dependent gene suppression. Mol Cell Biol 32: 4998– 5008. doi: 10.1128/MCB.00726-12. 23045394 PMC3510542 sourceNot checked yet

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Genes involved in the TCA cycle and glutamate biosynthesis (ACO1, IDH1, IDH2), as well as genes with iron sulfur cluster-dependent functions (ACO1, ISA1), were transcribed to a lesser degree under iron starvation conditions upon SEF1 deletion.

“Genes involved in the TCA cycle and in glutamate biosynthesis in a broader sense (ACO1, IDH1, IDH2), as well as genes with iron sulfur cluster-dependent functions (ACO1, ISA1), were transcribed to a lesser degree under iron starvation conditions upon SEF1 deletion.”

Growth of the hap4Δ and hap5Δ mutants was impaired on the nonfermentative carbon sources glycerol, lactate, ethanol, and acetate.

“Instead, growth of the hap4Δ and hap5Δ mutants was impaired on the nonfermentative carbon sources glycerol, lactate, ethanol, and acetate, which require respiration for energy generation, indicating a role for these genes in respiration similar to that in S. cerevisiae.”

A search for this motif in intergenic regions of all known C. glabrata ORFs detected additional iron metabolism-associated genes such as AFT1, SEF1, ACO1, and CCC1.

“A search for this motif in the intergenic regions of all known C. glabrata ORFs (see Dataset S4 in the supplemental material) detected additional iron metabolism-associated genes such as AFT1, SEF1, ACO1, and CCC1 (Fig. 4c).”

Candidate regulator knockout strains other than aft1Δ and sef1Δ did not show any growth defects under low-iron conditions.

“These candidate regulator knockout strains were included in the growth screen along with aft1Δ and sef1Δ mutants but did not show any growth defects under low-iron conditions (see Dataset S1 in the supplemental material).”

Under replete-iron conditions, some genes coding for iron-requiring enzymes (CCP1, CYT1, HEM15, CCC1, ACO1) showed a tendency of increased transcription in the sef1Δ mutant compared to the WT.

“Under replete-iron conditions, interestingly, some genes coding for iron-requiring enzymes (CCP1, CYT1, HEM15, CCC1, ACO1) showed a tendency of increased transcription in the sef1Δ mutant compared to the WT (see Dataset S3c in the supplemental material), pointing to a possible role of Sef1 in the “fine-tuning” of iron consumption processes.”

These genes were significantly less transcribed in the aft1Δ mutant than in the WT after 4 h.

“However, these genes were significantly less transcribed in the aft1Δ mutant than in the WT after 4 h.”

A large set of genes involved in iron-consuming processes (e.g., CYC1, COX6, CCP1, CCC1, HEM15) were strongly downregulated in WT cells upon iron starvation but not in aft1Δ cells.

“Remarkably, there was a large set of genes involved in iron-consuming processes (e.g., CYC1, COX6, CCP1, CCC1, HEM15), and those genes were strongly downregulated in the WT cells upon iron starvation but not in aft1Δ cells.”

At 4 h after a shift from 5 μM FeCl3 to iron-free medium, genes involved in iron uptake processes (FTR1, FET3, SIT1) and iron recycling processes (FTH1, HMX1, SMF3) were strongly upregulated in the WT.

“At 4 h after a shift from 5 µM FeCl3 to iron-free medium, genes involved in iron uptake processes (FTR1, FET3, SIT1) and iron recycling processes (FTH1, HMX1, SMF3) were strongly upregulated in the WT (Fig. 4a).”

Transcriptional downregulation was verified by qRT-PCR for ACO1, IDH2, and ISA1.

“Transcriptional downregulation was verified by quantitative reverse transcription-PCR (qRT-PCR) for the aconitase gene ACO1, the isocitrate dehydrogenase gene IDH2, and the gene encoding the iron sulfur cluster protein Isa1.”

Deletion of SEF1 had only subtle effects on the transcription profile under iron deprivation conditions.

“Despite its phenotypic effects on growth and iron content, deletion of SEF1 had only subtle effects on the transcription profile under iron deprivation conditions.”

Many genes associated with iron uptake or recycling contain one or more copies of the Aft1 consensus-binding motif PyPuCACCCPu in their promoter regions.

