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

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.

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

Medical Mycology ◽

10.1093/mmy/myaa118 ◽

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.

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Multi-resistance to non-azole fungicides in Aspergillus fumigatus TR 34 /L98H azole resistant isolates.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00642-21 ◽

2021 ◽

Author(s):

I Gonzalez-Jimenez ◽

R Garcia-Rubio ◽

S Monzon ◽

J Lucio ◽

I Cuesta ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Plant Pathogens ◽

Crop Protection ◽

Resistance Mechanisms ◽

Azole Resistance ◽

Sequencing Analysis ◽

Human Fungal Pathogen ◽

Evolution Pattern ◽

Environmental Setting ◽

Resistant Strains

Drug resistance is a worldwide problem affecting all pathogens. The human fungal pathogen Aspergillus fumigatus coexists in the environment with other fungi targeted by crop protection compounds being unintentionally exposed to the selective pressure of multiple antifungal classes leading to the selection of resistant strains. A. fumigatus azole resistant isolates are emerging in both the clinical and environmental setting. Since their approval, azole drugs have dominated the clinical treatment for aspergillosis infections, and the agriculture fungicide market. However, other antifungal classes are used for crop protection including benzimidazoles (MBC), strobilurins (QoIs) and succinate dehydrogenase inhibitors (SDHIs). Mutations responsible for resistance to these fungicides have been widely researched in plant pathogens, but it has not been explored in A. fumigatus . In this work, the genetic basis underlying resistance to MBCs, QoIs and SDHIs were studied in azole susceptible and resistant A. fumigatus strains. E198A/Q and F200Y mutations in the β-tubulin conferred resistance to MBCs, G143A and F129L substitutions in the Cytochrome b to QoIs and H270R/Y mutations in SdhB to SDHIs. Characterization of the susceptibility to azoles showed a correlation between strains resistant to these fungicides and the ones with TR-based azole resistance mechanisms. Whole genome sequencing analysis showed a genetic relationship among fungicide multi resistant strains, which grouped together into subclusters that only included strains carrying the TR-based azole resistance mechanisms, indicating a common ancestor/evolution pattern and confirming the environmental origin of this type of azole resistant A. fumigatus .

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Epidemiology and Molecular Characterizations of Azole Resistance in Clinical and Environmental Aspergillus fumigatus Isolates from China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01005-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5878-5884 ◽

Cited By ~ 32

Author(s):

Yong Chen ◽

Zhongyi Lu ◽

Jingjun Zhao ◽

Ziying Zou ◽

Yanwen Gong ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Antifungal Susceptibility ◽

Public Health Problem ◽

Wide Distribution ◽

Resistance Mechanisms ◽

Azole Resistance ◽

Common Mutation ◽

Content Type ◽

Environmental Resistance ◽

Resistant Strains

ABSTRACTAzole resistance inAspergillus fumigatushas emerged as a worldwide public health problem. We sought here to demonstrate the occurrence and characteristics of azole resistance inA. fumigatusfrom different parts of China. A total of 317 clinical and 144 environmentalA. fumigatusisolates from 12 provinces were collected and subjected to screening for azole resistance. Antifungal susceptibility,cyp51Agene sequencing, and genotyping were carried out for all suspected azole-resistant isolates and a subset of azole-susceptible isolates. As a result, 8 (2.5%) clinical and 2 (1.4%) environmentalA. fumigatusisolates were identified as azole resistant. Five azole-resistant strains exhibit the TR34/L98H mutation, whereas four carry the TR34/L98H/S297T/F495I mutation in thecyp51Agene. Genetic typing and phylogenetic analysis showed that there was a worldwide clonal expansion of the TR34/L98H isolates, while the TR34/L98H/S297T/F495I isolates from China harbored a distinct genetic background with resistant isolates from other countries. High polymorphisms existed in thecyp51Agene that produced amino acid changes among azole-susceptibleA. fumigatusisolates, with N248K being the most common mutation. These data suggest that the wide distribution of azole-resistantA. fumigatusmight be attributed to the environmental resistance mechanisms in China.

