scholarly journals Chromosomal Resistance to Metronidazole in Clostridioides difficile can be Mediated By Epistasis Between Iron Homeostasis and Oxidoreductases

2020 ◽  
Author(s):  
Aditi Deshpande ◽  
Xiaoqian Wu ◽  
Wenwen Huo ◽  
Kelli L. Palmer ◽  
Julian G. Hurdle
Keyword(s):  
Iron Homeostasis ◽  
De Novo ◽  
Synonymous Codon ◽  
Point Mutations ◽  
Resistance Mechanisms ◽  
Intracellular Iron ◽  
Iron Sulfur Cluster ◽  
Clostridioides Difficile ◽  
Genetic Mechanisms ◽  
Level Resistance

AbstractChromosomal resistance to metronidazole has emerged in clinical Clostridioides difficile, but the genetic mechanisms remain unclear. This is further hindered by the inability to generate spontaneous metronidazole-resistant mutants in the lab to aid genetic studies. We therefore constructed a mismatch repair mutator, in non-toxigenic ATCC 700057, to unbiasedly survey the mutational landscape for de novo resistance mechanisms. In separate experimental evolutions, the mutator adopted a deterministic path to resistance, with truncation of ferrous iron transporter FeoB1 as a first-step mechanism of low level resistance. Allelic deletion of feoB1 in ATCC 700057 reduced intracellular iron content, appearing to shift cells toward flavodoxin-mediated oxidoreductase reactions, which are less favorable for metronidazole’s cellular action. Higher level resistance evolved from sequential acquisition of mutations to catalytic domains of pyruvate-ferredoxin oxidoreductase (PFOR encoded by nifJ); a synonymous codon change to xdhA1 (xanthine dehydrogenase subunit A), likely affecting its translation; and lastly, frameshift and point mutations that inactivated the iron-sulfur cluster regulator (IscR). Gene silencing of nifJ, xdhA1 or iscR with catalytically dead Cas9 revealed that resistance involving these genes only occurred when feoB1 was inactivated i.e. resistance was only seen in an feoB1-deletion mutant and not the isogenic wild-type parent. These findings show that metronidazole resistance in C. difficile is complex, involving multi-genetic mechanisms that could intersect with iron-dependent metabolic pathways.

scholarly journals Chromosomal Resistance to Metronidazole in Clostridioides difficile Can Be Mediated by Epistasis between Iron Homeostasis and Oxidoreductases

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Aditi Deshpande ◽  
Xiaoqian Wu ◽  
Wenwen Huo ◽  
Kelli L. Palmer ◽  
Julian G. Hurdle
Keyword(s):  
Iron Homeostasis ◽  
De Novo ◽  
Synonymous Codon ◽  
Point Mutations ◽  
Resistance Mechanisms ◽  
Intracellular Iron ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Metronidazole Resistance ◽  
Level Resistance

ABSTRACT Chromosomal resistance to metronidazole has emerged in clinical Clostridioides difficile isolates, but the genetic mechanisms remain unclear. This is further hindered by the inability to generate spontaneous metronidazole-resistant mutants in the lab to interpret genetic variations in clinical isolates. We therefore constructed a mismatch repair mutator in nontoxigenic ATCC 700057 to survey the mutational landscape for de novo resistance mechanisms. In separate experimental evolutions, the mutator adopted a deterministic path to resistance, with truncation of the ferrous iron transporter FeoB1 as a first-step mechanism of low-level resistance. Deletion of feoB1 in ATCC 700057 reduced the intracellular iron content, appearing to shift cells toward flavodoxin-mediated oxidoreductase reactions, which are less favorable for metronidazole’s cellular action. Higher-level resistance evolved from sequential acquisition of mutations to catalytic domains of pyruvate-ferredoxin/flavodoxin oxidoreductase (PFOR; encoded by nifJ), a synonymous codon change to putative xdh (xanthine dehydrogenase; encoded by CD630_31770), likely affecting mRNA stability, and last, frameshift and point mutations that inactivated the iron-sulfur cluster regulator (IscR). Gene silencing of nifJ, xdh, or iscR with catalytically dead Cas9 revealed that resistance involving these genes occurred only when feoB1 was inactivated; i.e., resistance was seen only in the feoB1 deletion mutant and not in the isogenic wild-type (WT) parent. Interestingly, metronidazole resistance in C. difficile infection (CDI)-associated strains carrying mutations in nifJ was reduced upon gene complementation. This observation supports the idea that mutation in PFOR is one mechanism of metronidazole resistance in clinical strains. Our findings indicate that metronidazole resistance in C. difficile is complex, involving multigenetic mechanisms that could intersect with iron-dependent and oxidoreductive metabolic pathways.

