Understanding the 7‐<scp>C</scp>ys module amplification of <scp><i>C</i></scp><i>. neoformans</i> metallothioneins: how high capacity <scp>C</scp>u‐binding polypeptides are built to neutralize host nutritional immunity

Espart, Anna; Gil-Moreno, Selene; Palacios, Òscar; Capdevila, Mercè; Atrian, Sı́lvia

doi:10.1111/mmi.13171

Cited by 4 publications

(9 citation statements)

References 43 publications

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“…First, the RmMT gene encoded 106 amino acids, longer than general MT (ca. 60 aa) , but shorter than TmMT (257 aa) of Tremella mesenterica , CnMT1 (122 aa) and CnMT2 (183 aa) of Cryptoccocus neoformans . Second, 16 cysteine residues were scattered throughout the whole sequence and its content in RmMT was about 15%, much lower than typical MTs (ca.…”

Section: Discussionmentioning

confidence: 89%

“…Now, many MTs have been characterized in yeasts, including Cup1 and Crs5 in Saccharomyces cerevisiae , MT‐I and MT‐II in Candida glabrata , MTPI‐II in Yarrowia lipolytica , Zym1 in Schizosaccharomyces pombe , and CnMT1 and CnMT2 in Cryptococcus neoformans . Whereas, to the best of our knowledge, no relevant study of the MTs from the Antarctic yeast Rhodotorula mucilaginosa was published.…”

Section: Introductionmentioning

confidence: 99%

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Cloning and functional characterization of a novel metallothionein gene in Antarctic sea‐ice yeast (Rhodotorula mucilaginosa)

Kan

Zhou

et al. 2019

J Basic Microbiol

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Metallothionein (MT) is a low-molecular-weight protein with a high metal binding capacity and plays a key role in organism adaptation to heavy metals. In this study, a metallothionein gene was successfully cloned and sequenced from Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5. Nucleotide sequencing and analysis revealed that the gene had four exons interrupted by three introns. MTs complementary DNA (named as RmMT) had an open reading frame of 321 bp encoding a 106 amino acid protein with a predicted molecular weight of 10.3 kDa and pI of 8.49. The number of amino acids and distribution of cysteine residues indicated that RmMT was a novel family of fungal MTs. Quantitative real-time polymerase chain reaction analysis showed that RmMT expression was elevated under copper-induced stress. TheRmMT gene was transferred into E. coli and the RmMT expressing bacteria showed improved tolerance to copper ion and increased accumulation of heavy metals, such as Cu 2+ , Pb 2+ , Zn 2+ , Cd 2+ , and Ag + . Moreover, in vitro studies, purified recombinant RmMT demonstrated that it could be used as a good scavenger of superoxide anion, hydroxyl, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radicals. In summary, these results demonstrate that RmMT plays a key role in the tolerance and bioaccumulation of heavy metals. K E Y W O R D Sfunctional analysis, heavy metal, metallothionein, polar yeast

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Section: Discussionmentioning

confidence: 89%

Section: Introductionmentioning

confidence: 99%

Cloning and functional characterization of a novel metallothionein gene in Antarctic sea‐ice yeast (Rhodotorula mucilaginosa)

Kan

Zhou

et al. 2019

J Basic Microbiol

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“…The Saccharomyces cerevisiae 51.2cΔc5 strain ( MAT a, trp 1-1, ura 3-52, ade- , his- , CAN R , gal 1, leu2-3 , 112 met13 , cup1 Δ:: URA 3 crs5 Δ:: LEU2 ), derived from VC-sp6 [ 25 ] was used for copper tolerance assays as described in detail previously for the CnMT1-derived peptides [ 26 ]. The TmMT cDNA was inserted into the BamH I/ Xho I sites of the yeast vector p424-GPD, which contains TRP1 as a selection marker, the constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter for gene expression, and the cytochrome-c-oxidase (CYC1) transcriptional terminator [ 27 ].…”

Section: Methodsmentioning

confidence: 99%

“…Due to the pGEX vector cloning specificities, the recombinant TmMT exhibited two additional N-term residues (Gly-Ser), but they have been shown to have no effect on the MT metal-binding features [ 30 ]. Further details about the synthesis and purification procedures can be found in previous publications [ 17 , 23 , 26 ].…”

Section: Methodsmentioning

confidence: 99%

“…ICP-AES (inductively coupled plasma atomic emission spectroscopy) analysis of the purified metal-TmMT complexes was essentially performed as previously described for other MTs [ 17 , 23 , 26 ]. S, Zn, Cd and Cu contents were measured in a Polyscan 61E (Thermo Jarrell Ash) spectrometer, reading S at 182.040 nm, Zn at 213.856 nm, Cd at 228.802 and Cu at 324.803 nm.…”

