scholarly journals Regulation of Copper Toxicity by Candida albicansGPA2

2013 ◽  
Vol 12 (7) ◽  
pp. 954-961 ◽  
Author(s):  
Jennifer A. Schwartz ◽  
Karen T. Olarte ◽  
Jamie L. Michalek ◽  
Gurjinder S. Jandu ◽  
Sarah L. J. Michel ◽  
...  
Keyword(s):  
Copper Toxicity ◽  
Copper Homeostasis ◽  
Copper Resistance ◽  
Copper Accumulation ◽  
Copper Uptake ◽  
Α Subunit ◽  
Content Type ◽  
Copper Chelation ◽  
Excess Copper ◽  
Pka Pathway

ABSTRACTCopper is an essential nutrient that is toxic to cells when present in excess. The fungal pathogenCandida albicansemploys several mechanisms to survive in the presence of excess copper, but the molecular pathways that govern these responses are not completely understood. We report that deletion ofGPA2, which specifies a G-protein α subunit, confers increased resistance to excess copper and propose that the increased resistance is due to a combination of decreased copper uptake and an increase in copper chelation by metallothioneins. This is supported by our observations that agpa2Δ/Δ mutant has reduced expression of the copper uptake genes,CTR1andFRE7, and a marked decrease in copper accumulation following exposure to high copper levels. Furthermore, deletion ofGPA2results in an increased expression of the copper metallothionein gene,CRD2. Gpa2p functions upstream in the cyclic AMP (cAMP)-protein kinase A (PKA) pathway to govern hyphal morphogenesis. The copper resistance phenotype of thegpa2Δ/Δ mutant can be reversed by artificially increasing the intracellular concentration of cAMP. These results provide evidence for a novel role of the PKA pathway in regulation of copper homeostasis. Furthermore, the connection between the PKA pathway and copper homeostasis appears to be conserved in the pathogenCryptococcus neoformansbut not in the nonpathogenicSaccharomyces cerevisiae.

scholarly journals c-Type Cytochrome Assembly Is a Key Target of Copper Toxicity within the Bacterial Periplasm

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Anne Durand ◽  
Asma Azzouzi ◽  
Marie-Line Bourbon ◽  
Anne-Soisig Steunou ◽  
Sylviane Liotenberg ◽  
...  
Keyword(s):  
Radical Scavenging ◽  
Copper Toxicity ◽  
Chlorophyll Synthesis ◽  
Copper Tolerance ◽  
Wild Type ◽  
Tight Control ◽  
Content Type ◽  
Excess Copper ◽  
Rubrivivax Gelatinosus ◽  
First Time

ABSTRACT In the absence of a tight control of copper entrance into cells, bacteria have evolved different systems to control copper concentration within the cytoplasm and the periplasm. Central to these systems, the Cu+ ATPase CopA plays a major role in copper tolerance and translocates copper from the cytoplasm to the periplasm. The fate of copper in the periplasm varies among species. Copper can be sequestered, oxidized, or released outside the cells. Here we describe the identification of CopI, a periplasmic protein present in many proteobacteria, and show its requirement for copper tolerance in Rubrivivax gelatinosus. The ΔcopI mutant is more susceptible to copper than the Cu+ ATPase copA mutant. CopI is induced by copper, localized in the periplasm and could bind copper. Interestingly, copper affects cytochrome c membrane complexes (cbb3 oxidase and photosystem) in both ΔcopI and copA-null mutants, but the causes are different. In the copA mutant, heme and chlorophyll synthesis are affected, whereas in ΔcopI mutant, the decrease is a consequence of impaired cytochrome c assembly. This impact on c-type cytochromes would contribute also to the copper toxicity in the periplasm of the wild-type cells when they are exposed to high copper concentrations. IMPORTANCE Copper is an essential cation required as a cofactor in enzymes involved in vital processes such as respiration, photosynthesis, free radical scavenging, and pathogenesis. However, copper is highly toxic and has been implicated in disorders in all organisms, including humans, because it can catalyze the production of toxic reactive oxygen species and targets various biosynthesis pathways. Identifying copper targets, provides insights into copper toxicity and homeostatic mechanisms for copper tolerance. In this work, we describe for the first time a direct effect of excess copper on cytochrome c assembly. We show that excess copper specifically affects periplasmic and membrane cytochromes c, thus suggesting that the copper toxicity targets c-type cytochrome biogenesis.