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Hospital Environment as a Source of Azole-Resistant Aspergillus fumigatus Strains with TR34/L98H and G448S Cyp51A Mutations

Journal of Fungi ◽

10.3390/jof7010022 ◽

2021 ◽

Vol 7 (1) ◽

pp. 22

Author(s):

Irene Gonzalez-Jimenez ◽

Jose Lucio ◽

Maria Dolores Menéndez-Fraga ◽

Emilia Mellado ◽

Teresa Peláez

Keyword(s):

Treatment Failure ◽

Aspergillus Fumigatus ◽

High Resistance ◽

Resistance Mechanisms ◽

Hospital Environment ◽

Azole Resistance ◽

Therapy Failure ◽

Naive Patient ◽

Resistant Strains ◽

Effective Analysis

Azole-resistant Aspergillus fumigatus is an emerging worldwide problem with increasing reports of therapy failure cases produced by resistant isolates. A case of azole-resistant A. fumigatus hospital colonization in a patient is reported here. Investigations of the hospital environment led to the recovery of A. fumigatus strains harboring the TR34/L98H and the G448S Cyp51A azole resistance mechanisms. Isolate genotyping showed that one strain from the environment was isogenic with the patient strains. These are the first environmental A. fumigatus azole resistant strains collected in a hospital in Spain; it supports the idea of the hospital environment as a source of dissemination and colonization/infection by azole resistant A. fumigatus in patients. The isolation of an azole-resistant strain from an azole-naïve patient is an interesting finding, suggesting that an effective analysis of clinical and environmental sources must be done to detect azole resistance in A. fumigatus. The emergence and spread of these resistance mechanisms in A. fumigatus is of major concern because it confers high resistance to voriconazole and is associated with treatment failure in patients with invasive aspergillosis.

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Interrogation of Related Clinical Pan-Azole-Resistant Aspergillus fumigatus Strains: G138C, Y431C, and G434C Single Nucleotide Polymorphisms incyp51A, Upregulation ofcyp51A, and Integration and Activation of TransposonAtf1in thecyp51APromoter

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00517-11 ◽

2011 ◽

Vol 55 (11) ◽

pp. 5113-5121 ◽

Cited By ~ 63

Author(s):

Ahmed M. Albarrag ◽

Michael J. Anderson ◽

Susan J. Howard ◽

Geoff D. Robson ◽

Peter A. Warn ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Resistance Mechanisms ◽

Start Codon ◽

Azole Resistance ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Content Type ◽

Transcription Start Sites ◽

Chronic Pulmonary Aspergillosis

ABSTRACTMultipleAspergillus fumigatusisolates from a patient with two aspergillomas complicating chronic pulmonary aspergillosis were pan-azole resistant. Microsatellite typing was identical for all isolates despite major phenotypic and some growth rate differences. Three differentcyp51Amutations were found (G138C, Y431C, and G434C), of which the first two were demonstrated by heterologous expression in a hypersusceptibleSaccharomyces cerevisiaestrain to be at least partly responsible for elevated MICs.cyp51Aandcyp51Bgene duplication was excluded, but increased expression ofcyp51Awas demonstrated in three isolates selected for additional study (7-to 13-fold increases). In the isolate with the greatestcyp51Aexpression, anAft1transposon was found inserted 370 bp upstream of the start codon of thecyp51Agene, an integration location never previously demonstrated inAspergillus. Two transcription start sites were identified at 49 and 136 bp upstream of the start codon. The role of theAft1transposon, if any, in modulatingcyp51Aexpression remains to be established. Increased mRNA expression of the transportersAfuMDR1andAfuMDR4also was demonstrated in some isolates, which could contribute to azole resistance or simply represent a stress response. The diversity of confirmed and possible azole resistance mechanisms demonstrated in a single series of isogenic isolates is remarkable, indicating the ability ofA. fumigatusto adapt in the clinical setting.

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

Frontiers in Fungal Biology ◽

10.3389/ffunb.2020.617338 ◽

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.

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A Cyp51B Mutation Contributes to Azole Resistance in Aspergillus fumigatus

Journal of Fungi ◽

10.3390/jof6040315 ◽

2020 ◽

Vol 6 (4) ◽

pp. 315

Author(s):

Irene Gonzalez-Jimenez ◽

Jose Lucio ◽

Jorge Amich ◽

Isabel Cuesta ◽

Rafael Sanchez Arroyo ◽

...

