Insights in the molecular mechanisms of an azole stress adapted laboratory-generated Aspergillus fumigatus strain

2021 ◽  
Author(s):  
Marion Aruanno ◽  
Samantha Gozel ◽  
Isabelle Mouyna ◽  
Josie E Parker ◽  
Daniel Bachmann ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Molecular Mechanisms ◽  
Drug Transporters ◽  
Major Facilitator Superfamily ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Drug Transporter

Abstract Aspergillus fumigatus is the main cause of invasive aspergillosis, for which azole drugs are the first-line therapy. Emergence of pan-azole resistance among A. fumigatus is concerning and has been mainly attributed to mutations in the target gene (cyp51A). However, azole resistance may also result from other mutations (hmg1, hapE) or other adaptive mechanisms. We performed microevolution experiment exposing an A. fumigatus azole-susceptible strain (Ku80) to sub-minimal inhibitory concentration of voriconazole to analyze emergence of azole resistance. We obtained a strain with pan-azole resistance (Ku80R), which was partially reversible after drug relief, and without mutations in cyp51A, hmg1, and hapE. Transcriptomic analyses revealed overexpression of the transcription factor asg1, several ATP-binding cassette (ABC) and major facilitator superfamily transporters and genes of the ergosterol biosynthesis pathway in Ku80R. Sterol analysis showed a significant decrease of the ergosterol mass under voriconazole exposure in Ku80, but not in Ku80R. However, the proportion of the sterol compounds was similar between both strains. To further assess the role of transporters, we used the ABC transporter inhibitor milbemycine oxime (MLB). MLB inhibited transporter activity in both Ku80 and Ku80R and demonstrated some potentiating effect on azole activity. Criteria for synergism were reached for MLB and posaconazole against Ku80. Finally, deletion of asg1 revealed some role of this transcription factor in controlling drug transporter expression, but had no impact on azole susceptibility. This work provides further insight in mechanisms of azole stress adaptation and suggests that drug transporters inhibition may represent a novel therapeutic target. Lay Summary A pan-azole-resistant strain was generated in vitro, in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity.

scholarly journals The C2H2 Transcription Factor SltA Contributes to Azole Resistance by Coregulating the Expression of the Drug Target Erg11A and the Drug Efflux Pump Mdr1 in Aspergillus fumigatus

2021 ◽  
Author(s):  
Wenlong Du ◽  
Pengfei Zhai ◽  
Tingli Wang ◽  
Michael J Bromley ◽  
Yuanwei Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Fungal Pathogens ◽  
Efflux Pump ◽  
Antifungal Drugs ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Drug Efflux ◽  
Mobility Shift ◽  
Drug Efflux Pump

The emergence of azole-resistant fungal pathogens has posed a great threat to public health worldwide. Although the molecular mechanism of azole resistance has been extensively investigated, the potential regulators of azole resistance remain largely unexplored. Here we identified a new function of the fungal specific C2H2 zinc finger transcription factor SltA (involved in salt-tolerance pathway) in the regulation of azole resistance of the human fungal pathogen Aspergillus fumigatus. Lack of SltA results in an itraconazole hypersusceptibility phenotype. Transcriptional profiling combined with LacZ reporter analysis and electrophoretic mobility shift assays (EMSA) demonstrate that SltA is involved in its own transcriptional regulation and also regulates the expression of genes related to ergosterol biosynthesis (erg11A, erg13A and erg24A) and drug efflux pumps (mdr1, mfsC and abcE) by directly binding to the conserved 5’-AGGCA-3’ motif in their promoter regions, and this binding is dependent on the conserved cysteine and histidine within the C2H2 DNA binding domain of SltA. Moreover, overexpression of erg11A or mdr1 rescues sltA deletion defects under itraconazole conditions, suggesting that erg11A and mdr1 are related to sltA-mediated itraconazole resistance. Most importantly, deletion of SltA in laboratory-derived and clinical azole-resistant isolates significantly attenuates drug resistance. Collectively, we have identified a new function of the transcription factor SltA in regulating azole resistance by coordinately mediating the key azole target Erg11A and the drug efflux pump Mdr1, and targeting SltA may provide a potential strategy for intervention of clinical azole-resistant isolates to improve the efficiency of currently approved antifungal drugs.

