Biology subjects

Maufrais, C.

Publications and source records attributed to Maufrais, C..

16 recordsLinked to original sources

bioRxiv2026

A Multiplex Droplet Digital PCR Assay for Chromosome Copy Number Determination in Candida albicans

Chromosome copy number variation (CNV) is a major contributor to genome plasticity and adaptation in Candida albicans, a leading fungal pathogen of humans. Aneuploidy, defined as deviations from the normal diploid chromosome set, rapidly alters gene dosage, enabling tolerance to host-imposed and antifungal stress. Accurate detection and quantification of chromosomal copy number changes are thus essential to dissect the mechanisms by which C. albicans adapts and evolves. Here, we describe the development, optimization, and validation of a six-color, 16-plex droplet digital PCR assay for simultaneous quantification of all C. albicans chromosome arms in a single reaction. Each target is detected by a unique dual-color or single-color combination of probes, enabling high-order multiplexing through binary fluorescence encoding. Following optimization of probe concentrations, PCR cycling parameters, genomic DNA extraction and pre-treatment with restriction enzymes, the assay provides accurate, reproducible chromosome-level copy number estimates that correlate closely with WGS results across euploid and aneuploid isolates. Compared to whole-genome sequencing, the assay is rapid, cost-effective, and scalable, requiring minimal DNA input and allowing high-throughput analysis of large isolate collections. The 16-plex assay thus provides a platform for dissecting genome instability and adaptive evolution in C. albicans. Article SummaryWe developed and validated a 16-plex droplet digital PCR assay that estimates chromosome dosage across the entire genome of the human fungal pathogen C. albicans in a single reaction. The assay uses six fluorescent colors and unique color combinations to track one marker on each chromosome arm, enabling rapid detection of aneuploidy (extra or missing chromosomes). Results closely matched whole-genome sequencing for isolates with simple aneuploid forms and detected low-frequency trisomic clones in mixed populations. With optimized DNA preparation, this method provides a practical tool for screening genome instability in research and clinical settings.

microbiology↗

bioRxiv2025

Sphingolipid Homeostasis, Mitochondrial Activity, and PKA Signaling Drive an Azole-Tolerant State

Antifungal therapies frequently fail, resulting in persistent infections caused by the important opportunistic pathogen, Candida albicans, which is highly tolerant to azole drugs. Tolerance arises from a subpopulation of cells that survive prolonged drug exposure, yet the underlying mechanisms remain poorly defined. Here, transcription factor mutant library screens identified Mnl1 as a key repressor of tolerance. Loss of MNL1 elevated cAMP levels and activated the PKA pathway, linking Mnl1 to growth control and stress responses. Additionally, mnl1 cells exhibited altered sphingolipid composition, implicating Mnl1 in regulating membrane permeability, a key determinant of drug efflux and membrane integrity. Increased tolerance in mnl1 cells was also linked to increased mitochondrial function. Thus, Mnl1 regulates tolerance through coordinated control of sphingolipid metabolism, mitochondrial activity, and PKA signaling, highlighting that tolerant cells adopt a distinct physiological state primed to withstand antifungal stress.

microbiology↗

bioRxiv2025

Genome-guided manipulation of regulators of morphogenesis in a C. albicans strain with low virulence is not sufficient to trigger a high-virulence phenotype

As a member of the microbiome, C. albicans colonizes the oral cavity and other mucosal surfaces of the human body. While commensalism is tightly controlled by the host immune system, the fungal determinants enabling the fungus to colonize the host mucosa without causing tissue damage and inflammation remain less clear. In search of genetic determinants that may underly the commensal lifestyle of the low-virulent C. albicans isolate 101, we identified a small sequence duplication in one of the BRG1 allele resulting in a truncated loss-of-function allele (BRG1TRUNC). Replacing BRG1TRUNC by the full-length allele (BRG1FL) resulted in a modest increase in filamentation, but did not alter the phenotype of the fungus in the oral mucosa of experimentally colonized mice. Spontaneous outgrowth of highly virulent variants of the 101 strain with the BRG1TRUNC alleles replaced by a full-length allele identified the Cyr1-cAMP-PKA signalling pathway as a modulator of fungal virulence. Although the Glu-to-Lys mutation in CYR1 (CYR1E1541K), which converts Cyr1 into a hyperactive adenylate cyclase, greatly increased filamentation, the expression of hyphae-associated genes, and host cell damage when tested in vitro, it was insufficient to increase virulence of C. albicans strain 101 in the oral mucosa in vivo, irrespective of the BRG1 status. Together, this highlights that the low-virulent nature of strain 101 is firmly anchored and cannot be overcome by manipulating BRG1 and CYR1, two genes with known roles in C. albicans virulence. IMPORTANCEDuring homeostasis, the fungus Candida albicans establishes mutualistic interactions with its human host. It can however also adopt a pathogenic state and cause infections with diverse clinical manifestions that pose a significant challenge for diagnosis and therapy. Understanding the fungal determinants that underlie C. albicans colonization under steady state conditions may thus provide new avenues for modulating the fungus-host interaction in candidiasis patients to restore homeostasis. Here, we report gene variants that distinguish high-virulent from low-virulent C. albicans. Gene-exchange mutants provided evidence for the contribution of a BRG1 loss-of-function allele and a CYR1 gain-of-function mutation toward fungal pathogenicity. However, in vivo in an experimental model of C. albicans oral colonization, none of these gene variants individually or in combination were sufficient to change the virulence profile of the fungus. These findings indicate that C. albicans mucosal colonization is regulated by a complex gene network rather than by single genetic determinants.