scholarly journals Novel Metal Cation Resistance Systems from Mutant Fitness Analysis of Denitrifying Pseudomonas stutzeri

2016 ◽  
Vol 82 (19) ◽  
pp. 6046-6056 ◽  
Author(s):  
Brian J. Vaccaro ◽  
W. Andrew Lancaster ◽  
Michael P. Thorgersen ◽  
Grant M. Zane ◽  
Adam D. Younkin ◽  
...  
Keyword(s):  
Metal Ion ◽  
Sequence Data ◽  
Copper Toxicity ◽  
Pseudomonas Stutzeri ◽  
Hypothetical Protein ◽  
Protein Family ◽  
Copper Resistance ◽  
Transport Systems ◽  
Content Type ◽  
New Genes

ABSTRACTMetal ion transport systems have been studied extensively, but the specificity of a given transporter is often unclear from amino acid sequence data alone. In this study, predicted Cu2+and Zn2+resistance systems inPseudomonas stutzeristrain RCH2 are compared with those experimentally implicated in Cu2+and Zn2+resistance, as determined by using a DNA-barcoded transposon mutant library. Mutant fitness data obtained under denitrifying conditions are combined with regulon predictions to yield a much more comprehensive picture of Cu2+and Zn2+resistance in strain RCH2. The results not only considerably expand what is known about well-established metal ion exporters (CzcCBA, CzcD, and CusCBA) and their accessory proteins (CzcI and CusF), they also reveal that isolates with mutations in some predicted Cu2+resistance systems do not show decreased fitness relative to the wild type when exposed to Cu2+. In addition, new genes are identified that have no known connection to Zn2+(corB,corC, Psest_3226, Psest_3322, and Psest_0618) or Cu2+resistance (Mrp antiporter subunit gene, Psest_2850, and Psest_0584) but are crucial for resistance to these metal cations. Growth of individual deletion mutants lackingcorB,corC, Psest_3226, or Psest_3322 confirmed the observed Zn-dependent phenotypes. Notably, to our knowledge, this is the first time a bacterial homolog of TMEM165, a human gene responsible for a congenital glycosylation disorder, has been deleted and the resulting strain characterized. Finally, the fitness values indicate Cu2+- and Zn2+-based inhibition of nitrite reductase and interference with molybdenum cofactor biosynthesis for nitrate reductase. These results extend the current understanding of Cu2+and Zn2+efflux and resistance and their effects on denitrifying metabolism.IMPORTANCEIn this study, genome-wide mutant fitness data inP. stutzeriRCH2 combined with regulon predictions identify several proteins of unknown function that are involved in resisting zinc and copper toxicity. For zinc, these include a member of the UPF0016 protein family that was previously implicated in Ca2+/H+antiport and a human congenital glycosylation disorder, CorB and CorC, which were previously linked to Mg2+transport, and Psest_3322 and Psest_0618, two proteins with no characterized homologs. Experiments using mutants lacking Psest_3226, Psest_3322,corB,corC, orczcIverified their proposed functions, which will enable future studies of these little-characterized zinc resistance determinants. Likewise, Psest_2850, annotated as an ion antiporter subunit, and the conserved hypothetical protein Psest_0584 are implicated in copper resistance. Physiological connections between previous studies and phenotypes presented here are discussed. Functional and mechanistic understanding of transport proteins improves the understanding of systems in which members of the same protein family, including those in humans, can have different functions.

scholarly journals Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

2014 ◽  
Vol 81 (3) ◽  
pp. 1024-1031 ◽  
Author(s):  
Bhagyalakshmi Kalidass ◽  
Muhammad Farhan Ul-Haque ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
Jeremy D. Semrau
Keyword(s):  
Methane Monooxygenase ◽  
High Specificity ◽  
Copper Uptake ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
Sds Page ◽  
Genes Encoding ◽  
Key Factor ◽  
Membrane Bound

ABSTRACTIt is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that inMethylosinus trichosporiumOB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced byM. trichosporiumOB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and activein situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

scholarly journals The Extracellular Domain of the β2Integrin β Subunit (CD18) Is Sufficient forEscherichia coliHemolysin andAggregatibacter actinomycetemcomitansLeukotoxin Cytotoxic Activity

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Laura C. Ristow ◽  
Vy Tran ◽  
Kevin J. Schwartz ◽  
Lillie Pankratz ◽  
Andrew Mehle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Integrin Signaling ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Content Type ◽  
Animal Pathogens ◽  
Tract Infections