scholarly journals Antifungal Resistance: Specific Focus on Multidrug Resistance in Candida auris and Secondary Azole Resistance in Aspergillus fumigatus

2018 ◽  
Vol 4 (4) ◽  
pp. 129 ◽  
Author(s):  
Sevtap Arikan-Akdagli ◽  
Mahmoud Ghannoum ◽  
Jacques Meis
Keyword(s):  
Aspergillus Fumigatus ◽  
Molecular Mechanisms ◽  
Multidrug Resistant ◽  
Fungal Species ◽  
Antifungal Resistance ◽  
Current Status ◽  
Clinical Implications ◽  
Azole Resistance ◽  
Candida Auris

Antifungal resistance is a topic of concern, particularly for specific fungal species and drugs. Among these are the multidrug-resistant Candida auris and azole-resistant Aspergillus fumigatus. While the knowledge on molecular mechanisms of resistance is now accumulating, further data are also available for the clinical implications and the extent of correlation of in vitro resistance to clinical outcomes. This review article summarizes the epidemiology of C. auris infections, animal models focusing on the activity of novel antifungal compounds in C. auris infections, virulence factors, and the mechanisms of antifungal resistance for this multi-resistant Candida species. Regarding A. fumigatus, the significance of azoles in the treatment of A. fumigatus infections, reference methods available for the detection of resistance in vitro, molecular mechanisms of secondary azole resistance, routes of acquisition, and clinical implications of in vitro resistance are covered to provide guidance for the current status of azole resistance in A. fumigatus.

scholarly journals Are Point Mutations in HMG-CoA Reductases (Hmg1 and Hmg2) a Step towards Azole Resistance in Aspergillus fumigatus?

Molecules ◽  
2021 ◽  
Vol 26 (19) ◽  
pp. 5975
Author(s):  
Irene Gonzalez-Jimenez ◽  
Jose Lucio ◽  
Alejandra Roldan ◽  
Laura Alcazar-Fuoli ◽  
Emilia Mellado
Keyword(s):  
Aspergillus Fumigatus ◽  
Structural Changes ◽  
Survival Rates ◽  
Point Mutations ◽  
Resistance Mechanisms ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Recent Emergence ◽  
Resistant Strains

Invasive aspergillosis, mainly caused by Aspergillus fumigatus, can lead to severe clinical outcomes in immunocompromised individuals. Antifungal treatment, based on the use of azoles, is crucial to increase survival rates. However, the recent emergence of azole-resistant A. fumigatus isolates is affecting the efficacy of the clinical therapy and lowering the success rate of azole strategies against aspergillosis. Azole resistance mechanisms described to date are mainly associated with mutations in the azole target gene cyp51A that entail structural changes in Cyp51A or overexpression of the gene. However, strains lacking cyp51A modifications but resistant to clinical azoles have recently been detected. Some genes have been proposed as new players in azole resistance. In this study, the gene hmg1, recently related to azole resistance, and its paralogue hmg2 were studied in a collection of fifteen azole-resistant strains without cyp51A modifications. Both genes encode HMG-CoA reductases and are involved in the ergosterol biosynthesis. Several mutations located in the sterol sensing domain (SSD) of Hmg1 (D242Y, G307D/S, P309L, K319Q, Y368H, F390L and I412T) and Hmg2 (I235S, V303A, I312S, I360F and V397C) were detected. The role of these mutations in conferring azole resistance is discussed in this work.

scholarly journals Genome-Wide Association for Itraconazole Sensitivity in Non-resistant Clinical Isolates of Aspergillus fumigatus

2021 ◽  
Vol 1 ◽  
Author(s):  
Shu Zhao ◽  
Wenbo Ge ◽  
Akira Watanabe ◽  
Jarrod R. Fortwendel ◽  
John G. Gibbons
Keyword(s):  
Aspergillus Fumigatus ◽  
Complex Traits ◽  
Molecular Mechanisms ◽  
Clinical Isolates ◽  
Genome Wide Association ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Genome Wide ◽  
Population Structure Analysis ◽  
Recent Emergence