“We found many genes associated with iron uptake or recycling containing one or more copies of this binding motif in their promoter regions (marked with an asterisk [*] in Fig. 4a).”

Intracellular iron content was decreased only in the aft1Δ and sef1Δ strains among C. glabrata regulator knockout strains.

“Finally, we determined the intracellular iron content for all C. glabrata regulator knockout strains and found decreased iron content only in the aft1Δ and sef1Δ strains (Fig. 2e).”

Category 3 contained 36 mutants with growth defects under low-iron conditions that were rescued by iron addition.

“Most relevant to this work, category 3 contained 36 mutants with growth defects under low-iron conditions that were rescued by iron addition (Fig. 1c).”

In silico screening of the C. glabrata genome identified seven candidate orthologs of known iron-associated regulators in C. albicans and S. cerevisiae.

“Therefore, we screened the C. glabrata genome in silico for potential orthologs of the known iron-associated regulators in C. albicans and S. cerevisiae and identified seven candidates.”

The S. cerevisiae sef1Δ mutant results were indistinguishable from the WT results under all conditions.

“In contrast, the S. cerevisiae sef1Δ mutant results were indistinguishable from the WT results under all conditions (Fig. 3b).”

Category 1 contained 31 mutants with growth defects under specifically near-toxic iron conditions.

“Category 1 contained 31 mutants with growth defects under specifically near-toxic iron conditions (Fig. 1a).”

Growth curve analysis showed a defect under low-iron conditions (5 μM and 10 μM FeCl3) for a C. glabrata sef1Δ mutant which was restored to wild-type levels by addition of 100 μM FeCl3.

“Growth curve analysis showed growth representative of a defect under low-iron conditions (5 µM and 10 µM FeCl3) for a C. glabrata sef1Δ mutant which was restored to wild-type (WT) levels by addition of 100 µM FeCl3 (Fig. 3a).”

GO-term analysis of category 3 revealed enrichment of genes involved in transcription regulation, chromatin remodeling, and iron assimilation.

“GO-term analysis revealed an enrichment of genes involved in transcription regulation, chromatin remodeling (CYC8, PHO23), and—as expected—iron assimilation.”

The iron acquisition genes FTR1 and FET3 as well as AFT1 and SEF1 were among the category 3 mutants.

“These included the iron acquisition genes FTR1 and FET3 as well as AFT1 and SEF1, encoding orthologs of activators of iron acquisition in S. cerevisiae and C. albicans, respectively.”

The motif 5′-TTATTTATT-3′ was located in the 3′ UTR of 36 of 106 ORFs downregulated in WT at 4 h but with at least 2-fold higher expression in the aft1Δ mutant.

“We found the motif 5′-TTATTTATT-3′ to be located in the 3′ untranscribed region (UTR) of 36 of these ORFs (E value 1.7 × 10−5) in single or multiple copies.”

GO-term analysis revealed an enrichment of processes associated with cell wall organization and integrity in category 1.

“Gene ontology (GO)-term analysis revealed an enrichment of processes associated with cell wall organization and integrity in this category.”

Expression of SEF1 was elevated in both aft1Δ and cth2Δ strains of C. glabrata.

“Furthermore, expression of SEF1 was elevated in both aft1Δ and cth2Δ strains of C. glabrata.”

Under iron deprivation, mRNA levels of FTR1, SIT1, HMX1, and FTH1 are consistently elevated in the cth2Δ mutant.

“As the mRNA levels of all of these genes were also consistently elevated in the cth2Δ mutant”

In addition to upregulation of iron acquisition genes, the study observed marked downregulation of genes involved in iron consumption processes.

“In addition to the upregulation of iron acquisition genes, we also observed a marked downregulation of genes involved in iron consumption processes.”

C. glabrata has no ortholog of CTH1.

“Interestingly, C. glabrata has no ortholog of CTH1.”

A reduction of aft1Δ survival is observable also at later time points, albeit without reaching the statistical significance levels of the earlier samples.

“A reduction of aft1Δ survival was observable also at later time points, albeit without reaching the statistical significance levels of the earlier samples.”

Under iron deprivation, transcript levels of iron consumption genes CCP1, CYT1, HEM15, and CCC1 are much higher in the aft1Δ mutant than in WT.

