Publications by authors named "Dominique Sanglard"

126 Publications

Novel and mutations associated with azole resistance in .

Antimicrob Agents Chemother 2021 Feb 22. Epub 2021 Feb 22.

Institute of Microbiology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.

is a novel species that has spread in all continents causing nosocomial outbreaks of invasive candidiasis. has the ability to develop resistance to all antifungal drug classes. Notably, many isolates are resistant to the azole drug fluconazole, a standard therapy of invasive candidiasis.Azole resistance in can result from mutations in the azole target gene and/or overexpression of the efflux pump Cdr1. is a transcription factor controlling expression in The role of homologs in ( and ) remains to be better defined.In this study, we compared sequences of , and between a fluconazole-susceptible and five fluconazole-resistant isolates of clade IV. Among four of the resistant isolates, we identified a similar genotype with concomitant mutations in (F444L) and (S611P). The simultaneous deletion of tandemly arranged a/b resulted in a decrease of minimal inhibitory concentration (MIC) for fluconazole. Introduction of the and mutations separately and/or combined in the wild-type azole susceptible isolate resulted in a significant increase of azole resistance with a cumulative effect of the two combined mutations. Interestingly, expression was not significantly affected by deletion or by the presence of the S611P mutation, suggesting the existence of Tac1-dependent and Cdr1-independent azole resistance mechanisms.We demonstrated the role of two previously unreported mutations responsible for azole resistance in , which were a common signature among four azole-resistant isolates of clade IV.
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http://dx.doi.org/10.1128/AAC.02663-20DOI Listing
February 2021

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

Med Mycol 2021 Feb 7. Epub 2021 Feb 7.

Institute of Microbiology, Lausanne University Hospital and University of Lausanne, 1011 Lausanne, Switzerland.

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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http://dx.doi.org/10.1093/mmy/myaa118DOI Listing
February 2021

Camphor and Eucalyptol-Anticandidal Spectrum, Antivirulence Effect, Efflux Pumps Interference and Cytotoxicity.

Int J Mol Sci 2021 Jan 6;22(2). Epub 2021 Jan 6.

Department of Plant Physiology, Institute for Biological Research "Siniša Stanković"-National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia.

represents one of the most common fungal pathogens. Due to its increasing incidence and the poor efficacy of available antifungals, finding novel antifungal molecules is of great importance. Camphor and eucalyptol are bioactive terpenoid plant constituents and their antifungal properties have been explored previously. In this study, we examined their ability to inhibit the growth of different species in suspension and biofilm, to block hyphal transition along with their impact on genes encoding for efflux pumps ( and ), ergosterol biosynthesis (), and cytotoxicity to primary liver cells. Camphor showed excellent antifungal activity with a minimal inhibitory concentration of 0.125-0.35 mg/mL while eucalyptol was active in the range of 2-23 mg/mL. The results showed camphor's potential to reduce fungal virulence traits, that is, biofilm establishment and hyphae formation. On the other hand, camphor and eucalyptol treatments upregulated was positively regulated after eucalyptol application while camphor downregulated it. Neither had an impact on expression. The beneficial antifungal activities of camphor were achieved with an amount that was non-toxic to porcine liver cells, making it a promising antifungal compound for future development. The antifungal concentration of eucalyptol caused cytotoxic effects and increased expression of efflux pump genes, which suggests that it is an unsuitable antifungal candidate.
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http://dx.doi.org/10.3390/ijms22020483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825113PMC
January 2021

Flavones, Flavonols, and Glycosylated Derivatives-Impact on Growth and Virulence, Expression of and , Cytotoxicity.

Pharmaceuticals (Basel) 2020 Dec 30;14(1). Epub 2020 Dec 30.

Department of Plant Physiology, Institute for Biological Research "Siniša Stanković", National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia.

Due to the high incidence of fungal infections worldwide, there is an increasing demand for the development of novel therapeutic approaches. A wide range of natural products has been extensively studied, with considerable focus on flavonoids. The antifungal capacity of selected flavones (luteolin, apigenin), flavonols (quercetin), and their glycosylated derivatives (quercitrin, isoquercitrin, rutin, and apigetrin) along with their impact on genes encoding efflux pumps () and ergosterol biosynthesis enzyme () has been the subject of this study. Cytotoxicity of flavonoids towards primary liver cells has also been addressed. Luteolin, quercitrin, isoquercitrin, and rutin inhibited growth of with the minimal inhibitory concentration of 37.5 µg/mL. The application of isoquercitrin has reduced biofilm establishing capacities for 76%, and hyphal formation by yeast. In vitro treatment with apigenin, apigetrin, and quercitrin has downregulated . Contrary to rutin and apigenin, isoquercitrin has upregulated . Except apigetrin and quercitrin (90 µg/mL and 73 µg/mL, respectively inhibited 50% of the net cell growth), the examined flavonoids did not exhibit cytotoxicity. The reduction of both fungal virulence and expression of antifungal resistance-linked genes was the most pronounced for apigenin and apigetrin; these results indicate flavonoids' indispensable capacity for further development as part of an anticandidal therapy or prevention strategy.
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http://dx.doi.org/10.3390/ph14010027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824033PMC
December 2020

Revealing the astragalin mode of anticandidal action.