Aspergillus fumigatus is a potentially lethal opportunistic pathogen that infects over ~200,000 people and causes ~100,000 deaths per year globally. Treating A. fumigatus infections is particularly challenging because of the recent emergence of azole-resistance. The majority of studies focusing on the molecular mechanisms underlying azole resistance have examined azole-resistant isolates. However, isolates that are susceptible to azoles also display variation in their sensitivity, presenting a unique opportunity to identify genes contributing to azole sensitivity. Here, we used genome-wide association (GWA) analysis to identify loci involved in azole sensitivity by analyzing the association between 68,853 SNPs and itraconazole (ITCZ) minimum inhibitory concentration (MIC) in 76 clinical isolates of A. fumigatus from Japan. Population structure analysis suggests the presence of four distinct populations, with ITCZ MICs distributed relatively evenly across populations. We independently conducted GWA when treating ITCZ MIC as a quantitative trait and a binary trait, and identified two SNPs with strong associations in both analyses. These SNPs fell within the coding regions of Afu2g02220 and Afu2g02140. We functionally validated Afu2g02220 by knocking it out using a CRISPR/Cas9 approach, because orthologs of this gene are involved in sterol modification and ITCZ targets the ergosterol biosynthesis pathway. Knockout strains displayed no difference in growth compared to the parent strain in minimal media, yet a minor but consistent inhibition of growth in the presence of 0.15 μg/ml ITCZ. Our results suggest that GWA paired with efficient gene deletion is a powerful and unbiased strategy for identifying the genetic basis of complex traits in A. fumigatus.

scholarly journals Switching on pluripotency: a perspective on the biological requirement of Nanog

2011 ◽  
Vol 366 (1575) ◽  
pp. 2222-2229 ◽  
Author(s):  
Thorold W. Theunissen ◽  
José C. R. Silva
Keyword(s):  
Transcription Factor ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Induced Pluripotency ◽  
Somatic Cell Reprogramming ◽  
Evolutionarily Conserved

Pluripotency is a transient cellular state during early development which can be recreated in vitro by direct reprogramming. The molecular mechanisms driving entry into and exit from the pluripotent state are the subject of intense research interest. Here, we review the role of the homeodomain-containing transcription factor Nanog in mammalian embryology and induced pluripotency. Nanog was originally thought to be confined to the maintenance of pluripotency, but recent insights from genetic studies uncovered a new biological function. Embryonic stem cells deficient in Nanog alleles are more prone to differentiate but do not lose pluripotency per se . Instead, Nanog is transiently required for the specification of the naive pluripotent epiblast and development of primordial germ cells. Nanog is also essential to finalize somatic cell reprogramming during induction of pluripotency. We propose that this unique transcription factor acts as a molecular switch to turn on the naive pluripotent programme in mammalian cells. In this context, the capacity of Nanog to resist differentiation can be regarded as recapitulation of effects normally associated with the specification of pluripotency. Pertinent questions are how Nanog specifies naive pluripotency and whether this mechanism is evolutionarily conserved.

scholarly journals Microfluidic tumor-on-a-chip model to evaluate the role of tumor environmental stress on NK cell exhaustion

2021 ◽  
Vol 7 (8) ◽  
pp. eabc2331 ◽  
Author(s):  
Jose M. Ayuso ◽  
Shujah Rehman ◽  
Maria Virumbrales-Munoz ◽  
Patrick H. McMinn ◽  
Peter Geiger ◽  
...  
Keyword(s):  
Nk Cells ◽  
Immune Cells ◽  
Molecular Mechanisms ◽  
Nk Cell ◽  
Checkpoint Inhibitors ◽  
Waste Product ◽  
Extended Period ◽  
Cell Exhaustion