Section: Methodsmentioning

confidence: 99%

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The Fungus Tremella mesenterica Encodes the Longest Metallothionein Currently Known: Gene, Protein and Metal Binding Characterization

et al. 2016

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Fungal Cu-thioneins, and among them, the paradigmatic Neurospora crassa metallothionein (MT) (26 residues), were once considered as the shortest MTs -the ubiquitous, versatile metal-binding proteins- among all organisms, and thus representatives of their primeval forms. Nowadays, fungal MTs of diverse lengths and sequence features are known, following the huge heterogeneity of the Kingdom of Fungi. At the opposite end of N. crassa MT, the recently reported Cryptococcus neoformans CnMT1 and CnMT2 (122 and 186 aa) constitute the longest reported fungal MTs, having been identified as virulence factors of this pathogen. CnMTs are high-capacity Cu-thioneins that appear to be built by tandem amplification of a basic unit, a 7-Cys segment homologous to N. crassa MT. Here, we report the in silico, in vivo and in vitro study of a still longer fungal MT, belonging to Tremella mesenterica (TmMT), a saprophytic ascomycete. The TmMT gene has 10 exons, and it yields a 779-bp mature transcript that encodes a 257 residue-long protein. This MT is also built by repeated fragments, but of variable number of Cys: six units of the 7-Cys building blocks-CXCX3CSCPPGXCXCAXCP-, two fragments of six Cys, plus three Cys at the N-terminus. TmMT metal binding abilities have been analyzed through the spectrophotometric and spectrometric characterization of its recombinant Zn-, Cd- and Cu-complexes. Results allow it to be unambiguous classified as a Cu-thionein, also of extraordinary coordinating capacity. According to this feature, when the TmMT cDNA is expressed in MT-devoid yeast cells, it is capable of restoring a high Cu tolerance level. Since it is not obvious that T. mesenterica shares the same physiological needs for a high capacity Cu-binding protein with C. neoformans, the existence of this peculiar MT might be better explained on the basis of a possible role in Cu-handling for the Cu-enzymes responsible in lignin degradation pathways.

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Fluconazole induces ROS in Cryptococcus neoformans and contributes to DNA damage in vitro

et al. 2018

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Pathogenic basidiomycetous yeast, Cryptococcus neoformans, causes fatal meningitis in immunocompromised individuals. Fluconazole (FLC) is a fungistatic drug commonly administered to treat cryptococcosis. Unfortunately, FLC-resistant strains characterized by various degree of chromosomal instability were isolated from clinical patients. Importantly, the underlying mechanisms that lead to chromosomal instability in FLC-treated C. neoformans remain elusive. Previous studies in fungal and mammalian cells link chromosomal instability to the reactive oxygen species (ROS). This study provides the evidence that exposure of C. neoformans to FLC induces accumulation of intracellular ROS, which correlates with plasma membrane damage. FLC caused transcription changes of oxidative stress related genes encoding superoxide dismutase (SOD1), catalase (CAT3), and thioredoxin reductase (TRR1). Strikingly, FLC contributed to an increase of the DNA damage in vitro, when complexed with iron or copper in the presence of hydrogen peroxide. Strains with isogenic deletion of copper response protein metallothionein were more susceptible to FLC. Addition of ascorbic acid (AA), an anti-oxidant at 10 mM, reduced the inhibitory effects of FLC. Consistent with potential effects of FLC on DNA integrity and chromosomal segregation, FLC treatment led to elevated transcription of RAD54 and repression of cohesin-encoding gene SCC1. We propose that FLC forms complexes with metals and contributes to elevated ROS, which may lead to chromosomal instability in C. neoformans.

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Understanding the 7‐Cys module amplification of C. neoformans metallothioneins: how high capacity Cu‐binding polypeptides are built to neutralize host nutritional immunity

Cited by 4 publications

References 43 publications

Cloning and functional characterization of a novel metallothionein gene in Antarctic sea‐ice yeast (Rhodotorula mucilaginosa)

Cloning and functional characterization of a novel metallothionein gene in Antarctic sea‐ice yeast (Rhodotorula mucilaginosa)

The Fungus Tremella mesenterica Encodes the Longest Metallothionein Currently Known: Gene, Protein and Metal Binding Characterization

Fluconazole induces ROS in Cryptococcus neoformans and contributes to DNA damage in vitro

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