Keyword(s):

Gene Expression ◽

Aspergillus Fumigatus ◽

Novel Mutation ◽

Resistance Mechanisms ◽

The Other ◽

Therapeutic Options ◽

Azole Resistance ◽

The Novel ◽

Clinical Strains

The emergence and spread of Aspergillus fumigatus azole resistance has been acknowledged worldwide. The main problem of azole resistance is the limited therapeutic options for patients suffering aspergillosis. Azole resistance mechanisms have been mostly linked to the enzyme Cyp51A, a target of azole drugs, with a wide variety of modifications responsible for the different resistance mechanisms described to date. However, there are increasing reports of A. fumigatus strains showing azole resistance without Cyp51A modifications, and thus, novel resistance mechanisms are being explored. Here, we characterized two isogenic A. fumigatus clinical strains isolated two years apart from the same patient. Both strains were resistant to clinical azoles but showed different azole resistance mechanisms. One strain (CM8940) harbored a previously described G54A mutation in Cyp51A while the other strain (CM9640) had a novel G457S mutation in Cyp51B, the other target of azoles. In addition, this second strain had a F390L mutation in Hmg1. CM9640 showed higher levels of gene expression of cyp51A, cyp51B and hmg1 than the CM8940 strain. The role of the novel mutation found in Cyp51B together with the contribution of a mutation in Hmg1 in azole resistance is discussed.

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Point mutation or overexpression of A. fumigatus cyp51B, encoding lanosterol 14α-sterol demethylase, leads to triazole resistance.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01252-21 ◽

2021 ◽

Author(s):

Mariana Handelman ◽

Zohar Meir ◽

Jennifer Scott ◽

Yona Shadkchan ◽

Wei Liu ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Point Mutation ◽

Clinical Isolate ◽

Recipient Strain ◽

Antifungal Treatment ◽

Ergosterol Biosynthesis ◽

Common Cause ◽

Clinical Resistance

Aspergillus fumigatus is the most common cause of invasive fungal mold infections in immunocompromised individuals. Current antifungal treatment relies heavily on the triazole antifungals which inhibit fungal Erg11/Cyp51 activity and subsequent ergosterol biosynthesis. However, resistance, due primarily to cyp51 mutation, is rapidly increasing. A. fumigatus contains two Cyp51 isoenzymes, Cyp51A and Cyp51B. Overexpression and mutation of Cyp51A is a major cause of triazole resistance in A. fumigatus . The role of Cyp51B in generating resistance is unclear. Here we show that overexpression or mutation of cyp51B results in triazole resistance. We demonstrate that introduction of a G457S Cyp51B mutation identified in a resistant clinical isolate, results in voriconazole resistance in the naïve recipient strain. Our results indicate that mutations in cyp51B resulting in clinical resistance do exist and should be monitored.

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Multiplecyp51A-Based Mechanisms Identified in Azole-Resistant Isolates of Aspergillus fumigatus from China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00003-15 ◽

2015 ◽

Vol 59 (7) ◽

pp. 4321-4325 ◽

Cited By ~ 30

Author(s):

Musang Liu ◽

Rong Zeng ◽

Lili Zhang ◽

Dongmei Li ◽

Guixia Lv ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Clinical Isolates ◽

Resistant Isolate ◽

Azole Resistance ◽

Content Type ◽

Resistant Strains

ABSTRACTSeventy-twoA. fumigatusclinical isolates from China were investigated for azole resistance based on mutations ofcyp51A. 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.

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An Early Response to Environmental Stress Involves Regulation of OmpX and OmpF, Two Enterobacterial Outer Membrane Pore-Forming Proteins

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01481-06 ◽

2007 ◽

Vol 51 (9) ◽

pp. 3190-3198 ◽

Cited By ~ 45

Author(s):

Myrielle Dupont ◽

Chloë E. James ◽

Jacqueline Chevalier ◽

Jean-Marie Pagès

Keyword(s):

Outer Membrane ◽

Early Response ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Membrane Pore ◽

Bacterial Response ◽

External Stresses ◽

Resistant Strains ◽

Pore Forming Proteins

ABSTRACT Bacterial adaptation to external stresses and toxic compounds is a key step in the emergence of multidrug-resistant strains that are a serious threat to human health. Although some of the proteins and regulators involved in antibiotic resistance mechanisms have been described, no information is available to date concerning the early bacterial response to external stresses. Here we report that the expression of ompX, encoding an outer membrane protein, is increased during early exposure to drugs or environmental stresses. At the same time, the level of ompF porin expression is noticeably affected. Because of the role of these proteins in membrane permeability, these data suggest that OmpF and OmpX are involved in the control of the penetration of antibiotics such as β-lactams and fluoroquinolones through the enterobacterial outer membrane. Consequently, the early control of ompX and ompF induced by external stresses may represent a preliminary response to antibiotics, thus triggering the initial bacterial line of defense against antibiotherapy.

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