ABSTRACTTheEscherichia colihemolysin (HlyA) is a pore-forming exotoxin associated with severe complications of human urinary tract infections. HlyA is the prototype of the repeats-in-toxin (RTX) family, which includes LtxA fromAggregatibacter actinomycetemcomitans, a periodontal pathogen. The existence and requirement for a host cell receptor for these toxins are controversial. We performed an unbiased forward genetic selection in a mutant library of human monocytic cells, U-937, for host factors involved in HlyA cytotoxicity. The top candidate was the β2integrin β subunit. Δβ2cell lines are approximately 100-fold more resistant than wild-type U-937 cells to HlyA, but remain sensitive to HlyA at high concentrations. Similarly, Δβ2cells are more resistant than wild-type U-937 cells to LtxA, as Δβ2cells remain LtxA resistant even at >1,000-fold-higher concentrations of the toxin. Loss of any single β2integrin α subunit, or even all four α subunits together, does not confer resistance to HlyA. HlyA and LtxA bind to the β2subunit, but not to αL, αM, or αXin far-Western blots. Genetic complementation of Δβ2cells with either β2or β2with a cytoplasmic tail deletion restores HlyA and LtxA sensitivity, suggesting that β2integrin signaling is not required for cytotoxicity. Finally, β2mutations do not alter sensitivity to unrelated pore-forming toxins, as wild-type or Δβ2cells are equally sensitive toStaphylococcus aureusα-toxin andProteus mirabilisHpmA. Our studies show two RTX toxins use the β2integrin β subunit alone to facilitate cytotoxicity, but downstream integrin signaling is dispensable.IMPORTANCEUrinary tract infections are one of the most common bacterial infections worldwide. UropathogenicEscherichia colistrains are responsible for more than 80% of community-acquired urinary tract infections. Although we have known for nearly a century that severe infections stemming from urinary tract infections, including kidney or bloodstream infections are associated with expression of a toxin, hemolysin, from uropathogenicEscherichia coli, how hemolysin functions to enhance virulence is unknown. Our research defines the interaction of hemolysin with the β2integrin, a human white cell adhesion molecule, as a potential therapeutic target during urinary tract infections. TheE. colihemolysin is the prototype for a toxin family (RTX family) produced by a wide array of human and animal pathogens. Our work extends to the identification and characterization of the receptor for an additional member of the RTX family, suggesting that this interaction may be broadly conserved throughout the RTX toxin family.

scholarly journals Altered copper homeostasis underlies sensitivity of hepatocellular carcinoma to copper chelation

Metallomics ◽  
2020 ◽  
Author(s):  
Caroline I. Davis ◽  
Xingxing Gu ◽  
Ryan M. Kiefer ◽  
Martina Ralle ◽  
Terence P. Gade ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Genetic Manipulation ◽  
Copper Homeostasis ◽  
Glycolytic Metabolism ◽  
Copper Chelation ◽  
Oncogenic Pathways ◽  
Pharmacologic Inhibition

Bioavailable Cu fuels oncogenic pathways that drive tumorigenesis. Intriguingly, genetic manipulation or pharmacologic inhibition of intracellular Cu diminishes hypoxia-induced glycolytic metabolism and attenuates HCC tumorigenic properties.

scholarly journals Current Biomedical Use of Copper Chelation Therapy

2020 ◽  
Vol 21 (3) ◽  
pp. 1069 ◽  
Author(s):  
Silvia Baldari ◽  
Giuliana Di Rocco ◽  
Gabriele Toietta
Keyword(s):  
Clinical Trials ◽  
Idiopathic Pulmonary Fibrosis ◽  
Genetic Disease ◽  
Copper Concentration ◽  
Chelating Agents ◽  
Chelation Therapy ◽  
Biological Processes ◽  
Copper Chelation ◽  
Excess Copper ◽  
Parkinson’S Diseases

Copper is an essential microelement that plays an important role in a wide variety of biological processes. Copper concentration has to be finely regulated, as any imbalance in its homeostasis can induce abnormalities. In particular, excess copper plays an important role in the etiopathogenesis of the genetic disease Wilson’s syndrome, in neurological and neurodegenerative pathologies such as Alzheimer’s and Parkinson’s diseases, in idiopathic pulmonary fibrosis, in diabetes, and in several forms of cancer. Copper chelating agents are among the most promising tools to keep copper concentration at physiological levels. In this review, we focus on the most relevant compounds experimentally and clinically evaluated for their ability to counteract copper homeostasis deregulation. In particular, we provide a general overview of the main disorders characterized by a pathological increase in copper levels, summarizing the principal copper chelating therapies adopted in clinical trials.