microbiology↗

bioRxiv2025

Comprehensive mapping of the 5' and 3' untranslated regions of Aspergillus fumigatus reveals new insights into gene regulation

In the twenty years since the first genome sequencing of Aspergillus fumigatus, the field has seen an explosion in both the number of sequenced genomes and our molecular understanding of this ubiquitous human fungal pathogen. Despite an improved knowledge of the A. fumigatus genome, we still know little about the transcriptome, with key regulatory sequences like the untranslated regions of mRNA based only on in silico predictions and bulk-RNA-seq. Here, we provide an improved description of 5' and 3' untranslated regions of A. fumigatus poly(A)-enriched RNA through experimental mapping of transcription start sites and polyadenylation sites using 5' and 3' End-Seq. We assigned high-quality 5' ends to 2,747 genes (average length 126 nt), 3' ends to 7,079 genes (average length 268 nt), and improved our understanding of the regulatory landscape of A. fumigatus gene expression. We leveraged the refined 5' UTRs to identify upstream open reading frames and binding sites for important RNA binding proteins like the translational regulator Ssd1 and the 3' UTRs to define binding sites for PUF proteins known to contribute to mRNA localization and regulation. Although a single isoform typically dominated expression, we observed 148 instances of alternative start sites and 1,675 alternative stop sites. Interestingly, we detected multiple examples of premature transcriptional termination, including the first evidence for promoter-proximal premature transcriptional termination in a member of the Eurotiomycetes. Ultimately, we provide a resource to the Aspergillus community and an accurate starting point for unravelling the complexities of gene regulation in an important human pathogen.

microbiology↗

bioRxiv2025

Polygenetic Determinants of Azole Resistance, Tolerance, and Heteroresistance in Candida albicans

Azole antifungals are widely used to treat Candida infections, yet therapeutic failures are common. In addition to resistance, fungal populations can exhibit tolerance and heteroresistance, subpopulation-driven drug responses that can contribute to treatment failure. Here, we performed a genome-wide association study (GWAS) on 557 genetically diverse C. albicans isolates to identify genetic loci associated with fluconazole susceptibility, tolerance, and heteroresistance. We uncovered a complex, polygenic architecture underlying all three traits, involving novel loci linked to stress responses, cell cycle, and genome integrity. Notably, canonical resistance genes were absent from our top associations, reflecting either their low frequency or lineage specificity. Functional validation of several candidate genes confirmed distinct genetic determinants for each drug response. Heritability estimates and conditional analyses revealed that susceptibility and heteroresistance are governed by partially overlapping but largely independent loci, with some genetic variants displaying opposing effects on different traits. This limited overlap suggests that each phenotype represents a distinct evolutionary strategy for drug evasion, which may not be simultaneously addressed by a single therapeutic approach. Our findings underscore the polygenic nature of adaptation to antifungal drugs and expand our understanding of drug response mechanisms in C. albicans. SIGNIFICANCEDespite advances in antifungal therapies, treatment failures in Candida albicans infections remain common and poorly understood. Standard clinical diagnostic tools rely on susceptibility testing to predict treatment outcomes, yet many treatment-refractory infections are caused by strains that appear susceptible in laboratory tests. This study provides the first genome-wide dissection of three distinct drug responses - susceptibility, tolerance, and heteroresistance - across a global population of C. albicans isolates. By identifying novel genetic contributors beyond canonical resistance genes, our findings shift the current paradigm toward a more nuanced, polygenic model of antifungal adaptation. These results underscore the importance of integrating tolerance and heteroresistance into clinical and research frameworks and point to new molecular pathways that could be leveraged to enhance antifungal strategies.