EXCLI J 2020 29;19:1436-1445. Epub 2020 Oct 29.

Department of Plant Physiology, Institute for Biological Research "Siniša Stankovic"- National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia.

Due to limited arsenal of systemically available antifungal agents, infections caused by are difficult to treat and the emergence of drug-resistant strains present a major challenge to the clinicians worldwide. Hence further exploration of potential novel and effective antifungal drugs is required. In this study we have explored the potential of a flavonoid, astragalin, in controlling the growth of in both planktonic and biofilm forms by microdilution method; and in regulating the morphological switch between yeast and hyphal growth. Astragalin ability to interfere with membrane integrity, ergosterol synthesis and its role in the regulation of genes encoding for efflux pumps has been addressed. In our study, astragalin treatment produced good antimicrobial and significant antibiofilm activity. Anticandidal activity of astragalin was not related to downregulation, neither to direct binding to CYP51 enzyme nor was linked to membrane ergosterol assembly. Instead, astragalin treatment resulted in reduced expression of and also affected cell membrane integrity without causing cytotoxic effect on human gingival fibroblast cells. Considering that astragalin-mediated decreased expression of efflux pumps increases the concentration of antifungal drug inside the fungal cells, a combinatorial treatment with this agent could be explored as a novel therapeutic option for candidiasis.
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http://dx.doi.org/10.17179/excli2020-2987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726490PMC
October 2020

Identification and Characterization of Mediators of Fluconazole Tolerance in .

Front Microbiol 2020 11;11:591140. Epub 2020 Nov 11.

Department of Laboratory, Institute of Microbiology, Lausanne University Hospital, Lausanne, Switzerland.

is an important human pathogen and a major concern in intensive care units around the world. infections are associated with a high mortality despite the use of antifungal treatments. One of the causes of therapeutic failures is the acquisition of antifungal resistance by mutations in the genome. Fluconazole (FLC) is one of the most widely used antifungal and mechanisms of FLC resistance occurring by mutations have been extensively investigated. However, some clinical isolates are known to be able to survive at high FLC concentrations without acquiring resistance mutations, a phenotype known as tolerance. Mechanisms behind FLC tolerance are not well studied, mainly due to the lack of a proper way to identify and quantify tolerance in clinical isolates. We proposed here culture conditions to investigate FLC tolerance as well as an easy and efficient method to identity and quantify tolerance to FLC. The screening of strain collections revealed that FLC tolerance is pH- and strain-dependent, suggesting the involvement of multiple mechanisms. Here, we addressed the identification of FLC tolerance mediators in by an overexpression strategy focusing on 572 genes. This strategy led to the identification of two transcription factors, and . is a C2H2-type transcription factor that is part of the calcineurin-dependent pathway in , while is a GATA-type transcription factor of unknown function in . Overexpression of each gene resulted in an increase of FLC tolerance, however, only the deletion of in clinical FLC-tolerant strains consistently decreased their FLC tolerance. Transcription profiling of clinical isolates with variable levels of FLC tolerance confirmed a calcineurin-dependent signature in these isolates when exposed to FLC.
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http://dx.doi.org/10.3389/fmicb.2020.591140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686038PMC
November 2020

New Data on the Activity of Fenticonazole against Fluconazole-Resistant Species.

Antimicrob Agents Chemother 2020 11 17;64(12). Epub 2020 Nov 17.

Dipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, Rome, Italy.

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http://dx.doi.org/10.1128/AAC.01459-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674059PMC
November 2020

Signaling Pathways Governing the Caspofungin Paradoxical Effect in Aspergillus fumigatus.

mBio 2020 08 18;11(4). Epub 2020 Aug 18.

Institute of Microbiology, Department of Laboratory, Lausanne University Hospital, Lausanne, Switzerland

is responsible for a wide range of diseases affecting several million people worldwide. Currently, a few families of antifungals are available to fight aspergillosis, and we are facing a worrisome increase in resistance to azoles, the drugs used for both first-line treatment and prophylaxis of invasive aspergillosis. In this context, some of the latest antifungals, i.e., echinocandins, have gained attention. Even though acquired resistance to echinocandins is yet uncommon in clinical isolates, some strains exhibit another characteristic that relies on their capacity to grow at suprainhibitory echinocandin concentrations This intriguing phenomenon, especially observed with caspofungin and now referred to as the caspofungin paradoxical effect (CPE), relies on molecular mechanisms that were hitherto little understood. Here, we discuss the recent key findings of Valero and colleagues published in (C. Valero, A. C. Colabardini, J. Chiaratto, L. Pardeshi, et al., mBio 11:e00816-20, 2020, https://doi.org/10.1128/mBio.00816-20) that will allow a better understanding of the complex regulatory pathway involved in governing the response of to caspofungin.
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http://dx.doi.org/10.1128/mBio.01816-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439473PMC
August 2020

Single yeast cell nanomotions correlate with cellular activity.