“but transcript levels were much higher in the aft1Δ mutant.”

Lack of CTH2 does not influence the already low expression levels of iron acquisition genes under iron-replete conditions.

“Consistently, lack of CTH2 did not influence their already low expression levels under iron-replete conditions (see Dataset S3b in the supplemental material).”

Cth2 has a moderate effect on mRNA levels of iron acquisition genes in C. glabrata.

“Additionally, we have also observed a moderate effect of Cth2 on the mRNA levels of iron acquisition genes in C. glabrata.”

Deletion of CTH2 does not affect survival in human blood at any time point.

“whereas, remarkably, deletion of CTH2 did not affect survival at any time point.”

The sef1Δ mutant shows significantly attenuated survival early during exposure to human blood, at 1 h and with a tendency already seen at 0.5 h.

“Survival of the sef1Δ mutant was likewise significantly attenuated early during exposure to blood (at 1 h and with the tendency already seen at 0.5 h)”

Growth of a mutant lacking CTH2 was strongly impaired under low-iron conditions in liquid culture.

“Growth of a mutant lacking CTH2 was strongly impaired under low-iron conditions in liquid culture (Fig. 4d), and the mutant showed susceptibility to BPS and alkaline growth conditions (Fig. 4e).”

CTH2 transcription was highly upregulated upon iron starvation in the WT but not in the aft1Δ mutant.

“In agreement with this, CTH2 transcription was highly upregulated upon iron starvation in the WT but not in the aft1Δ mutant (Fig. 4a).”

Under iron deprivation, mRNA levels of iron consumption genes CCP1, CYT1, HEM15, and CCC1 are downregulated in the WT.

“the mRNA levels of these genes were downregulated upon iron deprivation in the WT”

The study confirmed important roles of FTR1, FET3, YFH1, and ATM1 in iron homeostasis.

“We were able to confirm the important roles of various iron-associated genes described previously in C. glabrata, such as FTR1, FET3, and YFH1 (40), or in C. albicans and S. cerevisiae, such as ATM1 (29, 31).”

• cites [40] Srivastava VK, Suneetha KJ, Kaur R 2014 A systematic analysis reveals an essential role for high-affinity iron uptake system, haemolysin and CFEM domain-containing protein in iron homoeostasis and virulence in Candida glabrata. Biochem J 463: 103– 114. doi: 10.1042/BJ20140598. 24987864 sourceNot checked yet

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• cites [29] Lan CY, Rodarte G, Murillo LA, Jones T, Davis RW, Dungan J, Newport G, Agabian N 2004 Regulatory networks affected by iron availability in Candida albicans. Mol Microbiol 53: 1451– 1469. doi: 10.1111/j.1365-2958.2004.04214.x. 15387822 sourceNot checked yet

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• cites [31] Ueta R, Fujiwara N, Iwai K, Yamaguchi-Iwai Y 2012 Iron-induced dissociation of the Aft1p transcriptional regulator from target gene promoters is an initial event in iron-dependent gene suppression. Mol Cell Biol 32: 4998– 5008. doi: 10.1128/MCB.00726-12. 23045394 PMC3510542 sourceNot checked yet

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Under iron deprivation, lack of CTH2 results in a significant and comparable increase in mRNA levels of iron consumption genes CCP1, CYT1, HEM15, and CCC1.

“Similarly, lack of CTH2 resulted in a significant and comparable increase in mRNA levels.”

Under iron-replete conditions, iron consumption processes are mainly upregulated in the WT.

“Under iron-replete conditions, the iron consumption processes were mainly upregulated in the WT.”

Lack of AFT1 abolished expression of CTH2.

“In fact, qRT-PCR showed that lack of AFT1 abolished expression of CTH2 (Fig. 5a).”

The aft1Δ mutant shows a clear and significant reduction in early survival in human blood.

“There, the aft1Δ mutant showed a clear and significant reduction in early survival in blood.”

In the C. glabrata aft1Δ mutant, iron acquisition genes FTR1, SIT1, HMX1, and FTH1 lack induction under iron deprivation, as confirmed by qRT-PCR.