scholarly journals The Yeast Scaffold Proteins Isu1p and Isu2p Are Required inside Mitochondria for Maturation of Cytosolic Fe/S Proteins

2004 ◽  
Vol 24 (11) ◽  
pp. 4848-4857 ◽  
Author(s):  
Jana Gerber ◽  
Karina Neumann ◽  
Corinna Prohl ◽  
Ulrich Mühlenhoff ◽  
Roland Lill
Keyword(s):  
Iron Homeostasis ◽  
De Novo ◽  
Mitochondrial Targeting ◽  
Protein Maturation ◽  
Iron Sulfur Cluster ◽  
Subcellular Compartment ◽  
S Protein ◽  
Iron Sulfur ◽  
Protein Biogenesis ◽  
S Proteins

ABSTRACT Iron-sulfur (Fe/S) proteins are located in mitochondria, cytosol, and nucleus. Mitochondrial Fe/S proteins are matured by the iron-sulfur cluster (ISC) assembly machinery. Little is known about the formation of Fe/S proteins in the cytosol and nucleus. A function of mitochondria in cytosolic Fe/S protein maturation has been noted, but small amounts of some ISC components have been detected outside mitochondria. Here, we studied the highly conserved yeast proteins Isu1p and Isu2p, which provide a scaffold for Fe/S cluster synthesis. We asked whether the Isu proteins are needed for biosynthesis of cytosolic Fe/S clusters and in which subcellular compartment the Isu proteins are required. The Isu proteins were found to be essential for de novo biosynthesis of both mitochondrial and cytosolic Fe/S proteins. Several lines of evidence indicate that Isu1p and Isu2p have to be located inside mitochondria in order to perform their function in cytosolic Fe/S protein maturation. We were unable to mislocalize Isu1p to the cytosol due to the presence of multiple, independent mitochondrial targeting signals in this protein. Further, the bacterial homologue IscU and the human Isu proteins (partially) complemented the defects of yeast Isu protein-depleted cells in growth rate, Fe/S protein biogenesis, and iron homeostasis, yet only after targeting to mitochondria. Together, our data suggest that the Isu proteins need to be localized in mitochondria to fulfill their functional requirement in Fe/S protein maturation in the cytosol.

Genomic mechanisms of resistance to neoadjuvant leuprolide plus abiraterone in locally advanced prostate cancer.

2017 ◽  
Vol 35 (6_suppl) ◽  
pp. 98-98
Author(s):  
Adam G. Sowalsky ◽  
Huihui Ye ◽  
Rachel J. Schaefer ◽  
Olga S. Voznesensky ◽  
Zhenwei Zhang ◽  
...  
Keyword(s):  
De Novo ◽  
Residual Disease ◽  
Locally Advanced ◽  
Point Mutations ◽  
Residual Tumor ◽  
Single Copy ◽  
Resistance Mechanisms ◽  
Copy Number Variations ◽  
Abiraterone Acetate ◽  
Phase 2 Trial