Sci Adv 2020 Jun 24;6(26):eaba3139. Epub 2020 Jun 24.

International Joint Research Group BioNanotechnology & NanoMedicine (NANO), Vrije Universiteit Brussel-Ecole Polytechnique de Lausanne (EPFL), B-1050 Brussels, Belgium-B-1015 Lausanne, Switzerland.

Living single yeast cells show a specific cellular motion at the nanometer scale with a magnitude that is proportional to the cellular activity of the cell. We characterized this cellular nanomotion pattern of nonattached single yeast cells using classical optical microscopy. The distribution of the cellular displacements over a short time period is distinct from random motion. The range and shape of such nanomotion displacement distributions change substantially according to the metabolic state of the cell. The analysis of the nanomotion frequency pattern demonstrated that single living yeast cells oscillate at relatively low frequencies of around 2 hertz. The simplicity of the technique should open the way to numerous applications among which antifungal susceptibility tests seem the most straightforward.
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http://dx.doi.org/10.1126/sciadv.aba3139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314535PMC
June 2020

Erratum for Kannan et al., "Comparative Genomics for the Elucidation of Multidrug Resistance in Candida lusitaniae.

mBio 2020 Feb 11;11(1). Epub 2020 Feb 11.

Institute of Microbiology, Department of Laboratory, Lausanne University Hospital, Lausanne, Switzerland

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http://dx.doi.org/10.1128/mBio.03403-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018650PMC
February 2020

Machine Learning Approach for Fluconazole Resistance Detection Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Front Microbiol 2019 14;10:3000. Epub 2020 Jan 14.

Microbiology Institute, University Hospital Lausanne, Lausanne, Switzerland.

causes life-threatening systemic infections in immunosuppressed patients. These infections are commonly treated with fluconazole, an antifungal agent targeting the ergosterol biosynthesis pathway. Current Antifungal Susceptibility Testing (AFST) methods are time-consuming and are often subjective. Moreover, they cannot reliably detect the tolerance phenomenon, a breeding ground for the resistance. An alternative to the classical AFST methods could use Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass spectrometry (MS). This tool, already used in clinical microbiology for microbial species identification, has already offered promising results to detect antifungal resistance on non-azole tolerant yeasts. Here, we propose a machine-learning approach, adapted to MALDI-TOF MS data, to qualitatively detect fluconazole resistance in the azole tolerant species . MALDI-TOF MS spectra were acquired from 33 clinical strains isolated from 15 patients. Those strains were exposed for 3 h to 3 fluconazole concentrations (256, 16, 0 μg/mL) and with (5 μg/mL) or without cyclosporin A, an azole tolerance inhibitor, leading to six different experimental conditions. We then optimized a protein extraction protocol allowing the acquisition of high-quality spectra, which were further filtered through two quality controls. The first one consisted of discarding not identified spectra and the second one selected only the most similar spectra among replicates. Quality-controlled spectra were divided into six sets, following the sample preparation's protocols. Each set was then processed through an R based script using pre-defined housekeeping peaks allowing peak spectra positioning. Finally, 32 machine-learning algorithms applied on the six sets of spectra were compared, leading to 192 different pipelines of analysis. We selected the most robust pipeline with the best accuracy. This LDA model applied to the samples prepared in presence of tolerance inhibitor but in absence of fluconazole reached a specificity of 88.89% and a sensitivity of 83.33%, leading to an overall accuracy of 85.71%. Overall, this work demonstrated that combining MALDI-TOF MS and machine-learning could represent an innovative mycology diagnostic tool.
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http://dx.doi.org/10.3389/fmicb.2019.03000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971193PMC
January 2020

Large-scale genome mining allows identification of neutral polymorphisms and novel resistance mutations in genes involved in Candida albicans resistance to azoles and echinocandins.

J Antimicrob Chemother 2020 04;75(4):835-848

Unité Biologie et Pathogénicité Fongiques, Département de Mycologie, Institut Pasteur, USC2019 INRA, Paris, France.

Background: The genome of Candida albicans displays significant polymorphism. Point mutations in genes involved in resistance to antifungals may either confer phenotypic resistance or be devoid of phenotypic consequences.

Objectives: To catalogue polymorphisms in azole and echinocandin resistance genes occurring in susceptible strains in order to rapidly pinpoint relevant mutations in resistant strains.

Methods: Genome sequences from 151 unrelated C. albicans strains susceptible to fluconazole and caspofungin were used to create a catalogue of non-synonymous polymorphisms in genes involved in resistance to azoles (ERG11, TAC1, MRR1 and UPC2) or echinocandins (FKS1). The potential of this catalogue to reveal putative resistance mutations was tested in 10 azole-resistant isolates, including 1 intermediate to caspofungin. Selected mutations were analysed by mutagenesis experiments or mutational prediction effect.