“Microarray transcriptional profiling analysis of the aft1Δ mutant had indicated a lack of induction of iron acquisition genes under iron deprivation conditions, which we confirmed by qRT-PCR for FTR1, SIT1, HMX1, and FTH1 (Fig. 5b).”

Aft1 is an essential transcription factor of C. glabrata necessary for growth under low-iron conditions via activation of iron uptake and intracellular recycling.

“Strikingly, we identified Aft1 as an essential transcription factor of C. glabrata necessary for growth under low-iron conditions via activation of iron uptake and intracellular recycling.”

The highly conserved motif 5′-UUAUUUAUU-3′ is present in the 3′ UTR of target mRNAs of Cth2 in C. glabrata, as in S. cerevisiae.

“As in S. cerevisiae, we found the highly conserved motif 5′-UUAUUUAUU-3′ in the 3′ UTR of target mRNAs of Cth2.”

In C. glabrata, very specific changes in gene expression were observed for genes associated with the TCA cycle (ACO1, IDH1, IDH2) and for genes encoding known iron sulfur cluster-containing proteins (ACO1, ISA1) in the sef1Δ mutant.

“However, at least in C. glabrata, very specific changes in gene expression were observed for genes associated with the (iron-requiring) TCA cycle (ACO1, IDH1, IDH2) as well as for genes encoding known iron sulfur cluster-containing proteins (ACO1, ISA1).”

Most but not all decreases in mRNA level were dependent on the presence of Cth2.

“Further factors may play a role in this iron-level-dependent transcript degradation process, as most but not all decreases in the mRNA level were dependent on the presence of Cth2.”

Despite its phenotype, the transcriptome of the sef1Δ mutant did not differ strongly from that of the WT.

“Surprisingly, despite its phenotype, the transcriptome of the sef1Δ mutant did not differ strongly from that of the WT.”

C. glabrata lacks a second copy of CTH2.

“C. glabrata is lacking a second copy of CTH2”

Growth defects of the sef1Δ mutant under iron deprivation and alkaline pH, as well as in silico analysis, place Sef1 in the C. glabrata iron homeostasis regulatory network.

“Growth defects of the sef1Δ mutant under conditions of iron deprivation and of alkaline pH (with its concomitant low iron solubility), as well as the in silico analysis, place it firmly in the C. glabrata iron homeostasis regulatory network.”

In contrast to S. cerevisiae, the Cth2-binding motif is present in FTR1, FET3, and SIT1 transcripts in C. glabrata.

“In contrast to S. cerevisiae (24), the Cth2-binding motif is present in FTR1, FET3, and SIT1 transcripts”

• cites [24] Puig S, Askeland E, Thiele DJ 2005 Coordinated remodeling of cellular metabolism during iron deficiency through targeted mRNA degradation. Cell 120: 99– 110. doi: 10.1016/j.cell.2004.11.032. 15652485 sourceNot checked yet

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C. glabrata is much more closely related to Saccharomyces cerevisiae than to Candida albicans.

“In fact, C. glabrata is much more closely related to the normally nonpathogenic baker’s yeast Saccharomyces cerevisiae than to C. albicans (9), and their respective pathogenicity strategies must have evolved independently.”

• cites [9] Dujon B, Sherman D, Fischer G, Durrens P, Casaregola S, Lafontaine I, De Montigny J, Marck C, Neuvéglise C, Talla E, Goffard N, Frangeul L, Aigle M, Anthouard V, Babour A, Barbe V, Barnay S, Blanchin S, Beckerich JM, Beyne E, Bleykasten C, Boisrame A, Boyer J, Cattolico L, Confanioleri F, De Daruvar A, Despons L, Fabre E, Fairhead C, Ferry-Dumazet H, Groppi A, Hantraye F, Hennequin C, Jauniaux N, Joyet P, Kachouri R, Kerrest A, Koszul R, Lemaire M, Lesur I, Ma L, Muller H, Nicaud JM, Nikolski M, Oztas S, Ozier-Kalogeropoulos O, Pellenz S, Potier S, Richard GF, Straub ML 2004 Genome evolution i sourceNot checked yet

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C. glabrata is an evolutionary intermediate to SEF1-dependent fungal pathogens.

