Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

CRISPR/Cas9 Genome Editing To Demonstrate the Contribution of Cyp51A Gly138Ser to Azole Resistance in Aspergillus fumigatus.

ABSTRACT: A pan-azole-resistant Aspergillus fumigatus strain with the cyp51A mutations Gly138Ser and Asn248Lys was isolated from a patient receiving long-term voriconazole treatment. PCR fragments containing cyp51A with the mutations were introduced along with the Cas9 protein and single guide RNA into the azole-resistant/susceptible strains. Recombinant strains showed increased susceptibility via the replacement of Ser138 by glycine. Genetic recombination, which has been hampered thus far in clinical isolates, can now be achieved using CRISPR/Cas9 genome editing.

SUBMITTER: Umeyama T

PROVIDER: S-EPMC6125507 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

CRISPR/Cas9 Genome Editing To Demonstrate the Contribution of Cyp51A Gly138Ser to Azole Resistance in Aspergillus fumigatus.

Umeyama Takashi T Hayashi Yuta Y Shimosaka Hisaki H Inukai Tatsuya T Yamagoe Satoshi S Takatsuka Shogo S Hoshino Yasutaka Y Nagi Minoru M Nakamura Shigeki S Kamei Katsuhiko K Ogawa Kenji K Miyazaki Yoshitsugu Y

Antimicrobial agents and chemotherapy 20180827 9

A pan-azole-resistant Aspergillus fumigatus strain with the cyp51A mutations Gly138Ser and Asn248Lys was isolated from a patient receiving long-term voriconazole treatment. PCR fragments containing cyp51A with the mutations were introduced along with the Cas9 protein and single guide RNA into the azole-resistant/susceptible strains. Recombinant strains showed increased susceptibility via the replacement of Ser138 by glycine. Genetic recombination, which has been hampered thu ...[more]

PMID: 29914956

Similar Datasets

Uncovering New Mutations Conferring Azole Resistance in the Aspergillus fumigatus cyp51A Gene.

Project description:The opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in cytochromeP450 enzyme Cyp51. In this study, we indicated that in vitro azole situation results in emergence of azole-resistant mutations. There are previously identified azole-resistant cyp51A mutations (M220K, M220I, M220R, G54E and G54W mutations) and we successfully identified in this study two new mutations (N248K/V436A, Y433N substitution) conferring azole resistance among 18 independent stable azole-resistant isolates. The Galleria mellonella model of A. fumigatus infection experiment verified that Cyp51A mutations N248K/V436A and Y433N reduce efficacy of azole therapy. In addition, a predicted Cyp51A 3D structural model suggested that Y433N mutation causes the reduced affinities between drug target Cyp51A and azole antifungals. This study suggests that drug selection pressure make it possible to isolate unidentified cyp51A mutations conferring azole resistance in A. fumigatus.

| S-EPMC6986205 | biostudies-literature

A Novel Combination of CYP51A Mutations Confers Pan-Azole Resistance in Aspergillus fumigatus.

Project description:The treatment of invasive and chronic aspergillosis involves triazole drugs. Its intensive use has resulted in the selection of resistant isolates, and at present, azole resistance in Aspergillus fumigatus is considered an emerging threat to public health worldwide. The aim of this work is to uncover the molecular mechanism implicated in the azole resistance phenotype of three Aspergillus fumigatus clinical strains isolated from an Argentinian cystic fibrosis patient under long-term triazole treatment. Strain susceptibilities were assessed, and CYP51A gene sequences were analyzed. Two of the studied Aspergillus fumigatus strains harbored the TR34-L98H allele. These strains showed high MIC values for all tested triazoles (>16.00 μg/ml, 1.00 μg/ml, 1.00 μg/ml, and 2.00 μg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The third strain had a novel amino acid change (R65K) combined with the TR34-L98H mutations. This new mutation combination induces a pan-azole MIC augment compared with TR34-L98H mutants (>16 μg/ml, 4.00 μg/ml, 4.00 μg/ml, and 8.00 μg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The strain harboring the TR34-R65K-L98H allele showed no inhibition halo when voriconazole susceptibility was evaluated by disk diffusion. The effect of these mutations in the azole-resistant phenotype was confirmed by gene replacement experiments. Transformants harboring the TR34-L98H and TR34-R65K-L98H alleles mimicked the azole-resistant phenotype of the clinical isolates, while the incorporation of the TR34-R65K and R65K alleles did not significantly increase azole MIC values. This is the first report of the TR34-L98H allele in Argentina. Moreover, a novel CYP51A allele (TR34-R65K-L98H) that induces a pan-azole MIC augment is described.

| S-EPMC7526825 | biostudies-literature

Azole resistance profile of amino acid changes in Aspergillus fumigatus CYP51A based on protein homology modeling.

