scholarly journals Analysis of the Contribution of cyp51 Genes to Azole Resistance in Aspergillus Section Nigri with the CRISPR-Cas9 Technique

2021 ◽  
Vol 65 (5) ◽  
Author(s):  
Alba Pérez-Cantero ◽  
Adela Martin-Vicente ◽  
Josep Guarro ◽  
Jarrod R. Fortwendel ◽  
Javier Capilla
Keyword(s):  
Single Gene ◽  
Transcriptional Response ◽  
Azole Resistance ◽  
Gene Deletions ◽  
Content Type ◽  
Ergosterol Content ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Induced Changes ◽  
The Impact

ABSTRACT Cyp51 contribution to azole resistance has been broadly studied and characterized in Aspergillus fumigatus, whereas it remains poorly investigated in other clinically relevant species of the genus, such as those of section Nigri. In this work, we aimed to analyze the impact of cyp51 genes (cyp51A and cyp51B) on the voriconazole (VRC) response and resistance of Aspergillus niger and Aspergillus tubingensis. We generated CRISPR-Cas9 cyp51A and cyp51B knockout mutants from strains with different genetic backgrounds and diverse patterns of azole susceptibility. Single-gene deletions of cyp51 genes resulted in 2- to 16-fold decreases of the VRC MIC values, which were below the VRC epidemiological cutoff value (ECV) established by the Clinical and Laboratory Standards Institute (CLSI), irrespective of their parental strains’ susceptibilities. Gene expression studies in the tested species confirmed that cyp51A participates more actively than cyp51B in the transcriptional response of azole stress. However, ergosterol quantification revealed that both enzymes comparably impact the total ergosterol content within the cell, as basal- and VRC-induced changes to ergosterol content were similar in all cases. These data contribute to our understanding of Aspergillus azole resistance, especially in non-A. fumigatus species.

scholarly journals Azole Resistance Reduces Susceptibility to the Tetrazole Antifungal VT-1161

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Brian C. Monk ◽  
Mikhail V. Keniya ◽  
Manya Sabherwal ◽  
Rajni K. Wilson ◽  
Danyon O. Graham ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Efflux Pumps ◽  
Drug Binding ◽  
Full Length ◽  
Major Facilitator Superfamily ◽  
Azole Resistance ◽  
Drug Efflux ◽  
Content Type ◽  
Drug Efflux Pumps ◽  
The Impact

ABSTRACTTetrazole antifungals designed to target fungal lanosterol 14α-demethylase (LDM) appear to be effective against a range of fungal pathogens. In addition, a crystal structure of the catalytic domain ofCandida albicansLDM in complex with the tetrazole VT-1161 has been obtained. We have addressed concern about artifacts that might arise from crystallizing VT-1161 with truncated recombinant CYP51s and measured the impact on VT-1161 susceptibility of genotypes known to confer azole resistance. A yeast system was used to overexpress recombinant full-lengthSaccharomyces cerevisiaeLDM with a C-terminal hexahistidine tag (ScLDM6×His) for phenotypic analysis and crystallographic studies with VT-1161 or with the widely used triazole drug posaconazole (PCZ). We determined the effect of characterized mutations in LDM on VT-1161 activity and identified drug efflux pumps from fungi, including key fungal pathogens, that efflux VT-1161. The relevance of these yeast-based observations on drug efflux was verified using clinical isolates ofC. albicansandCandida glabrata. VT-1161 binding elicits a significant conformational difference between the full-length and truncated enzymes not found when posaconazole is bound. Susceptibility to VT-1161 is reduced by ATP-binding cassette (ABC) and major facilitator superfamily (MFS) drug efflux pumps, the overexpression of LDM, and mutations within the drug binding pocket of LDM that affect interaction with the tertiary alcohol of the drug.

scholarly journals New Insights into the Cyp51 Contribution to Azole Resistance in Aspergillus Section Nigri

2019 ◽  
Vol 63 (7) ◽  
Author(s):  
Alba Pérez-Cantero ◽  
Loida López-Fernández ◽  
Josep Guarro ◽  
Javier Capilla
Keyword(s):  
Resistance Mechanisms ◽  
Azole Resistance ◽  
Wild Type ◽  
Severe Condition ◽  
Content Type ◽  
Recommended Treatment ◽  
Highly Active ◽  
Expression Studies