scholarly journals β-Aminopeptidases: Insight into Enzymes without a Known Natural Substrate

2019 ◽  
Vol 85 (15) ◽  
Author(s):  
Marietta John-White ◽  
James Gardiner ◽  
Priscilla Johanesen ◽  
Dena Lyras ◽  
Geoffrey Dumsday
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gram Negative Bacteria ◽  
Diverse Range ◽  
Α Subunit ◽  
Gram Negative ◽  
Content Type ◽  
Proteolytic Resistance ◽  
Insight Into ◽  
Unique Capability

ABSTRACT β-Aminopeptidases have the unique capability to hydrolyze N-terminal β-amino acids, with varied preferences for the nature of β-amino acid side chains. This unique capability makes them useful as biocatalysts for synthesis of β-peptides and to kinetically resolve β-peptides and amides for the production of enantiopure β-amino acids. To date, six β-aminopeptidases have been discovered and functionally characterized, five from Gram-negative bacteria and one from a fungus, Aspergillus. Here we report on the purification and characterization of an additional four β-aminopeptidases, one from a Gram-positive bacterium, Mycolicibacterium smegmatis (BapAMs), one from a yeast, Yarrowia lipolytica (BapAYlip), and two from Gram-negative bacteria isolated from activated sludge identified as Burkholderia spp. (BapABcA5 and BapABcC1). The genes encoding β-aminopeptidases were cloned, expressed in Escherichia coli, and purified. The β-aminopeptidases were produced as inactive preproteins that underwent self-cleavage to form active enzymes comprised of two different subunits. The subunits, designated α and β, appeared to be tightly associated, as the active enzyme was recovered after immobilized-metal affinity chromatography (IMAC) purification, even though only the α-subunit was 6-histidine tagged. The enzymes were shown to hydrolyze chromogenic substrates with the N-terminal l-configurations β-homo-Gly (βhGly) and β3-homo-Leu (β3hLeu) with high activities. These enzymes displayed higher activity with H-βhGly-p-nitroanilide (H-βhGly-pNA) than previously characterized enzymes from other microorganisms. These data indicate that the new β-aminopeptidases are fully functional, adding to the toolbox of enzymes that could be used to produce β-peptides. Overexpression studies in Pseudomonas aeruginosa also showed that the β-aminopeptidases may play a role in some cellular functions. IMPORTANCE β-Aminopeptidases are unique enzymes found in a diverse range of microorganisms that can utilize synthetic β-peptides as a sole carbon source. Six β-aminopeptidases have been previously characterized with preferences for different β-amino acid substrates and have demonstrated the capability to catalyze not only the degradation of synthetic β-peptides but also the synthesis of short β-peptides. Identification of other β-aminopeptidases adds to this toolbox of enzymes with differing β-amino acid substrate preferences and kinetics. These enzymes have the potential to be utilized in the sustainable manufacture of β-amino acid derivatives and β-peptides for use in biomedical and biomaterial applications. This is important, because β-amino acids and β-peptides confer increased proteolytic resistance to bioactive compounds and form novel structures as well as structures similar to α-peptides. The discovery of new enzymes will also provide insight into the biological importance of these enzymes in nature.

Comparative effects of ascobin and glutathione on copper homeostasis and oxidative stress metabolism in mitigation of copper toxicity in rice

Plant Biology ◽  
2021 ◽  
Author(s):  
M. Tahjib‐Ul‐Arif ◽  
A. A. M. Sohag ◽  
M. G. Mostofa ◽  
M. A. S. Polash ◽  
A. G. M. S. U. Mahamud ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Copper Toxicity ◽  
Copper Homeostasis ◽  
And Oxidative Stress

scholarly journals Copper-Induced Expression of a Transmissible Lipoprotein Intramolecular Transacylase Alters Lipoprotein Acylation and the Toll-Like Receptor 2 Response to Listeria monocytogenes