Project description:Molecular studies have shown that the majority of azole resistance in Aspergillus fumigatus is associated with amino acid substitutions in the cyp51A gene. To obtain insight into azole resistance mutations, the cyp51A gene of 130 resistant and 76 susceptible A. fumigatus isolates was sequenced. Out of 130 azole-resistant isolates, 105 contained a tandem repeat of 34 bp in the promoter region and a leucine-to-histidine substitution in codon 98 (designated TR/L98H). Additionally, in 12 of these TR/L98H resistant isolates, the mutations S297T and F495I were found, and in 1 isolate, the mutation F495I was found. In eight azole-resistant isolates, known azole resistance mutations were detected in codon G54, G138, or M220. In three azole-susceptible isolates, the mutation E130D, L252L, or S400I was found and in 13 azole-susceptible isolates but also in 1 azole-resistant isolate, the mutations F46Y, G98G, M172V, N248T, D255E, L358L, E427K, and C454C were found. All of the nonsynonymous mutations, apart from the mutations in codons G54, G138, and M220 and L98H, were located at the periphery of the protein, as determined by a structural model of the A. fumigatus Cyp51A protein, and were predicted neither to interact with azole compounds nor to affect structural integrity. Therefore, this wide diversity of mutations in the cyp51A gene in azole-susceptible A. fumigatus isolates is not correlated with azole resistance. Based on the Cyp51A protein homology model, the potential correlation of a mutation to azole resistance can be predicted.

| S-EPMC2876375 | biostudies-literature

Screening and Characterization of a Non-cyp51A Mutation in an Aspergillus fumigatus cox10 Strain Conferring Azole Resistance.

Project description:The rapid and global emergence of azole resistance in the human pathogen Aspergillus fumigatus has drawn attention. Thus, a thorough understanding of its mechanisms of drug resistance requires extensive exploration. In this study, we found that the loss of the putative calcium-dependent protein-encoding gene algA causes an increased frequency of azole-resistant A. fumigatus isolates. In contrast to previously identified azole-resistant isolates related to cyp51A mutations, only one isolate carries a point mutation in cyp51A (F219L mutation) among 105 independent stable azole-resistant isolates. Through next-generation sequencing (NGS), we successfully identified a new mutation (R243Q substitution) conferring azole resistance in the putative A. fumigatus farnesyltransferase Cox10 (AfCox10) (AFUB_065450). High-performance liquid chromatography (HPLC) analysis verified that the decreased absorption of itraconazole in related Afcox10 mutants is the primary reason for itraconazole resistance. Moreover, a complementation experiment by reengineering the mutation in a parental wild-type background strain demonstrated that both the F219L and R243Q mutations contribute to itraconazole resistance in an algA-independent manner. These data collectively suggest that the loss of algA results in an increased frequency of azole-resistant isolates with a non-cyp51A mutation. Our findings indicate that there are many unexplored non-cyp51A mutations conferring azole resistance in A. fumigatus and that algA defects make it possible to isolate drug-resistant alleles. In addition, our study suggests that genome-wide sequencing combined with alignment comparison analysis is an efficient approach to identify the contribution of single nucleotide polymorphism (SNP) diversity to drug resistance.

| S-EPMC5192120 | biostudies-literature

Non-cyp51A Azole-Resistant Aspergillus fumigatus Isolates with Mutation in HMG-CoA Reductase.

Project description:The recent increase in azole-resistant Aspergillus fumigatus is a global concern. Identifying the mutations that confer azole resistance is essential for developing novel methods for prompt diagnosis and effective drug treatment. We screened A. fumigatus clinical isolates for novel mutations conferring azole resistance. We compared the genomic sequences of susceptible and resistant isolates without mutations in cyp51A (non-cyp51A) and found mutations in hmg1 and erg6 involved in ergosterol biosynthesis. We also found the novel mutations in these genes in azole-resistant isolates with different genetic backgrounds. The resistant isolates with mutations in hmg1 showed increased intracellular ergosterol levels compared with susceptible isolates. This finding supports the concept that the ergosterol level is a determinant for resistance to any class of azoles. Multiple isolates with increased resistance to azole possessed a mutation in hmg1, indicating that this mutation is widely present in non-cyp51A azole-resistant A. fumigatus.

| S-EPMC6154143 | biostudies-literature

A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51A alterations.

Project description:Fourteen Aspergillus fumigatus clinical isolates that exhibited a pattern of reduced susceptibility to triazole drugs were analyzed. The sequences of the cyp51A gene from all isolates showed the presence of a point mutation at t364a, which led to the substitution of leucine 98 for histidine (L98H), together with the presence of two copies of a 34-bp sequence in tandem in the promoter of the cyp51A gene. Quantitative expression analysis (real-time PCR) showed up to an eightfold increase in the level of expression of the cyp51A gene compared to that by the susceptible strain. Three PCR fragments of one azole-resistant strain (strain CM2627) that included the promoter with the tandem repeat and part of cyp51A with the t364a mutation or PCR fragments with only one of the modifications were used to replace the cyp51A gene of an azole drug-susceptible A. fumigatus wild-type strain (strain CM237). Only transformants which had incorporated the tandem repeat in the promoter of the cyp51A gene and the L98H amino acid substitution exhibited similarly reduced patterns of susceptibility to all triazole agents and similarly increased levels of cyp51A expression, confirming that the combination of both alterations was responsible for the azole-resistant phenotype.

| S-EPMC1891382 | biostudies-literature

Multiple cyp51A-based mechanisms identified in azole-resistant isolates of Aspergillus fumigatus from China.