“However, unlike the case with S. cerevisiae, a Sef1 ortholog is required for full growth under iron limitation conditions, making C. glabrata an evolutionary intermediate to SEF1-dependent fungal pathogens.”

C. glabrata uses a regulation network largely consisting of components of the S. cerevisiae regulon but also of elements of other pathogenic fungi.

“Here, we show that C. glabrata uses a regulation network largely consisting of components of the S. cerevisiae regulon but also of elements of other pathogenic fungi.”

Cth2 degrades mRNAs encoding iron-requiring enzymes in C. glabrata.

“Specifically, similarly to baker’s yeast, Aft1 is the main positive regulator under iron starvation conditions, while Cth2 degrades mRNAs encoding iron-requiring enzymes.”

C. glabrata has evolved an iron homeostasis system which seems to be unique within the pathogenic fungi.

“Therefore, C. glabrata has evolved an iron homeostasis system which seems to be unique within the pathogenic fungi.”

Sef1 seems to regulate specific iron-associated processes such as the TCA cycle under iron restriction conditions.

“Additionally, Sef1 seems to regulate specific iron-associated processes such as the TCA cycle under iron restriction conditions.”

Aft1 and Sef1 have different regulatory functions under iron starvation conditions.

“Aft1 and Sef1 have different regulatory functions under iron starvation conditions.”

S. cerevisiae Cth2, along with its paralog Cth1, targets mRNA of iron-dependent genes for degradation via binding to this motif.

“S. cerevisiae Cth2 (ScCth2), along with its paralog Cth1, was found specifically to target mRNA of iron-dependent genes for degradation via binding to this motif (22, 24), whereas the corresponding Cth2 protein in C. glabrata was still uncharacterized.”

• cites [22] Shakoury-Elizeh M, Tiedeman J, Rashford J, Ferea T, Demeter J, Garcia E, Rolfes R, Brown PO, Botstein D, Philpott CC 2004 Transcriptional remodeling in response to iron deprivation in Saccharomyces cerevisiae. Mol Biol Cell 15: 1233– 1243. doi: 10.1091/mbc.E03-09-0642. 14668481 PMC363115 sourceNot checked yet

  Not checked in the free run.

• cites [24] Puig S, Askeland E, Thiele DJ 2005 Coordinated remodeling of cellular metabolism during iron deficiency through targeted mRNA degradation. Cell 120: 99– 110. doi: 10.1016/j.cell.2004.11.032. 15652485 sourceNot checked yet

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These data indicate a potential role for Sef1 in TCA cycle regulation.

“These data indicate a potential role for Sef1 in TCA cycle regulation (Fig. 4b).”

C. glabrata is the first known pathogen engaging an Aft1-dependent system as the main iron regulation strategy.

“To our knowledge, C. glabrata is the first known pathogen engaging an Aft1-dependent system as the main iron regulation strategy.”

Cth2 is strictly needed for downregulating iron consumption processes under iron restriction conditions.

“Taking the results together, Cth2 is strictly needed for downregulating iron consumption processes under iron restriction conditions.”

Both Aft1 and Sef1 are required for full survival in human blood, especially during the early interaction.

“Hence, both Aft1 and Sef1 seem to be required for full survival in blood, especially during the early interaction.”

In both C. glabrata and S. cerevisiae, Cth2 target transcripts code especially for proteins with a role in iron consumption.

“In both species, these are transcripts coding especially for proteins with a role in iron consumption (24).”

• cites [24] Puig S, Askeland E, Thiele DJ 2005 Coordinated remodeling of cellular metabolism during iron deficiency through targeted mRNA degradation. Cell 120: 99– 110. doi: 10.1016/j.cell.2004.11.032. 15652485 sourceNot checked yet

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C. glabrata Cth2 is a potential Aft1-induced mediator of mRNA degradation of iron-associated genes, a group which includes SEF1.

“Taking the results together, we have identified C. glabrata Cth2 as a potential Aft1-induced mediator of mRNA degradation of iron-associated genes, a group which includes SEF1.”

Most candidate genes identified in this study have not been associated previously with iron homeostasis in C. glabrata.

“However, most of our candidate genes have not been associated previously with iron homeostasis in C. glabrata.”

The data point to continuous degradation of highly transcribed mRNAs involved in iron uptake processes.