Solid tumors generate a suppressive environment that imposes an overwhelming burden on the immune system. Nutrient depletion, waste product accumulation, hypoxia, and pH acidification severely compromise the capacity of effector immune cells such as T and natural killer (NK) cells to destroy cancer cells. However, the specific molecular mechanisms driving immune suppression, as well as the capacity of immune cells to adapt to the suppressive environment, are not completely understood. Thus, here, we used an in vitro microfluidic tumor-on-a-chip platform to evaluate how NK cells respond to the tumor-induced suppressive environment. The results demonstrated that the suppressive environment created by the tumor gradually eroded NK cell cytotoxic capacity, leading to compromised NK cell surveillance and tumor tolerance. Further, NK cell exhaustion persisted for an extended period of time after removing NK cells from the microfluidic platform. Last, the addition of checkpoint inhibitors and immunomodulatory agents alleviated NK cell exhaustion.

scholarly journals Regulatory Role of microRNAs Targeting the Transcription Co-Factor ZNF521 in Normal Tissues and Cancers

2021 ◽  
Vol 22 (16) ◽  
pp. 8461
Author(s):  
Emanuela Chiarella ◽  
Annamaria Aloisio ◽  
Stefania Scicchitano ◽  
Heather Mandy Bond ◽  
Maria Mesuraca
Keyword(s):  
Transcription Factor ◽  
Molecular Mechanisms ◽  
Transitional Cell ◽  
Biological Functions ◽  
Aberrant Expression ◽  
Normal Tissues ◽  
Novel Mirna ◽  
Mirna Expression Profiling ◽  
Mirna Deregulation

Powerful bioinformatics tools have provided a wealth of novel miRNA–transcription factor networks crucial in controlling gene regulation. In this review, we focus on the biological functions of miRNAs targeting ZNF521, explaining the molecular mechanisms by which the dysregulation of this axis contributes to malignancy. ZNF521 is a stem cell-associated co-transcription factor implicated in the regulation of hematopoietic, neural, and mesenchymal stem cells. The aberrant expression of ZNF521 transcripts, frequently associated with miRNA deregulation, has been detected in several tumors including pancreatic, hepatocellular, gastric, bladder transitional cell carcinomas as well as in breast and ovarian cancers. miRNA expression profiling tools are currently identifying a multitude of miRNAs, involved together with oncogenes and TFs in the regulation of oncogenesis, including ZNF521, which may be candidates for diagnostic and prognostic biomarkers of cancer.

scholarly journals Progression and Differentiation of Alveolar Rhabdomyosarcoma Is Regulated by PAX7 Transcription Factor—Significance of Tumor Subclones

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1870
Author(s):  
Klaudia Skrzypek ◽  
Grażyna Adamek ◽  
Marta Kot ◽  
Bogna Badyra ◽  
Marcin Majka
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Migration Activity ◽  
Id Proteins ◽  
Rh30 Cell ◽  
Str Profile ◽  
And Migration

Rhabdomyosarcoma (RMS), is the most frequent soft tissue tumor in children that originates from disturbances in differentiation process. Mechanisms leading to the development of RMS are still poorly understood. Therefore, by analysis of two RMS RH30 cell line subclones, one subclone PAX7 negative, while the second one PAX7 positive, and comparison with other RMS cell lines we aimed at identifying new mechanisms crucial for RMS progression. RH30 subclones were characterized by the same STR profile, but different morphology, rate of proliferation, migration activity and chemotactic abilities in vitro, as well as differences in tumor morphology and growth in vivo. Our analysis indicated a different level of expression of adhesion molecules (e.g., from VLA and ICAM families), myogenic microRNAs, such as miR-206 and transcription factors, such as MYOD, MYOG, SIX1, and ID. Silencing of PAX7 transcription factor with siRNA confirmed the crucial role of PAX7 transcription factor in proliferation, differentiation and migration of RMS cells. To conclude, our results suggest that tumor cell lines with the same STR profile can produce subclones that differ in many features and indicate crucial roles of PAX7 and ID proteins in the development of RMS.

scholarly journals Analysis of a Preliminary microRNA Expression Signature in a Human Telangiectatic Osteogenic Sarcoma Cancer Cell Line