Results: In the susceptible strains, we identified 126 amino acid substitutions constituting the catalogue of phenotypically neutral polymorphisms. By excluding these neutral substitutions, we identified 22 additional substitutions in the 10 resistant strains. Among these substitutions, 10 had already been associated with resistance. The remaining 12 were in Tac1p (n = 6), Upc2p (n = 2) and Erg11p (n = 4). Four out of the six homozygous substitutions in Tac1p (H263Y, A790V, H839Y and P971S) conferred increases in azole MICs, while no effects were observed for those in Upc2p. Additionally, two homozygous substitutions (Y64H and P236S) had a predicted conformation effect on Erg11p.

Conclusions: By establishing a catalogue of neutral polymorphisms occurring in genes involved in resistance to antifungal drugs, we provide a useful resource for rapid identification of mutations possibly responsible for phenotypic resistance in C. albicans.
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http://dx.doi.org/10.1093/jac/dkz537DOI Listing
April 2020

Comparative Genomics for the Elucidation of Multidrug Resistance in Candida lusitaniae.

mBio 2019 12 24;10(6). Epub 2019 Dec 24.

Institute of Microbiology, Department of Laboratory, Lausanne University Hospital, Lausanne, Switzerland

Multidrug resistance (MDR) has emerged in hospitals due to the use of several agents administered in combination or sequentially to the same individual. We reported earlier MDR in during therapy with amphotericin B (AmB), azoles, and candins. Here, we used comparative genomic approaches between the initial susceptible isolate and 4 other isolates with different MDR profiles. From a total of 18 nonsynonymous single nucleotide polymorphisms (NSS) in genome comparisons with the initial isolate, six could be associated with MDR. One of the single nucleotide polymorphisms (SNPs) occurred in a putative transcriptional activator () resulting in a V668G substitution in isolates resistant to azoles and 5-fluorocytosine (5-FC). We demonstrated by genome editing that acted by upregulation of (a multidrug transporter) in the presence of the V668G substitution. itself mediated not only azole resistance but also 5-FC resistance, which represents a novel resistance mechanism for this drug class. Three other distinct NSS occurred in (a glucan synthase gene that is targeted by candins) in three candin-resistant isolates. Last, two other NSS in and (ergosterol biosynthesis) resulting in nonsense mutations were revealed in AmB-resistant isolates, one of which accumulated the two NSS. AmB-resistant isolates lacked ergosterol and exhibited sterol profiles, consistent with and defects. In conclusion, this genome analysis combined with genetics and metabolomics helped decipher the resistance profiles identified in this clinical case. MDR isolates accumulated six different mutations conferring resistance to all antifungal agents used in medicine. This case study illustrates the capacity of to rapidly adapt under drug pressure within the host. Antifungal resistance is an inevitable phenomenon when fungal pathogens are exposed to antifungal drugs. These drugs can be grouped in four distinct classes (azoles, candins, polyenes, and pyrimidine analogs) and are used in different clinical settings. Failures in therapy implicate the sequential or combined use of these different drug classes, which can result in some cases in the development of multidrug resistance (MDR). MDR is particularly challenging in the clinic since it drastically reduces possible treatment alternatives. In this study, we report the rapid development of MDR in in a patient, which became resistant to all known antifungal agents used until now in medicine. To understand how MDR developed in , whole-genome sequencing followed by comparative genome analysis was undertaken in sequential MDR isolates. This helped to detect all specific mutations linked to drug resistance and explained the different MDR patterns exhibited by the clinical isolates.
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http://dx.doi.org/10.1128/mBio.02512-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935856PMC
December 2019

Persistence of in the Oral Mucosa Induces a Curbed Inflammatory Host Response That Is Independent of Immunosuppression.

Front Immunol 2019 27;10:330. Epub 2019 Feb 27.

Section of Immunology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland.

Controlled immune activation in response to commensal microbes is critical for the maintenance of stable colonization and prevention of microbial overgrowth on epithelial surfaces. Our understanding of the host mechanisms that regulate bacterial commensalism has increased substantially, however, much less data exist regarding host responses to members of the fungal microbiota on colonized surfaces. Using a murine model of oropharyngeal candidiasis, we have recently shown that differences in immune activation in response to diverse natural isolates of are associated with different outcomes of the host-fungal interaction. Here we applied a genome-wide transcriptomic approach to show that rapid induction of a strong inflammatory response characterized by neutrophil-associated genes upon colonization inversely correlated with the ability of the fungus to persist in the oral mucosa. Surprisingly, persistent fungal isolates showed no signs of a compensatory regulatory immune response. By combining RNA-seq data, genetic mouse models, and co-infection experiments, we show that attenuation of the inflammatory response at the onset of infection with a persistent isolate is not a consequence of enhanced immunosuppression. Importantly, depletion of regulatory T cells or deletion of the immunoregulatory cytokine IL-10 did not alter host-protective type 17 immunity nor did it impair fungal survival in the oral mucosa, indicating that persistence of in the oral mucosa is not a consequence of suppressed antifungal immunity.
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http://dx.doi.org/10.3389/fimmu.2019.00330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400982PMC
July 2020

Finding the needle in a haystack: Mapping antifungal drug resistance in fungal pathogen by genomic approaches.

PLoS Pathog 2019 01 31;15(1):e1007478. Epub 2019 Jan 31.

Institute of Microbiology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.