98 Background: Blocking both gonadal and extragonadal androgens with leuprolide and abiraterone acetate (AA) is approved by the FDA to treat mCRPC. Applying this treatment in the neoadjuvant setting reduced intratumoral testosterone levels in our Phase 2 trial, but pathologic CR's were rare and minimal residual disease was observed in some patients. Methods: We performed laser capture microdissection to isolate pure foci of residual tumor cells from 19 patients who underwent RP following 24 weeks of leuprolide plus AA. We also isolated 19 foci of matched benign glands as germline controls. In 15 of the 19 cases, we dissected 2 spatially-distinct foci of residual tumor. We then performed whole exome sequencing to assess somatic mutations and copy number variations (CNVs). Results: A diversity of genomic resistance mechanisms were observed. Resistance to AA as predicted by mutation of the ligand binding domain of AR was observed in only one case. In contrast, the majority residual tumor foci harbored CNVs coinciding with gain of oncogenes or loss of tumor suppressor genes. Importantly, only a limited number of alterations were shared between foci from the same case, as most mutations were unshared. Shared CNVs frequently included arm-level single copy losses of 10q ( PTEN), 13q ( BRCA2), 5q ( CHD1), 17p ( TP53), 16q ( ZFHX3) and 8p ( NKX3-1), and gain of 8q ( MYC). Mutations not shared by foci in the same case included further biallelic inactivation of PTEN, BRCA2 and TP53, as well as point mutations and single copy losses of BRCA1 and RB1. Distinct, focal gains included AR, PIK3CA and BRAF, and mutations also accumulated in KMT2B, KMT2C and KMT2D. Conclusions: By sampling multiple foci of residual tumor, we identified mutations that likely emerged by subclonal selection by ADT, which cooperated with the shared, clonal mutations that contributed to de novo development of the index lesion. Strikingly, the spectrum of alterations mimics mCRPC with enrichment for alterations affecting cell cycle, DNA damage repair and chromatin modifier pathways. If these alterations permit the tumor to evade AR directed therapy and subsequently mediate relapse, adjuvant therapies targeted to these mutations may increase survival. Clinical trial information: NCT00924469.

scholarly journals Copper Stress Affects Iron Homeostasis by Destabilizing Iron-Sulfur Cluster Formation in Bacillus subtilis

2010 ◽  
Vol 192 (10) ◽  
pp. 2512-2524 ◽  
Author(s):  
Shashi Chillappagari ◽  
Andreas Seubert ◽  
Hein Trip ◽  
Oscar P. Kuipers ◽  
Mohamed A. Marahiel ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Iron Homeostasis ◽  
Cluster Formation ◽  
Cellular Growth ◽  
Copper Stress ◽  
Intracellular Iron ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Copper Excess

ABSTRACT Copper and iron are essential elements for cellular growth. Although bacteria have to overcome limitations of these metals by affine and selective uptake, excessive amounts of both metals are toxic for the cells. Here we investigated the influences of copper stress on iron homeostasis in Bacillus subtilis, and we present evidence that copper excess leads to imbalances of intracellular iron metabolism by disturbing assembly of iron-sulfur cofactors. Connections between copper and iron homeostasis were initially observed in microarray studies showing upregulation of Fur-dependent genes under conditions of copper excess. This effect was found to be relieved in a csoR mutant showing constitutive copper efflux. In contrast, stronger Fur-dependent gene induction was found in a copper efflux-deficient copA mutant. A significant induction of the PerR regulon was not observed under copper stress, indicating that oxidative stress did not play a major role under these conditions. Intracellular iron and copper quantification revealed that the total iron content was stable during different states of copper excess or efflux and hence that global iron limitation did not account for copper-dependent Fur derepression. Strikingly, the microarray data for copper stress revealed a broad effect on the expression of genes coding for iron-sulfur cluster biogenesis (suf genes) and associated pathways such as cysteine biosynthesis and genes coding for iron-sulfur cluster proteins. Since these effects suggested an interaction of copper and iron-sulfur cluster maturation, a mutant with a conditional mutation of sufU, encoding the essential iron-sulfur scaffold protein in B. subtilis, was assayed for copper sensitivity, and its growth was found to be highly susceptible to copper stress. Further, different intracellular levels of SufU were found to influence the strength of Fur-dependent gene expression. By investigating the influence of copper on cluster-loaded SufU in vitro, Cu(I) was found to destabilize the scaffolded cluster at submicromolar concentrations. Thus, by interfering with iron-sulfur cluster formation, copper stress leads to enhanced expression of cluster scaffold and target proteins as well as iron and sulfur acquisition pathways, suggesting a possible feedback strategy to reestablish cluster biogenesis.