2021 ◽  
Vol 22 (3) ◽  
pp. 1163
Author(s):  
Gaia Palmini ◽  
Cecilia Romagnoli ◽  
Simone Donati ◽  
Roberto Zonefrati ◽  
Gianna Galli ◽  
...  
Keyword(s):  
Cell Line ◽  
Molecular Mechanisms ◽  
Osteogenic Sarcoma ◽  
Cancer Cell Line ◽  
Microscopic Features ◽  
In Vitro Establishment ◽  
Profile Characteristic ◽  
First Time

Telangiectatic osteosarcoma (TOS) is an aggressive variant of osteosarcoma (OS) with distinctive radiographic, gross, microscopic features, and prognostic implications. Despite several studies on OS, we are still far from understanding the molecular mechanisms of TOS. In recent years, many studies have demonstrated not only that microRNAs (miRNAs) are involved in OS tumorigenesis, development, and metastasis, but also that the presence in high-grade types of OS of cancer stem cells (CSCs) plays an important role in tumor progression. Despite these findings, nothing has been described previously about the expression of miRNAs and the presence of CSCs in human TOS. Therefore, we have isolated/characterized a putative CSC cell line from human TOS (TOS-CSCs) and evaluated the expression levels of several miRNAs in TOS-CSCs using real-time quantitative assays. We show, for the first time, the existence of CSCs in human TOS, highlighting the in vitro establishment of this unique stabilized cell line and an identification of a preliminary expression of the miRNA profile, characteristic of TOS-CSCs. These findings represent an important step in the study of the biology of one of the most aggressive variants of OS and the role of miRNAs in TOS-CSC behavior.

scholarly journals Endothelin 1-induced retinal ganglion cell death is largely mediated by JUN activation

2020 ◽  
Vol 11 (9) ◽  
Author(s):  
Olivia J. Marola ◽  
Stephanie B. Syc-Mazurek ◽  
Gareth R. Howell ◽  
Richard T. Libby
Keyword(s):  
Transcription Factor ◽  
Molecular Mechanisms ◽  
Ganglion Cells ◽  
Retinal Vessel ◽  
Vessel Diameter ◽  
Retinal Ganglion ◽  
Retinal Ganglion Cell Death ◽  
Ganglion Cell Death

Abstract Glaucoma is a neurodegenerative disease characterized by loss of retinal ganglion cells (RGCs), the output neurons of the retina. Multiple lines of evidence show the endothelin (EDN, also known as ET) system is important in glaucomatous neurodegeneration. To date, the molecular mechanisms within RGCs driving EDN-induced RGC death have not been clarified. The pro-apoptotic transcription factor JUN (the canonical target of JNK signaling) and the endoplasmic reticulum stress effector and transcription factor DNA damage inducible transcript 3 (DDIT3, also known as CHOP) have been shown to act downstream of EDN receptors. Previous studies demonstrated that JUN and DDIT3 were important regulators of RGC death after glaucoma-relevant injures. Here, we characterized EDN insult in vivo and investigated the role of JUN and DDIT3 in EDN-induced RGC death. To accomplish this, EDN1 ligand was intravitreally injected into the eyes of wildtype, Six3-cre+Junfl/fl (Jun−/−), Ddit3 null (Ddit3−/−), and Ddit3−/−Jun−/− mice. Intravitreal EDN1 was sufficient to drive RGC death in vivo. EDN1 insult caused JUN activation in RGCs, and deletion of Jun from the neural retina attenuated RGC death after EDN insult. However, deletion of Ddit3 did not confer significant protection to RGCs after EDN1 insult. These results indicate that EDN caused RGC death via a JUN-dependent mechanism. In addition, EDN signaling is known to elicit potent vasoconstriction. JUN signaling was shown to drive neuronal death after ischemic insult. Therefore, the effects of intravitreal EDN1 on retinal vessel diameter and hypoxia were explored. Intravitreal EDN1 caused transient retinal vasoconstriction and regions of RGC and Müller glia hypoxia. Thus, it remains a possibility that EDN elicits a hypoxic insult to RGCs, causing apoptosis via JNK-JUN signaling. The importance of EDN-induced vasoconstriction and hypoxia in causing RGC death after EDN insult and in models of glaucoma requires further investigation.

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