“Additionally, our data point to a continuous degradation of highly transcribed mRNAs involved in iron uptake processes.”

The observations indicate a tightly controlled and timed response to iron deprivation, probably depending on the increasing magnitude of intracellular iron deficiency.

“These observations indicate a tightly controlled and timed response to iron deprivation, probably depending on the increasing magnitude of intracellular iron deficiency.”

C. glabrata uses an Aft1-dominated system for iron homeostasis, like S. cerevisiae.

“Hence, we were able to show that C. glabrata, like its closest relative—the baker’s yeast S. cerevisiae—uses an Aft1-dominated system for iron homeostasis.”

Cth2 exerts posttranscriptional mRNA degradation on mRNA transcripts of highly expressed iron acquisition genes under iron starvation conditions.

“Cth2 seems to exert its posttranscriptional mRNA degradation on mRNA transcripts of highly expressed iron acquisition genes under iron starvation conditions.”

A shortage of iron represents a common challenge in most host niches of C. glabrata.

“A shortage of iron represents a common challenge in most host niches of C. glabrata (41, 42)”

• cites [41] Nevitt T 2011 War-Fe-re: iron at the core of fungal virulence and host immunity. Biometals 24: 547– 558. doi: 10.1007/s10534-011-9431-8. 21399939 sourceNot checked yet

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• cites [42] Pantopoulos K, Porwal SK, Tartakoff A, Devireddy L 2012 Mechanisms of mammalian iron homeostasis. Biochemistry 51: 5705– 5724. doi: 10.1021/bi300752r. 22703180 PMC3572738 sourceNot checked yet

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C. glabrata is the first example of a pathogenic fungus using its Cth2 ortholog to downregulate iron homeostasis-associated transcripts.

“To our knowledge, C. glabrata is thus the first example of a pathogenic fungus using its Cth2 ortholog to downregulate iron homeostasis-associated transcripts.”

The findings suggest a novel hybrid iron regulation system for C. glabrata combining features from S. cerevisiae (Aft1) and C. albicans (Sef1).

“These findings strongly suggest a novel hybrid iron regulation system for C. glabrata which combines features from the networks of S. cerevisiae (Aft1) and C. albicans (Sef1).”

Since Cth2 carries its own recognition motif, an autoregulatory loop seems likely.

“Since Cth2 carries its own recognition motif, an autoregulatory loop seems likely.”

C. albicans possesses a single Cth2 ortholog called Zfs1.

“Remarkably, C. albicans also possesses a single Cth2 ortholog called Zfs1.”

Zfs1 target transcripts are not related to iron homeostasis but rather to biofilm formation.

“However, Zfs1 target transcripts are not related to iron homeostasis but rather to biofilm formation (45).”

• cites [45] Wells ML, Washington OL, Hicks SN, Nobile CJ, Hartooni N, Wilson GM, Zucconi BE, Huang W, Li L, Fargo DC, Blackshear PJ 2015 Post-transcriptional regulation of transcript abundance by a conserved member of the tristetraprolin family in Candida albicans. Mol Microbiol 95: 1036– 1053. doi: 10.1111/mmi.12913. 25524641 PMC4557868 sourceNot checked yet

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The only other fungus with a functionally described Cth2 ortholog, S. pombe, displays no transcriptional alterations of its iron homeostasis genes when this gene is deleted.

“The only other fungus with a functionally described Cth2 ortholog, Schizosaccharomyces pombe, also displays no transcriptional alterations of its iron homeostasis genes when this gene is deleted (46, 47).”

• cites [46] Cuthbertson BJ, Liao Y, Birnbaumer L, Blackshear PJ 2008 Characterization of zfs1 as an mRNA-binding and -destabilizing protein in Schizosaccharomyces pombe. J Biol Chem 283: 2586– 2594. doi: 10.1074/jbc.M707154200. 18042546 PMC2736560 sourceNot checked yet

  Not checked in the free run.

• cites [47] Wells ML, Huang W, Li L, Gerrish KE, Fargo DC, Ozsolak F, Blackshear PJ 2012 Posttranscriptional regulation of cell-cell interaction protein-encoding transcripts by Zfs1p in Schizosaccharomyces pombe. Mol Cell Biol 32: 4206– 4214. doi: 10.1128/MCB.00325-12. 22907753 PMC3457339 sourceNot checked yet

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Cth2 mediates posttranscriptional degradation of specific transcripts carrying a conserved recognition motif.