ABSTRACT Invasive aspergillosis (IA) is a severe condition mainly caused by Aspergillus fumigatus, although other species of the genus, such as section Nigri members, can also be involved. Voriconazole (VRC) is the recommended treatment for IA; however, the prevalence of azole-resistant Aspergillus isolates has alarmingly increased in recent years, and the underlying resistance mechanisms in non-fumigatus species remain unclear. We have determined the in vitro susceptibility of 36 strains from section Nigri to VRC, posaconazole (POS), and itraconazole (ITC), and we have explored the role of Cyp51A and Cyp51B, both targets of azoles, in azole resistance. The three drugs were highly active; POS displayed the best in vitro activity, while ITC and VRC showed MICs above the established epidemiological cutoff values in 9 and 16% of the strains, respectively. Furthermore, expression studies of cyp51A and cyp51B in control condition and after VRC exposure were performed in 14 strains with different VRC susceptibility. We found higher transcription of cyp51A, which was upregulated upon VRC exposure, but no correlation between MICs and cyp51 transcription levels was observed. In addition, cyp51A sequence analyses revealed nonsynonymous mutations present in both, wild-type and non-wild-type strains of A. niger and A. tubingensis. Nevertheless, a few mutations were exclusively present in non-wild-type A. tubingensis strains. Altogether, our results suggest that azole resistance in section Nigri is not clearly explained by Cyp51A protein alteration or by cyp51 gene upregulation, which indicates that other mechanisms might be involved.

Human erythroid porphobilinogen deaminase exists in 2 splice variants

Blood ◽  
2001 ◽  
Vol 97 (3) ◽  
pp. 815-817 ◽  
Author(s):  
Alexander N. Gubin ◽  
Jeffery L. Miller
Keyword(s):  
Gene Product ◽  
Splice Variants ◽  
Single Gene ◽  
Specific Gene ◽  
Porphobilinogen Deaminase ◽  
Messenger Rnas ◽  
Alternate Splicing ◽  
Expression Studies ◽  
Endogenous Reference ◽  
Gene Expression Studies

Abstract Human porphobilinogen deaminase (PBGD) is, reportedly, encoded by 2 distinct messenger RNAs (mRNAs) transcribing from a single gene. The ubiquitous form of the PBGD gene product is often used as an endogenous reference in gene expression studies because it is pseudogene free and has minimal transcriptional variability among tissues. A distinct erythroid-specific gene product has also been described because of the alternate splicing of the gene. Here is reported the existence of an additional erythroid-specific isoform of PBGD mRNA in primary cells.

scholarly journals Identifying novel β-lactamase substrate activity through in silico prediction of antimicrobial resistance

2021 ◽  
Author(s):  
Kara K. Tsang ◽  
Finlay Maguire ◽  
Haley L. Zubyk ◽  
Sommer Chou ◽  
Arman Edalatmand ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Type Species ◽  
Multidrug Resistant ◽  
Content Type ◽  
Logistic Regression Models ◽  
Link Type ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Targeted Gene Expression ◽  
Phenotypic Methods

Diagnosing antimicrobial resistance (AMR) in the clinic is based on empirical evidence and current gold standard laboratory phenotypic methods. Genotypic methods have the potential advantages of being faster and cheaper, and having improved mechanistic resolution over phenotypic methods. We generated and applied rule-based and logistic regression models to predict the AMR phenotype from Escherichia coli and Pseudomonas aeruginosa multidrug-resistant clinical isolate genomes. By inspecting and evaluating these models, we identified previously unknown β-lactamase substrate activities. In total, 22 unknown β-lactamase substrate activities were experimentally validated using targeted gene expression studies. Our results demonstrate that generating and analysing predictive models can help guide researchers to the mechanisms driving resistance and improve annotation of AMR genes and phenotypic prediction, and suggest that we cannot solely rely on curated knowledge to predict resistance phenotypes.

scholarly journals Biosynthesis and Secretion of Indole-3-Acetic Acid and Its Morphological Effects on Tricholoma vaccinum-Spruce Ectomycorrhiza

2015 ◽  
Vol 81 (20) ◽  
pp. 7003-7011 ◽  
Author(s):  
Katrin Krause ◽  
Catarina Henke ◽  
Theodore Asiimwe ◽  
Andrea Ulbricht ◽  
Sandra Klemmer ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Mycorrhizal Fungi ◽  
Schizophyllum Commune ◽  
Content Type ◽  
Saprobic Fungi ◽  
Hartig Net ◽  
Expression Studies ◽  
Fungal Morphogenesis ◽  
Gene Expression Studies ◽  
Indole 3 Acetic Acid