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http://dx.doi.org/10.1371/journal.ppat.1007478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355021PMC
January 2019

Condition-specific series of metabolic sub-networks and its application for gene set enrichment analysis.

Bioinformatics 2019 07;35(13):2258-2266

Vital-IT Group, SIB Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland.

Motivation: Genome-scale metabolic networks and transcriptomic data represent complementary sources of knowledge about an organism's metabolism, yet their integration to achieve biological insight remains challenging.

Results: We investigate here condition-specific series of metabolic sub-networks constructed by successively removing genes from a comprehensive network. The optimal order of gene removal is deduced from transcriptomic data. The sub-networks are evaluated via a fitness function, which estimates their degree of alteration. We then consider how a gene set, i.e. a group of genes contributing to a common biological function, is depleted in different series of sub-networks to detect the difference between experimental conditions. The method, named metaboGSE, is validated on public data for Yarrowia lipolytica and mouse. It is shown to produce GO terms of higher specificity compared to popular gene set enrichment methods like GSEA or topGO.

Availability And Implementation: The metaboGSE R package is available at https://CRAN.R-project.org/package=metaboGSE.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6596900PMC
July 2019

Methodologies for and evaluation of efficacy of antifungal and antibiofilm agents and surface coatings against fungal biofilms.

Microb Cell 2018 06 14;5(7):300-326. Epub 2018 Jun 14.

Centre for Microbial and Plant Genetics, KU Leuven, Leuven, Belgium.

Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the and performance of anti-infective coatings and materials to prevent fungal biofilm-based infections.
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http://dx.doi.org/10.15698/mic2018.07.638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6035839PMC
June 2018

Worldwide emergence of resistance to antifungal drugs challenges human health and food security.

Science 2018 05;360(6390):739-742

Department of BioSciences, University of Exeter, Exeter EX4 4QD, UK.

The recent rate of emergence of pathogenic fungi that are resistant to the limited number of commonly used antifungal agents is unprecedented. The azoles, for example, are used not only for human and animal health care and crop protection but also in antifouling coatings and timber preservation. The ubiquity and multiple uses of azoles have hastened the independent evolution of resistance in many environments. One consequence is an increasing risk in human health care from naturally occurring opportunistic fungal pathogens that have acquired resistance to this broad class of chemicals. To avoid a global collapse in our ability to control fungal infections and to avoid critical failures in medicine and food security, we must improve our stewardship of extant chemicals, promote new antifungal discovery, and leverage emerging technologies for alternative solutions.
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http://dx.doi.org/10.1126/science.aap7999DOI Listing
May 2018

Azole Resistance of Environmental and Clinical Aspergillus fumigatus Isolates from Switzerland.

Antimicrob Agents Chemother 2018 04 27;62(4). Epub 2018 Mar 27.

Institute of Microbiology, University of Lausanne and University Hospital Center, Lausanne, Switzerland

is a ubiquitous opportunistic pathogen. This fungus can acquire resistance to azole antifungals due to mutations in the azole target (). Recently, mutations typical for environmental azole resistance acquisition (for example, TR/L98H) have been reported. These mutations can also be found in isolates recovered from patients. Environmental azole resistance acquisition has been reported on several continents. Here we describe, for the first time, the occurrence of azole-resistant isolates of environmental origin in Switzerland with mutations, and we show that these isolates can also be recovered from a few patients. While the TR/L98H mutation was dominant, a single azole-resistant isolate exhibited a mutation (G54R) that was reported only for clinical isolates. In conclusion, our study demonstrates that azole resistance with an environmental signature is present in environments and patients of Swiss origin and that mutations believed to be unique to clinical settings are now also observed in the environment.
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http://dx.doi.org/10.1128/AAC.02088-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913999PMC
April 2018

Extracellular enzymes and adhesive properties of medically important Candida spp. strains from landfill leachate.

Microb Pathog 2018 Mar 31;116:328-334. Epub 2018 Jan 31.

Water Research and Technologies Center (CERTE), Laboratory of Wastewater Treatment, University of Carthage, P.O. Box 273, 8020 Soliman, Tunis, Tunisia. Electronic address:

The virulence properties of Candida spp. presents in landfill leachate still unknown until today and they constitutes a serious source of potential danger for humans. We investigate the antifungal susceptibility, production of hydrolytic enzymes and biofilm formation on polystyrene as well as glass in Candida spp. strains isolated from a landfill leachate treatment station in Borj Chakir (Tunisia). 37 yeast strains were isolated belonging to the following species: C. robusta, C. lusitaniae, C. tropicalis, C. krusei. Most isolated yeast strains were resistant to Amphotericin B, produced several hydrolytic enzymes (67.56% produced phospholipase, 86.04% protease, 64.86% esterase) and most of them are able to degrade hemoglobin. All assayed Candida strains have been able to form biofilm on polystyrene depending on the species and strain of Candida. Landfills receiving clinical waste are a potential source of Candida ssp. strains with several virulence properties which allow them to survive in different aquatic biotopes.
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http://dx.doi.org/10.1016/j.micpath.2018.01.042DOI Listing
March 2018

Azole resistance in a mutant lacking the ABC transporter depends on TOR signaling.