microbiology↗

bioRxiv2024

Large circulation of a novel vesiculovirus in bats in the Mediterranean region

Bats are the natural reservoirs of a variety of emerging or re-emerging viruses. Among them, rabies virus (genus Lyssavirus, family Rhabdoviridae) is of the first and most iconic described in these animals. Since its first description, various new bat lyssaviruses have been regularly described. Apart from lyssaviruses, other bat rhabdoviruses have been also identified, including members of the Vesiculovirus, Ledantevirus and more recently Alphanemrhavirus and Tupavirus genera. However, the family Rhabdoviridae is one of the most abundant and diverse viral families, with 318 officially recognized species divided into 3 subfamilies and 46 different genera. Thus, the number of bat- associated rhabdoviruses is probably higher. In this study, we first developed and validated a combined nested RT-qPCR technique (pan-rhabdo RT-nqPCR) dedicated to the broad detection of animal rhabdovirus. After validation, this technique was used for a large retrospective screening of archival bat samples (n = 1962), including blood (n = 816), brain (n = 723) and saliva (n = 423). These samples were collected from various bat species over a period of 12 years (2007-2019) in 9 different countries in Europe and Africa. A total of 23 samples (1.2%) from Miniopterus schreibersii, Rhinolophus euryale and Rhinolophus ferrumequinum bat species was found positive for rhabdovirus infection, including 17 (2.1%) blood and 6 (1.4%) saliva samples, all collected from bats originated from the Mediterranean region. The complete virus genome sequences were obtained by next-generation sequencing for most of the positive samples. Molecular and phylogenetic analysis of these sequences demonstrated that these virus isolates, named Mediterranean bat virus (MBV), were closely related, and represented a new species Vesiculovirus mediterranean within the Vesiculovirus genus. MBV was more specifically related to the other bat vesiculoviruses previously described in China and North America, together clustering into a distinct group of bat viruses within this genus. Interestingly, our results suggest that MBV is widely distributed, at least in the West part of the Mediterranean region, where it can act as an arbovirus infecting and circulating in multiple bat species. These findings expand the host range and the viral diversity of bat vesiculoviruses and pave the way for further investigations to determine the route of transmission and the dynamic of diffusion of these viruses into bat colonies, as well as to evaluate their potential hazard for public health.

microbiology↗

bioRxiv2024

Step-wise evolution of azole resistance through copy number variation followed by KSR1 loss of heterozygosity in Candida albicans.

Antimicrobial drug resistance poses a global health threat, requiring a deeper understanding of the evolutionary processes that lead to its emergence in pathogens. Complex evolutionary dynamics involve multiple mutations that can result in cooperative or competitive (clonal interference) effects. Candida albicans, a major fungal pathogen, displays high rates of copy number variation (CNV) and loss of heterozygosity (LOH). CNV and LOH events involve large numbers of genes and could synergize during evolutionary adaptation. Understanding the contributions of CNV and LOH to antifungal drug adaptation is challenging, especially in the context of whole-population genome sequencing. Here, we document the sequential evolution of fluconazole tolerance and then resistance in a C. albicans isolate involving an initial CNV on chromosome 4, followed by an LOH on chromosome R that involves KSR1. Similar LOH events involving KSR1, which encodes a reductase involved in sphingolipid biosynthesis, were also detected in independently evolved fluconazole resistant isolates. We dissect the specific KSR1 codons that affect fluconazole resistance and tolerance. The combination of the chromosome 4 CNV and KSR1 LOH results in a >500-fold increase in azole resistance, illustrating a compelling example of rapid, yet step-wise, interplay between CNV and LOH in drug resistance evolution.

microbiology↗

bioRxiv2023

The Cryptocococcus extracellular vesicle transcriptome

Extracellular vesicles (EVs) are now recognized as key players in the biology of numerous organisms, including pathogenic fungi. However, studying EVs in these organisms remains challenging. The recent implementation of new protocols to purify EVs in the pathogenic yeast Cryptococcus neoformans has resulted in a more detailed description of their structure and protein composition. Although a few publications describing RNA molecules associated with EVs have already been published, we reasoned that these new protocols would be beneficial for gaining a deeper understanding of the EV transcriptome. We thus purified EVs and confirmed that some RNAs were associated with these EV extracts. Iodixanol gradient analyses also revealed that these RNAs co-sedimented with EVs. We then sequenced these RNAs in parallel with RNAs extracted from the very cells producing these EVs using different types of sequencing libraries. Our data confirm the presence of siRNAs and tRFs associated with EVs, some of which are enriched. We also identified some snoRNAs, which in Cryptococcus are mostly borne by coding gene or lncRNA introns.