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Krista M. Armbruster ◽  
Gloria Komazin ◽  
Timothy C. Meredith
Keyword(s):  
Listeria Monocytogenes ◽  
Bacterial Species ◽  
Acyl Chain ◽  
Constitutive Expression ◽  
Innate Immune ◽  
Copper Resistance ◽  
Toll Like Receptor ◽  
General Role ◽  
Content Type ◽  
Toll Like Receptor 2

ABSTRACT Bacterial lipoproteins are globular proteins anchored to the extracytoplasmic surfaces of cell membranes through lipidation at a conserved N-terminal cysteine. Lipoproteins contribute to an array of important cellular functions for bacteria, as well as being a focal point for innate immune system recognition through binding to Toll-like receptor 2 (TLR2) heterodimer complexes. Although lipoproteins are conserved among nearly all classes of bacteria, the presence and type of α-amino-linked acyl chain are highly variable and even strain specific within a given bacterial species. The reason for lyso-lipoprotein formation and N-acylation variability in general is presently not fully understood. In Enterococcus faecalis, lipoproteins are anchored by an N-acyl-S-monoacyl-glyceryl cysteine (lyso form) moiety installed by a chromosomally encoded lipoprotein intramolecular transacylase (Lit). Here, we describe a mobile genetic element common to environmental isolates of Listeria monocytogenes and Enterococcus spp. encoding a functional Lit ortholog (Lit2) that is cotranscribed with several well-established copper resistance determinants. Expression of Lit2 is tightly regulated, and induction by copper converts lipoproteins from the diacylglycerol-modified form characteristic of L. monocytogenes type strains to the α-amino-modified lyso form observed in E. faecalis. Conversion to the lyso form through either copper addition to media or constitutive expression of lit2 decreases TLR2 recognition when using an activated NF-κB secreted embryonic alkaline phosphatase reporter assay. While lyso formation significantly diminishes TLR2 recognition, lyso-modified lipoprotein is still predominantly recognized by the TLR2/TLR6 heterodimer. IMPORTANCE The induction of lipoprotein N-terminal remodeling in response to environmental copper in Gram-positive bacteria suggests a more general role in bacterial cell envelope physiology. N-terminal modification by lyso formation, in particular, simultaneously modulates the TLR2 response in direct comparison to their diacylglycerol-modified precursors. Thus, use of copper as a frontline antimicrobial control agent and ensuing selection raises the potential of diminished innate immune sensing and enhanced bacterial virulence.

scholarly journals Regulation of murine copper homeostasis by members of the COMMD protein family

2020 ◽  
Vol 14 (1) ◽  
pp. dmm045963
Author(s):  
Amika Singla ◽  
Qing Chen ◽  
Kohei Suzuki ◽  
Jie Song ◽  
Alina Fedoseienko ◽  
...  
Keyword(s):  
Copper Homeostasis ◽  
Copper Accumulation ◽  
Copper Transporter ◽  
Copper Excretion ◽  
Copper Absorption ◽  
Hepatic Copper ◽  
High Copper ◽  
Molecular Machinery ◽  
And Function

ABSTRACTCopper is an essential transition metal for all eukaryotes. In mammals, intestinal copper absorption is mediated by the ATP7A copper transporter, whereas copper excretion occurs predominantly through the biliary route and is mediated by the paralog ATP7B. Both transporters have been shown to be recycled actively between the endosomal network and the plasma membrane by a molecular machinery known as the COMMD/CCDC22/CCDC93 or CCC complex. In fact, mutations in COMMD1 can lead to impaired biliary copper excretion and liver pathology in dogs and in mice with liver-specific Commd1 deficiency, recapitulating aspects of this phenotype. Nonetheless, the role of the CCC complex in intestinal copper absorption in vivo has not been studied, and the potential redundancy of various COMMD family members has not been tested. In this study, we examined copper homeostasis in enterocyte-specific and hepatocyte-specific COMMD gene-deficient mice. We found that, in contrast to effects in cell lines in culture, COMMD protein deficiency induced minimal changes in ATP7A in enterocytes and did not lead to altered copper levels under low- or high-copper diets, suggesting that regulation of ATP7A in enterocytes is not of physiological consequence. By contrast, deficiency of any of three COMMD genes (Commd1, Commd6 or Commd9) resulted in hepatic copper accumulation under high-copper diets. We found that each of these deficiencies caused destabilization of the entire CCC complex and suggest that this might explain their shared phenotype. Overall, we conclude that the CCC complex plays an important role in ATP7B endosomal recycling and function.

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