ABSTRACTFungus-derived indole-3-acetic acid (IAA), which is involved in development of ectomycorrhiza, affects both partners, i.e., the tree and the fungus. The biosynthesis pathway, excretion from fungal hyphae, the induction of branching in fungal cultures, and enhanced Hartig net formation in mycorrhiza were shown. Gene expression studies, incorporation of labeled compounds into IAA, heterologous expression of a transporter, and bioinformatics were applied to study the effect of IAA on fungal morphogenesis and on ectomycorrhiza.Tricholoma vaccinumproduces IAA from tryptophan via indole-3-pyruvate, with the last step of this biosynthetic pathway being catalyzed by an aldehyde dehydrogenase. The geneald1was found to be highly expressed in ectomycorrhiza and induced by indole-3-acetaldehyde. The export of IAA from fungal cells is supported by the multidrug and toxic extrusion (MATE) transporter Mte1 found inT. vaccinum. The addition of IAA and its precursors induced elongated cells and hyphal ramification of mycorrhizal fungi; in contrast, in saprobic fungi such asSchizophyllum commune, IAA did not induce morphogenetic changes. Mycorrhiza responded by increasing its Hartig net formation. The IAA of fungal origin acts as a diffusible signal, influencing root colonization and increasing Hartig net formation in ectomycorrhiza.

scholarly journals Large-scale gene expression studies of the endometrium: what have we learnt?

Reproduction ◽  
2006 ◽  
Vol 132 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Rob Sherwin ◽  
Rob Catalano ◽  
Andrew Sharkey
Keyword(s):  
Large Scale ◽  
Dna Microarrays ◽  
Disease States ◽  
Complex Interactions ◽  
Expression Studies ◽  
Proliferation And Differentiation ◽  
Potential Applications ◽  
Underlying Causes ◽  
Gene Expression Studies ◽  
The Impact

The endometrium is a dynamic tissue that undergoes coordinated changes under the influence of steroid hormones. This results in proliferation and differentiation culminating in a receptive state, followed by menstruation and endometrial repair. These functions involve complex interactions between the epithelium, stroma and leucocytes in the endometrium. Understanding the underlying causes of endometrial disorders, such as infertility, endometriosis and heavy menstrual bleeding, therefore represents a considerable challenge. Recently developed techniques, such as differential display and DNA microarrays permit the abundance of thousands of mRNA transcripts within cells or tissues to be measured simultaneously. This provides a new approach to understanding the complex interactions that underlie both healthy and disease states. Responses of the endometrium to hormones or drugs can be studied and the response of the system as an integrated whole can be assessed. Comparisons of endometrium from healthy women and those with endometrial dysfunction have advanced our understanding of key areas of endometrial physiology, including infertility, receptivity, endometriosis and cancer. Using this approach, novel genes controlling specific endometrial functions like receptivity have been identified for functional testing. This paper will review the impact of these techniques for transcript profiling on our understanding of selected areas of endometrial biology and discuss the potential applications in future.

scholarly journals Glucocorticoid receptor activation induces gene-specific transcriptional memory and universally reversible changes in chromatin accessibility

2021 ◽  
Author(s):  
Melissa Bothe ◽  
René Buschow ◽  
Sebastiaan H. Meijsing
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Repression ◽  
Single Gene ◽  
Transcriptional Response ◽  
Receptor Activation ◽  
Chromatin Accessibility ◽  
Airway Epithelial Cells ◽  
Transcriptional Responses ◽  
Transcriptional Memory ◽  
Induced Changes

AbstractGlucocorticoids are stress hormones that elicit cellular responses by binding to the glucocorticoid receptor (GR), a ligand-activated transcription factor. The exposure of cells to this hormone induces wide-spread changes in the chromatin landscape and gene expression. Previous studies have suggested that some of these changes are reversible whereas others persist even when the hormone is no longer around. However, when we examined chromatin accessibility in human airway epithelial cells after hormone washout, we found that the hormone-induced changes were universally reversed after one day. Reversibility of hormone-induced changes are found for GR-occupied opening sites and also for closing sites that typically lack GR occupancy. These closing sites are enriched near repressed genes, suggesting that transcriptional repression by GR does not require nearby GR binding. Mirroring what we say in terms of chromatin accessibility, we found that transcriptional responses to hormone are universally reversable. Moreover, priming of cells by a previous exposure to hormone, in general, did not alter the transcriptional response to a subsequent encounter of the same cue. Interestingly, despite the short-lived nature of hormone-induced changes in the chromatin landscape, we identified a single gene, ZBTB16, that displays transcriptional memory manifesting itself as a more robust transcriptional response upon repeated hormone stimulation. Single-cell analysis revealed that the more robust response is driven by a higher probability of primed cells to activate ZBTB16 and by a subset of cells that express the gene at levels that are higher than the induction levels observed for naïve cells. Although our study shows that hormone-induced changes are typically reversable, exposure to hormone can induce gene-specific changes in the response to subsequent exposures which may play a role in habituation to stressors and changes in glucocorticoid sensitivity.