microbiology↗

bioRxiv2023

Metabolic reprogramming during Candida albicans planktonic-biofilm transition is modulated by the ZCF15 and ZCF26 paralogs

Candida albicans is a commensal of the human microbiota that can form biofilms on implanted medical devices. These biofilms are tolerant to antifungals and to the host immune system. To identify novel genes modulating C. albicans biofilm formation, we performed a large-scale screen with 2454 C. albicans doxycycline-dependent overexpression strains and identified 16 genes whose overexpression significantly hampered biofilm formation. Among those, overexpression of the ZCF15 and ZCF26 paralogs that encode transcription factors and have orthologs only in biofilm-forming species of the Candida clade, caused impaired biofilm formation both in vitro and in vivo. Interestingly, overexpression of ZCF15 specifically impeded biofilm formation without any defect in hyphal growth. Transcript profiling, transcription factor binding, and phenotypic microarray analyses conducted upon overexpression of ZCF15 and ZCF26 demonstrated their direct role in reprogramming cellular metabolism by regulating glycolytic cycle and tricarboxylic acid cycle genes. Taken together, this study has identified a new set of biofilm regulators, including ZCF15 and ZCF26, that appear to control biofilm development through their specific role in metabolic remodeling.

microbiology↗

bioRxiv2023

Tur1 regulates alternative TSS usage in Cryptococcus

Alternative transcription start site (TSS) usage regulation has been identified as a major means of gene expression regulation in metazoans. However, in fungi, its impact remains elusive as its study has thus far been restricted to model yeasts. Here, we first re-analysed TSS-seq data to define genuine TSS clusters in two species of pathogenic Cryptococcus. We identified two types of TSS clusters associated with specific DNA sequence motifs. Our analysis also revealed that alternative TSS usage regulation in response to environmental cues is widespread in Cryptococcus, altering gene expression and protein targeting. Importantly, we performed a forward genetic screen to identify a unique transcription factor (TF) named Tur1, which regulates alternative TSS (altTSS) usage genome-wide when cells switch from exponential phase to stationary phase. ChiP-Seq and DamID-Seq analyses suggest that at some loci the role of Tur1 might be direct. Tur1 has been previously shown to be essential for virulence in C. neoformans. We demonstrated here that a tur1{Delta} mutant strain is more sensitive to superoxide stress and phagocytosed more efficiently by macrophages than the wild-type (WT) strain.

molecular biology↗

bioRxiv2023

Extracellular vesicles production regulates fluconazole resistance in Cryptococcus neoformans

Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novel Cryptococcus neoformans regulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover, in vitro spontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen. IMPORTANCEExtracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation but thier functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogen Cryptococcus neoformans. Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated in C. neoformans clinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.

microbiology↗

bioRxiv2021

Candida albicans Isolates 529L and CHN1 Exhibit Stable Colonization of the Murine Gastrointestinal Tract

Candida albicans is a pathobiont that colonizes multiple niches in the body including the gastrointestinal (GI) tract, but is also responsible for both mucosal and systemic infections. Despite its prevalence as a human commensal, the murine GI tract is generally refractory to colonization with the C. albicans reference isolate SC5314. Here, we identify two C. albicans isolates, 529L and CHN1, that stably colonize the murine GI tract in three different animal facilities under conditions where SC5314 is lost from this niche. Analysis of the bacterial microbiota did not show notable differences between mice colonized with the three C. albicans strains. We compared the genotypes and phenotypes of these three strains and identified thousands of SNPs and multiple phenotypic differences, including their ability to grow and filament in response to nutritional cues. Despite striking filamentation differences under laboratory conditions, however, analysis of cell morphology in the GI tract revealed that the three isolates exhibited similar filamentation properties in this in vivo niche. Notably, we found that SC5314 is more sensitive to the antimicrobial peptide CRAMP, and the use of CRAMP-deficient mice increased the ability of SC5314 to colonize the GI tract relative to CHN1 and 529L. These studies provide new insights into how strain-specific differences impact C. albicans traits in the host and advance CHN1 and 529L as relevant strains to study C. albicans pathobiology in its natural host niche. IMPORTANCEUnderstanding how fungi colonize the GI tract is increasingly recognized as highly relevant to human health. The animal models used to study Candida albicans commensalism commonly rely on altering the host microbiome (via antibiotic treatment or defined diets) to establish successful GI colonization by the C. albicans reference isolate SC5314. Here, we characterize two C. albicans isolates that can colonize the murine GI tract without antibiotic treatment and can therefore be used as tools for studying fungal commensalism. Importantly, experiments were replicated in three different animal facilities and utilized three different mouse strains. Differential colonization between fungal isolates was not associated with alterations in the bacterial microbiome but rather with distinct responses to CRAMP, a host antimicrobial peptide. This work emphasizes the importance of C. albicans intra-species variation as well as host anti-microbial defense mechanisms in defining commensal interactions.