J Biol Chem 2018 01 20;293(2):412-432. Epub 2017 Nov 20.

From the School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India,

ATP-binding cassette (ABC) transporters help export various substrates across the cell membrane and significantly contribute to drug resistance. However, a recent study reported an unusual case in which the loss of an ABC transporter in , orf19.4531 (previously named ROA1), increases resistance against antifungal azoles, which was attributed to an altered membrane potential in the mutant strain. To obtain further mechanistic insights into this phenomenon, here we confirmed that the plasma membrane-localized transporter (renamed for consistency with nomenclature) could efflux xenobiotics such as berberine, rhodamine 123, and paraquat. Moreover, a null mutant, NKKY101, displayed increased susceptibility to these xenobiotics. Interestingly, fluorescence recovery after photobleaching (FRAP) results indicated that NKKY101 mutant cells exhibited increased plasma membrane rigidity, resulting in reduced azole accumulation and contributing to azole resistance. Transcriptional profiling revealed that ribosome biogenesis genes were significantly up-regulated in the NKKY101 mutant. As ribosome biogenesis is a well-known downstream phenomenon of target of rapamycin (TOR1) signaling, we suspected a link between ribosome biogenesis and TOR1 signaling in NKKY101. Therefore, we grew NKKY101 cells on rapamycin and observed TOR1 hyperactivation, which leads to Hsp90-dependent calcineurin stabilization and thereby increased azole resistance. This finding was supported by data from a mouse model of systemic infection in which NKKY101 cells led to higher fungal load after fluconazole challenge than wild-type cells. Taken together, our study uncovers a mechanism of azole resistance in , involving increased membrane rigidity and TOR signaling.
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http://dx.doi.org/10.1074/jbc.M117.807032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767851PMC
January 2018

A standardized toolkit for genetic engineering of CTG clade yeasts.

J Microbiol Methods 2018 01 16;144:152-156. Epub 2017 Nov 16.

Groupe d'Etude des Interactions Hôte-Pathogène (EA 3142), GEIHP, UNIV. Angers, Université Bretagne-Loire, Angers, France. Electronic address:

We have developed a series of synthetic constructs suitable to genetically manipulate a broad range of yeast species belonging to the fungal CTG clade. This molecular toolbox notably allows heterologous gene expression, single or dual fluorescence labeling and construction of luciferase-expressing strains for bioluminescence imaging.
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http://dx.doi.org/10.1016/j.mimet.2017.11.015DOI Listing
January 2018

Potential Use of MALDI-ToF Mass Spectrometry for Rapid Detection of Antifungal Resistance in the Human Pathogen Candida glabrata.

Sci Rep 2017 08 22;7(1):9099. Epub 2017 Aug 22.

Institute of Public Health (Section of Hygiene), Università Cattolica del Sacro Cuore, Fondazione Policlinico Universitario Agostino Gemelli, Largo F. Vito 1, 00168, Rome, Italy.

The echinocandins are relatively new antifungal drugs that represent, together with the older azoles, the recommended and/or preferred agents to treat candidaemia and other forms of invasive candidiasis in human patients. If "time is of the essence" to reduce the mortality for these infections, the administration of appropriate antifungal therapy could be accelerated by the timely reporting of laboratory test results. In this study, we attempted to validate a MALDI-ToF mass spectrometry-based assay for the antifungal susceptibility testing (AFST) of the potentially multidrug-resistant pathogen Candida glabrata against anidulafungin and fluconazole. The practical applicability of the assay, reported here as MS-AFST, was assessed with a panel of clinical isolates that were selected to represent phenotypically and genotypically/molecularly susceptible or resistant strains. The data show the potential of our assay for rapid detection of antifungal resistance, although the MS-AFST assay performed at 3 h of the in vitro antifungal exposure failed to detect C. glabrata isolates with echinocandin resistance-associated FKS2 mutations. However, cell growth kinetics in the presence of anidulafungin revealed important cues about the in vitro fitness of C. glabrata isolates, which may lead to genotypic or phenotypic antifungal testing in clinical practice.
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http://dx.doi.org/10.1038/s41598-017-09329-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5567316PMC
August 2017

Red-Shifted Firefly Luciferase Optimized for Bioluminescence Imaging.

Front Microbiol 2017 3;8:1478. Epub 2017 Aug 3.