Ethidium bromide exposure unmasks an antibiotic efflux system in Rhodococcus equi

2021 ◽  
Author(s):  
Elisa Rampacci ◽  
Maria Luisa Marenzoni ◽  
Rolando Cannalire ◽  
Donatella Pietrella ◽  
Stefano Sabatini ◽  
...  
Keyword(s):  
Ethidium Bromide ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Major Facilitator Superfamily ◽  
23S Rrna ◽  
Genetic Mechanisms ◽  
Major Facilitator ◽  
Antibiotic Efflux ◽  
Level Resistance ◽  
Mfs Transporter

Abstract Background This study introduces a newly created strain (Rhodococcus equiEtBr25) by exposing R. equi ATCC 33701 to ethidium bromide (EtBr), a substrate for MDR transporters. Such an approach allowed us to investigate the resulting phenotype and genetic mechanisms underlying the efflux-mediated resistance in R. equi. Methods R. equi ATCC 33701 was stimulated with increasing concentrations of EtBr. The antimicrobial susceptibility of the parental strain and R. equiEtBr25 was investigated in the presence/absence of efflux pump inhibitors (EPIs). EtBr efflux was evaluated by EtBr-agar method and flow cytometry. The presence of efflux pump genes was determined by conventional PCR before to quantify the expression of 30 genes coding for membrane transporters by qPCR. The presence of erm(46) and mutations in 23S rRNA, and gyrA/gyrB was assessed by PCR and DNA sequencing to exclude the occurrence of resistance mechanisms other than efflux. Results R. equi EtBr25 showed an increased EtBr efflux. Against this strain, the activity of EtBr, azithromycin and ciprofloxacin was more affected than that of rifampicin and azithromycin/rifampicin combinations. Resistances were reversed by combining the antimicrobials with EPIs. Gene expression analysis detected a marked up-regulation of REQ_RS13460 encoding for a Major Facilitator Superfamily (MFS) transporter. G→A transition occurred in the transcriptional repressor tetR/acrR adjacent to REQ_RS13460. Conclusions Exposure of R. equi to EtBr unmasked an efflux-mediated defence against azithromycin and ciprofloxacin, which seemingly correlates with the overexpression of a specific MFS transporter. This genotype may mirror an insidious low-level resistance of clinically important isolates that could be countered by EPI-based therapies.

scholarly journals Iron-sulfur cluster deficiency can be sensed by IRP2 and regulates iron homeostasis and sensitivity to ferroptosis independent of IRP1 and FBXL5

2021 ◽  
Vol 7 (22) ◽  
pp. eabg4302
Author(s):  
Erdem M. Terzi ◽  
Vladislav O. Sviderskiy ◽  
Samantha W. Alvarez ◽  
Gabrielle C. Whiten ◽  
Richard Possemato
Keyword(s):  
Protein Level ◽  
Iron Homeostasis ◽  
Tissue Level ◽  
Iron Starvation ◽  
Intracellular Iron ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Target Mrnas ◽  
Iron Sulfur ◽  
Irp1 And Irp2

Intracellular iron levels are strictly regulated to support homeostasis and avoid iron-mediated ROS production. Loss of iron-sulfur cluster (ISC) synthesis can increase iron loading and promote cell death by ferroptosis. Iron-responsive element-binding proteins IRP1 and IRP2 posttranscriptionally regulate iron homeostasis. IRP1 binding to target mRNAs is competitively regulated by ISC occupancy. However, IRP2 is principally thought to be regulated at the protein level via E3 ubiquitin ligase FBXL5–mediated degradation. Here, we show that ISC synthesis suppression can activate IRP2 and promote ferroptosis sensitivity via a previously unidentified mechanism. At tissue-level O2 concentrations, ISC deficiency enhances IRP2 binding to target mRNAs independent of IRP1, FBXL5, and changes in IRP2 protein level. Deletion of both IRP1 and IRP2 abolishes the iron-starvation response, preventing its activation by ISC synthesis inhibition. These findings will inform strategies to manipulate ferroptosis sensitivity and help illuminate the mechanism underlying ISC biosynthesis disorders, such as Friedreich’s ataxia.