Project description:Seventy-two A. fumigatus clinical isolates from China were investigated for azole resistance based on mutations of cyp51A. We identified four azole-resistant strains, among which we found three strains highly resistant to itraconazole, two of which exhibit the TR34/L98H/S297T/F495I mutation, while one carries only the TR34/L98H mutation. To our knowledge, the latter has not been found previously in China. The fourth multiazole-resistant isolate (with only moderate itraconazole resistance) carries a new G432A mutation.

| S-EPMC4468671 | biostudies-literature

The cytochrome P450 reductase CprA is a rate-limiting factor for Cyp51A-mediated azole resistance in Aspergillus fumigatus.

Project description:Azole antifungals remain the "gold standard" therapy for invasive aspergillosis. The world-wide emergence of isolates resistant to this drug class, however, developed into a steadily increasing threat to human health over the past years. In Aspergillus fumigatus, major mechanisms of resistance involve increased expression of cyp51A encoding one of two isoenzymes targeted by azoles. Yet, the level of resistance caused by cyp51A upregulation, driven by either clinically relevant tandem repeat mutations within its promoter or the use of high expressing heterologous promoters, is limited. Cytochrome P450 enzymes such as Cyp51A rely on redox partners that provide electrons for their activity. A. fumigatus harbors several genes encoding putative candidate proteins including two paralogous cytochrome P450 reductases, CprA and CprB, and the cytochrome b 5 CybE. In this work, we investigated the contribution of each cprA, cprB, and cybE overexpression to cyp51A-mediated resistance to different medical and agricultural azoles. Using the bidirectional promoter PxylP, we conditionally expressed these genes in combination with cyp51A, revealing cprA as the main limiting factor. Similar to this approach, we overexpressed cprA in an azole-resistant background strain carrying a cyp51A allele with TR34 in its promoter, which led to a further increase in its resistance. Employing sterol measurements, we demonstrate an enhanced eburicol turnover during upregulation of either cprA or cyp51A, which was even more pronounced during their simultaneous overexpression. In summary, our work suggests that mutations leading to increased Cyp51A activity through increased electron supply could be key factors that elevate azole resistance.

| S-EPMC10648939 | biostudies-literature

In Vivo Selection of a Unique Tandem Repeat Mediated Azole Resistance Mechanism (TR120) in Aspergillus fumigatus cyp51A, Denmark.

Project description:We report a fatal aspergillosis case in which STRAf typing and whole-genome sequencing substantiated in vivo emergence of an azole-resistant Aspergillus fumigatus with a 120-bp tandem repeat in the promoter region of cyp51A. This event, previously restricted to the environment, challenges current understanding of azole resistance development in A. fumigatus.

| S-EPMC6390761 | biostudies-literature

Aspergillus fumigatus Clinical Isolates Carrying CYP51A with TR34/L98H/S297T/F495I Substitutions Detected after Four-Year Retrospective Azole Resistance Screening in Brazil.

Project description:Azole antifungal resistance in Aspergillus fumigatus is a worldwide concern. As in most public hospitals in Brazil, antifungal susceptibility tests are not routinely performed for filamentous fungi at our institution. A 4-year retrospective azole antifungal resistance screening revealed two azole-resistant A. fumigatus clinical isolates carrying the CYP51A TR34 (34-bp tandem repeat)/L98H (change of L to H at position 98)/S297T/F495I resistance mechanism mutations, obtained from two unrelated patients. Broth microdilution antifungal susceptibility testing showed high MICs for itraconazole, posaconazole, and miconazole. Short tandem repeat (STR) typing analysis presented high levels of similarity between these two isolates and clinical isolates with the same mutations reported from the Netherlands, Denmark, and China, as well as environmental isolates from Taiwan. Our findings might indicate that active searching for resistant A. fumigatus is necessary. They also represent a concern considering that our hospital provides tertiary care assistance to immunocompromised patients who may be exposed to resistant environmental isolates. We also serve patients who receive prophylactic antifungal therapy or treatment for invasive fungal infections for years. In these two situations, isolates resistant to the antifungal in use may be selected within the patients themselves. We do not know the potential of this azole-resistant A. fumigatus strain to spread throughout our country. In this scenario, the impact on the epidemiology and use of antifungal drugs will significantly alter patient care, as in other parts of the world. In summary, this finding is an important contribution to alert hospital laboratories conducting routine microbiological testing to perform azole resistance surveillance and antifungal susceptibility tests of A. fumigatus isolates causing infection or colonization in patients at high risk for systemic aspergillosis.

| S-EPMC7038256 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data