Institute of Microbiology, University of LausanneLausanne, Switzerland.

is a major fungal pathogen causing life-threatening diseases in immuno-compromised patients. The efficacy of current drugs to combat infections is limited, as these infections have a 40-60% mortality rate. There is a real need for novel therapeutic approaches, but such advances require a detailed knowledge of and its pathogenesis. Additionally, any novel antifungal drugs against infections will need to be tested for their efficacy over time. Fungal pathogenesis and drug-mediated resolution studies can both be evaluated using non-invasive imaging technologies. In the work presented here, we used a codon-optimized firefly luciferase reporter system for detecting in mice. We adapted the firefly luciferase in order to improve its maximum emission intensity in the red light range (600-700 nm) as well as to improve its thermostability in mice. All non-invasive imaging of experimental animals was performed with a multimodal imaging system able to detect luminescent reporters and capture both reflectance and X-ray images. The modified firefly luciferase expressed in (Mut2) was found to significantly increase the sensitivity of bioluminescence imaging (BLI) in systemic infections as compared to unmodified luciferase (Mut0). The same modified bioluminescence reporter system was used in an oropharyngeal candidiasis model. In both animal models, fungal loads could be correlated to the intensity of emitted light. Antifungal treatment efficacies were also evaluated on the basis of BLI signal intensity. In conclusion, BLI with a red-shifted firefly luciferase was found to be a powerful tool for testing the fate of in various mice infection models.
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http://dx.doi.org/10.3389/fmicb.2017.01478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541039PMC
August 2017

Identification and Mode of Action of a Plant Natural Product Targeting Human Fungal Pathogens.

Antimicrob Agents Chemother 2017 09 24;61(9). Epub 2017 Aug 24.

Institute of Microbiology, University of Lausanne, and Lausanne University Hospital, Lausanne, Switzerland

is a major cause of fungal diseases in humans, and its resistance to available drugs is of concern. In an attempt to identify novel antifungal agents, we initiated a small-scale screening of a library of 199 natural plant compounds (i.e., natural products [NPs]). susceptibility profiling experiments identified 33 NPs with activity against (MICs ≤ 32 μg/ml). Among the selected NPs, the sterol alkaloid tomatidine was further investigated. Tomatidine originates from the tomato () and exhibited high levels of fungistatic activity against species (MICs ≤ 1 μg/ml) but no cytotoxicity against mammalian cells. Genome-wide transcriptional analysis of tomatidine-treated cells revealed a major alteration (upregulation) in the expression of ergosterol genes, suggesting that the ergosterol pathway is targeted by this NP. Consistent with this transcriptional response, analysis of the sterol content of tomatidine-treated cells showed not only inhibition of Erg6 (C-24 sterol methyltransferase) activity but also of Erg4 (C-24 sterol reductase) activity. A forward genetic approach in coupled with whole-genome sequencing identified 2 nonsynonymous mutations in (amino acids D249G and G132D) responsible for tomatidine resistance. Our results therefore unambiguously identified Erg6, a C-24 sterol methyltransferase absent in mammals, to be the main direct target of tomatidine. We tested the efficacy of tomatidine in a mouse model of systemic infection. Treatment with a nanocrystal pharmacological formulation successfully decreased the fungal burden in infected kidneys compared to the fungal burden achieved by the use of placebo and thus confirmed the potential of tomatidine as a therapeutic agent.
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http://dx.doi.org/10.1128/AAC.00829-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5571344PMC
September 2017

Comparative Genomics of Two Sequential Clinical Isolates.

G3 (Bethesda) 2017 08 7;7(8):2413-2426. Epub 2017 Aug 7.

Institute of Microbiology, University of Lausanne, CH-1011, Switzerland

is an important fungal pathogen which develops rapid antifungal resistance in treated patients. It is known that azole treatments lead to antifungal resistance in this fungal species and that multidrug efflux transporters are involved in this process. Specific mutations in the transcriptional regulator result in upregulation of the transporters. In addition, we showed that the mutations can contribute to enhance virulence in animal models. In this study, we were interested to compare genomes of two specific -related isolates, one of which was azole susceptible (DSY562) while the other was azole resistant (DSY565). DSY565 contained a mutation (L280F) and was isolated after a time-lapse of 50 d of azole therapy. We expected that genome comparisons between both isolates could reveal additional mutations reflecting host adaptation or even additional resistance mechanisms. The PacBio technology used here yielded 14 major contigs (sizes 0.18-1.6 Mb) and mitochondrial genomes from both DSY562 and DSY565 isolates that were highly similar to each other. Comparisons of the clinical genomes with the published CBS138 genome indicated important genome rearrangements, but not between the clinical strains. Among the unique features, several retrotransposons were identified in the genomes of the investigated clinical isolates. DSY562 and DSY565 each contained a large set of adhesin-like genes (101 and 107, respectively), which exceed by far the number of reported adhesins (63) in the CBS138 genome. Comparison between DSY562 and DSY565 yielded 17 nonsynonymous SNPs (among which the was the expected mutation) as well as small size indels in coding regions (11) but mainly in adhesin-like genes. The genomes contained a DNA mismatch repair allele of known to be involved in the so-called hyper-mutator phenotype of this yeast species and the number of accumulated mutations between both clinical isolates is consistent with the presence of a defect. In conclusion, this study is the first to compare genomes of sequential clinical isolates using the PacBio technology as an approach. The genomes of these isolates taken in the same patient at two different time points exhibited limited variations, even if submitted to the host pressure.
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http://dx.doi.org/10.1534/g3.117.042887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555451PMC
August 2017

The Swiss Society of Microbiology: Small Bugs, Big Questions and Cool Answers.