scholarly journals Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer’s disease

2021 ◽  
Author(s):  
Wen-Dai Bao ◽  
Pei Pang ◽  
Xiao-Ting Zhou ◽  
Fan Hu ◽  
Wan Xiong ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Memory Impairment ◽  
Iron Homeostasis ◽  
Critical Role ◽  
Gene Set Enrichment Analysis ◽  
Molecular Characteristics ◽  
Intracellular Iron

AbstractIron homeostasis disturbance has been implicated in Alzheimer’s disease (AD), and excess iron exacerbates oxidative damage and cognitive defects. Ferroptosis is a nonapoptotic form of cell death dependent upon intracellular iron. However, the involvement of ferroptosis in the pathogenesis of AD remains elusive. Here, we report that ferroportin1 (Fpn), the only identified mammalian nonheme iron exporter, was downregulated in the brains of APPswe/PS1dE9 mice as an Alzheimer’s mouse model and Alzheimer’s patients. Genetic deletion of Fpn in principal neurons of the neocortex and hippocampus by breeding Fpnfl/fl mice with NEX-Cre mice led to AD-like hippocampal atrophy and memory deficits. Interestingly, the canonical morphological and molecular characteristics of ferroptosis were observed in both Fpnfl/fl/NEXcre and AD mice. Gene set enrichment analysis (GSEA) of ferroptosis-related RNA-seq data showed that the differentially expressed genes were highly enriched in gene sets associated with AD. Furthermore, administration of specific inhibitors of ferroptosis effectively reduced the neuronal death and memory impairments induced by Aβ aggregation in vitro and in vivo. In addition, restoring Fpn ameliorated ferroptosis and memory impairment in APPswe/PS1dE9 mice. Our study demonstrates the critical role of Fpn and ferroptosis in the progression of AD, thus provides promising therapeutic approaches for this disease.

scholarly journals RNA-Seq reveals divergent gene expression between larvae with contrasting trophic modes in the poecilogonous polychaete Boccardia wellingtonensis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Álvaro Figueroa ◽  
Antonio Brante ◽  
Leyla Cárdenas
Keyword(s):  
De Novo ◽  
Juvenile Stage ◽  
Type I ◽  
Rna Seq ◽  
Mrna Synthesis ◽  
De Novo Transcriptome ◽  
Larval Stages ◽  
Divergent Gene ◽  
Genetic Mechanisms ◽  
Planktotrophic Larvae

AbstractThe polychaete Boccardia wellingtonensis is a poecilogonous species that produces different larval types. Females may lay Type I capsules, in which only planktotrophic larvae are present, or Type III capsules that contain planktotrophic and adelphophagic larvae as well as nurse eggs. While planktotrophic larvae do not feed during encapsulation, adelphophagic larvae develop by feeding on nurse eggs and on other larvae inside the capsules and hatch at the juvenile stage. Previous works have not found differences in the morphology between the two larval types; thus, the factors explaining contrasting feeding abilities in larvae of this species are still unknown. In this paper, we use a transcriptomic approach to study the cellular and genetic mechanisms underlying the different larval trophic modes of B. wellingtonensis. By using approximately 624 million high-quality reads, we assemble the de novo transcriptome with 133,314 contigs, coding 32,390 putative proteins. We identify 5221 genes that are up-regulated in larval stages compared to their expression in adult individuals. The genetic expression profile differed between larval trophic modes, with genes involved in lipid metabolism and chaetogenesis over expressed in planktotrophic larvae. In contrast, up-regulated genes in adelphophagic larvae were associated with DNA replication and mRNA synthesis.

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