“Subsequently, Cth2 mediates posttranscriptional degradation of specific transcripts carrying a conserved recognition motif.”

C. glabrata is not known to use hemoglobin or transferrin for iron acquisition, in contrast to C. albicans.

“Especially for C. glabrata, which (in contrast to C. albicans) is not known to use hemoglobin or transferrin for iron acquisition (8, 15)”

• cites [8] Brunke S, Hube B 2013 Two unlike cousins: Candida albicans and C. Glabrata infection strategies. Cell Microbiol 15: 701– 708. doi: 10.1111/cmi.12091. 23253282 PMC3654559 sourceNot checked yet

  Not checked in the free run.

• cites [15] Nevitt T, Thiele DJ 2011 Host iron withholding demands siderophore utilization for Candida glabrata to survive macrophage killing. PLoS Pathog 7: e01782-16. doi: 10.1371/journal.ppat.1001322. PMC3060170 21445236 sourceNot checked yet

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Other pathogenic fungi such as A. fumigatus, C. neoformans, and C. albicans generally rely on iron-dependent regulation via GATA factor orthologs and the CCAAT-binding complex.

“In contrast, other pathogenic fungi such as Aspergillus fumigatus, Cryptococcus neoformans, and C. albicans generally rely on iron-dependent regulation via GATA factor orthologs and the CCAAT-binding complex (12, 48–51).”

• cites [12] Schrettl M, Beckmann N, Varga J, Heinekamp T, Jacobsen ID, Jöchl C, Moussa TA, Wang S, Gsaller F, Blatzer M, Werner ER, Niermann WC, Brakhage AA, Haas H 2010 HapX-mediated adaption to iron starvation is crucial for virulence of Aspergillus fumigatus. PLoS Pathog 6: e01782-16. doi: 10.1371/journal.ppat.1001124. PMC2947994 20941352 sourceNot checked yet

  Not checked in the free run.

• cites [48] Baek YU, Li M, Davis DA 2008 Candida albicans ferric reductases are differentially regulated in response to distinct forms of iron limitation by the Rim101 and CBF transcription factors. Eukaryot Cell 7: 1168– 1179. doi: 10.1128/EC.00108-08. 18503007 PMC2446673 sourceNot checked yet

  Not checked in the free run.

• cites [49] Chen C, Noble SM 2012 Post-transcriptional regulation of the Sef1 transcription factor controls the virulence of Candida albicans in its mammalian host. PLoS Pathog 8: e01782-16. doi: 10.1371/journal.ppat.1002956. PMC3486892 23133381 sourceNot checked yet

  Not checked in the free run.

• cites [50] Jung WH, Saikia S, Hu G, Wang J, Fung CK, D’Souza C, White R, Kronstad JW 2010 HapX positively and negatively regulates the transcriptional response to iron deprivation in Cryptococcus neoformans. PLoS Pathog 6: e01782-16. doi: 10.1371/journal.ppat.1001209. PMC2991262 21124817 sourceNot checked yet

  Not checked in the free run.

• cites [51] Hortschansky P, Eisendle M, Al-Abdallah Q, Schmidt AD, Bergmann S, Thön M, Kniemeyer O, Abt B, Seeber B, Werner ER, Kato M, Brakhage AA, Haas H 2007 Interaction of HapX with the CCAAT-binding complex—a novel mechanism of gene regulation by iron. EMBO J 26: 3157– 3168. doi: 10.1038/sj.emboj.7601752. 17568774 PMC1914100 sourceNot checked yet

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Survival of C. glabrata in blood is likely dependent on tight and precise regulation of the balance between iron uptake and consumption.

“survival in this environment is likely dependent on tight and precise regulation of the balance between iron uptake and consumption.”

The presence of the Cth2-binding motif in FTR1, FET3, and SIT1 transcripts points to a novel role of Cth2 in posttranscriptional fine-tuning of iron uptake likely via increased turn-over of mRNA.

“pointing to a novel role in posttranscriptional fine-tuning of iron uptake likely via an increased turn-over of mRNA”