Chimia (Aarau) 2016 Dec;70(12):874-877

Chef de Service et Directeur de l'Institut de Microbiologie, Médecin chef des laboratoires de microbiologie diagnostique, Institut de microbiologie de l'Université de Lausanne, Département des laboratoires, Bureau IMUL 02-222, Bugnon 48, CH-1011 Lausanne, Switzerland.

The Swiss Society for Microbiology (SSM) represents around 700 scientists working in the fields of medical (human and veterinary), microbial biotechnology as well as fundamental, environmental, and food microbiology. Five sections: Clinical Microbiology, Environmental Microbiology, Mycology, Prokaryotic Biology, and Virology reflects the main interests of the membership.
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http://dx.doi.org/10.2533/chimia.2016.874DOI Listing
December 2016

High-Resolution Genetics Identifies the Lipid Transfer Protein Sec14p as Target for Antifungal Ergolines.

PLoS Genet 2016 Nov 17;12(11):e1006374. Epub 2016 Nov 17.

Novartis Institutes for BioMedical Research, Novartis Campus, Basel, Switzerland.

Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point.
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http://dx.doi.org/10.1371/journal.pgen.1006374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5147771PMC
November 2016

Dual action antifungal small molecule modulates multidrug efflux and TOR signaling.

Nat Chem Biol 2016 10 29;12(10):867-75. Epub 2016 Aug 29.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

There is an urgent need for new strategies to treat invasive fungal infections, which are a leading cause of human mortality. Here, we establish two activities of the natural product beauvericin, which potentiates the activity of the most widely deployed class of antifungal against the leading human fungal pathogens, blocks the emergence of drug resistance, and renders antifungal-resistant pathogens responsive to treatment in mammalian infection models. Harnessing genome sequencing of beauvericin-resistant mutants, affinity purification of a biotinylated beauvericin analog, and biochemical and genetic assays reveals that beauvericin blocks multidrug efflux and inhibits the global regulator TORC1 kinase, thereby activating the protein kinase CK2 and inhibiting the molecular chaperone Hsp90. Substitutions in the multidrug transporter Pdr5 that enable beauvericin efflux impair antifungal efflux, thereby impeding resistance to the drug combination. Thus, dual targeting of multidrug efflux and TOR signaling provides a powerful, broadly effective therapeutic strategy for treating fungal infectious disease that evades resistance.
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http://dx.doi.org/10.1038/nchembio.2165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030160PMC
October 2016

Upregulation of the Adhesin Gene EPA1 Mediated by PDR1 in Candida glabrata Leads to Enhanced Host Colonization.

mSphere 2016 Mar-Apr;1(2). Epub 2016 Mar 2.

Institute of Microbiology, University of Lausanne and University Hospital Center, Lausanne, Switzerland.

Candida glabrata is the second most common Candida species causing disseminated infection, after C. albicans. C. glabrata is intrinsically less susceptible to the widely used azole antifungal drugs and quickly develops secondary resistance. Resistance typically relies on drug efflux with transporters regulated by the transcription factor Pdr1. Gain-of-function (GOF) mutations in PDR1 lead to a hyperactive state and thus efflux transporter upregulation. Our laboratory has characterized a collection of C. glabrata clinical isolates in which azole resistance was found to correlate with increased virulence in vivo. Contributing phenotypes were the evasion of adhesion and phagocytosis by macrophages and an increased adhesion to epithelial cells. These phenotypes were found to be dependent on PDR1 GOF mutation and/or C. glabrata strain background. In the search for the molecular effectors, we found that PDR1 hyperactivity leads to overexpression of specific cell wall adhesins of C. glabrata. Further study revealed that EPA1 regulation, in particular, explained the increase in adherence to epithelial cells. Deleting EPA1 eliminates the increase in adherence in an in vitro model of interaction with epithelial cells. In a murine model of urinary tract infection, PDR1 hyperactivity conferred increased ability to colonize the bladder and kidneys in an EPA1-dependent way. In conclusion, this study establishes a relationship between PDR1 and the regulation of cell wall adhesins, an important virulence attribute of C. glabrata. Furthermore, our data show that PDR1 hyperactivity mediates increased adherence to host epithelial tissues both in vitro and in vivo through upregulation of the adhesin gene EPA1. IMPORTANCE Candida glabrata is an important fungal pathogen in human diseases and is also rapidly acquiring drug resistance. Drug resistance can be mediated by the transcriptional activator PDR1, and this results in the upregulation of multidrug transporters. Intriguingly, this resistance mechanism is associated in C. glabrata with increased virulence in animal models and also with increased adherence to specific host cell types. The C. glabrata adhesin gene EPA1 is a major contributor of virulence and adherence to host cells. Here, we show that EPA1 expression is controlled by PDR1 independently of subtelomeric silencing, a known EPA1 regulation mechanism. Thus, a relationship exists between PDR1, EPA1 expression, and adherence to host cells, which is critical for efficient virulence. Our results demonstrate that acquisition of drug resistance is beneficial for C. glabrata in fungus-host relationships. These findings further highlight the challenges of the therapeutic management of C. glabrata infections in human patients.
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http://dx.doi.org/10.1128/mSphere.00065-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863579PMC
June 2016