scholarly journals Method Enabling Gene Expression Studies of Pathogens in a Complex Food Matrix

2011 ◽  
Vol 77 (23) ◽  
pp. 8456-8458 ◽  
Author(s):  
Jette Kjeldgaard ◽  
Sidsel Henriksen ◽  
Marianne Thorup Cohn ◽  
Søren Aabo ◽  
Hanne Ingmer
Keyword(s):  
Gene Expression ◽  
Food Matrix ◽  
Simple Method ◽  
Bacterial Gene ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Bacterial Gene Expression ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Minced Meat

ABSTRACTWe describe a simple method for stabilizing and extracting high-quality prokaryotic RNA from meat. Heat and salt stress ofEscherichia coliandSalmonellaspp. in minced meat reproducibly induceddnaKandotsBexpression, respectively, as observed by quantitative reverse transcription-PCR (>5-fold relative changes). Thus, the method is applicable in studies of bacterial gene expression in a meat matrix.

scholarly journals Suboptimal Global Transcriptional Response Increases the Harmful Effects of Loss-of-Function Mutations

2020 ◽  
Author(s):  
Károly Kovács ◽  
Zoltán Farkas ◽  
Djordje Bajić ◽  
Dorottya Kalapis ◽  
Andreea Daraba ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Single Gene ◽  
Transcriptional Response ◽  
Gene Inactivation ◽  
Loss Of Function ◽  
Gene Deletions ◽  
Genomic Expression ◽  
Deleterious Gene ◽  
Functionally Diverse ◽  
Harmful Effects

Abstract The fitness impact of loss-of-function mutations is generally assumed to reflect the loss of specific molecular functions associated with the perturbed gene. Here, we propose that rewiring of the transcriptome upon deleterious gene inactivation is frequently nonspecific and mimics stereotypic responses to external environmental change. Consequently, transcriptional response to gene deletion could be suboptimal and incur an extra fitness cost. Analysis of the transcriptomes of ∼1,500 single-gene deletion Saccharomyces cerevisiae strains supported this scenario. First, most transcriptomic changes are not specific to the deleted gene but are rather triggered by perturbations in functionally diverse genes. Second, gene deletions that alter the expression of dosage-sensitive genes are especially harmful. Third, by elevating the expression level of downregulated genes, we could experimentally mitigate the fitness defect of gene deletions. Our work shows that rewiring of genomic expression upon gene inactivation shapes the harmful effects of mutations.

scholarly journals Genome Sequence of Thermotolerant Bacillus methanolicus: Features and Regulation Related to Methylotrophy and Production of l-Lysine and l-Glutamate from Methanol

2012 ◽  
Vol 78 (15) ◽  
pp. 5170-5181 ◽  
Author(s):  
Tonje M. B. Heggeset ◽  
Anne Krog ◽  
Simone Balzer ◽  
Alexander Wentzel ◽  
Trond E. Ellingsen ◽  
...  
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
System Level ◽  
Content Type ◽  
Physiological Diversity ◽  
Bacillus Methanolicus ◽  
Expression Studies ◽  
Genomic Analyses ◽  
Gene Expression Studies ◽  
Thermotolerant Bacterium ◽  
Insight Into

ABSTRACTBacillus methanolicuscan utilize methanol as its sole carbon and energy source, and the scientific interest in this thermotolerant bacterium has focused largely on exploring its potential as a biocatalyst for the conversion of methanol intol-lysine andl-glutamate. We present here the genome sequences of the importantB. methanolicusmodel strain MGA3 (ATCC 53907) and the alternative wild-type strain PB1 (NCIMB13113). The physiological diversity of these two strains was demonstrated by a comparative fed-batch methanol cultivation displaying highly different methanol consumption and respiration profiles, as well as major differences in theirl-glutamate production levels (406 mmol liter−1and 11 mmol liter−1, respectively). Both genomes are small (ca 3.4 Mbp) compared to those of other related bacilli, and MGA3 has two plasmids (pBM19 and pBM69), while PB1 has only one (pBM20). In particular, we focus here on genes representing biochemical pathways for methanol oxidation and concomitant formaldehyde assimilation and dissimilation, the important phosphoenol pyruvate/pyruvate anaplerotic node, the tricarboxylic acid cycle including the glyoxylate pathway, and the biosynthetic pathways forl-lysine andl-glutamate. Several unique findings were made, including the discovery of three different methanol dehydrogenase genes in each of the twoB. methanolicusstrains, and the genomic analyses were accompanied by gene expression studies. Our results provide new insight into a number of peculiar physiological and metabolic traits ofB. methanolicusand open up possibilities for system-level metabolic engineering of this bacterium for the production of amino acids and other useful compounds from methanol.

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