microbiology↗

bioRxiv2021

POPULATION GENOMIC ANALYSIS OF CRYPTOCOCCUS BRAZILIAN STRAINS REVEALS AN AFRICAN TYPE SUBCLADES DISTRIBUTION

The genomes of a large number of Cryptococcus neoformans isolates have been sequenced and analyzed in recent years. These genomes have been used to understand the global population structure of this opportunistic pathogen. However, only a small number of South American isolates have been considered in these studies, and the population structure of C. neoformans in this part of the world remains elusive. Here, we analyzed the genomic sequences of 53 Brazilian Cryptococcus isolates and deciphered the C. neoformans population structure in this country. Our data reveal an African-like structure that suggested repeated intercontinental transports from Africa to South America. We also identified a mutator phenotype in one VNBII Brazilian isolate, exemplifying how fast-evolving isolates can shape the Cryptococcus population structure. Finally, phenotypic analyses revealed wide diversity but not lineage specificity in the expression of classical virulence traits within the set of isolates.

genomics↗

bioRxiv2020

Application of an optimized annotation pipeline to the Cryptococcus deuterogattii genome reveals dynamic primary metabolic gene clusters and genomic impact of RNAi loss

Evaluating the quality of a de novo annotation of a complex fungal genome based on RNA-seq data remains a challenge. In this study, we sequentially optimized a Cufflinks-CodingQuary based bioinformatics pipeline fed with RNA-seq data using the manually annotated model pathogenic yeasts Cryptococcus neoformans and Cryptococcus deneoformans as test cases. Our results demonstrate that the quality of the annotation is sensitive to the quantity of RNA-seq data used and that the best quality is obtained with 5 to 10 million reads per RNA-seq replicate. We also demonstrated that the number of introns predicted is an excellent a priori indicator of the quality of the final de novo annotation. We then used this pipeline to annotate the genome of the RNAi-deficient species Cryptococcus deuterogattii strain R265 using RNA-seq data. Dynamic transcriptome analysis revealed that intron retention is more prominent in C. deuterogattii than in the other RNAi-proficient species C. neoformans and C. deneoformans. In contrast, we observed that antisense transcription was not higher in C. deuterogattii than in the two other Cryptococcus species. Comparative gene content analysis identified 21 clusters enriched in transcription factors and transporters that have been lost. Interestingly, analysis of the subtelomeric regions in these three annotated species identified a similar gene enrichment, reminiscent of the structure of primary metabolic clusters. Our data suggest that there is active exchange between subtelomeric regions, and that other chromosomal regions might participate in adaptive diversification of Cryptococcus metabolite assimilation potential.

microbiology↗

bioRxiv2019

Start codon context controls translation initiation in the fungal kingdom

Eukaryotic protein synthesis initiates at a start codon defined by an AUG and its surrounding Kozak sequence context, but studies of S. cerevisiae suggest this context is of little importance in fungi. We tested this concept in two pathogenic Cryptococcus species by genome-wide mapping of translation and of mRNA 5 and 3 ends. We observed that upstream open reading frames (uORFs) are a major contributor to translation repression, that uORF use depends on the Kozak sequence context of its start codon, and that uORFs with strong contexts promote nonsense-mediated mRNA decay. Numerous Cryptococcus mRNAs encode predicted dual-localized proteins, including many aminoacyl-tRNA synthetases, in which a leaky AUG start codon is followed by a strong Kozak context in-frame AUG, separated by mitochondrial-targeting sequence. Further analysis shows that such dual-localization is also predicted to be common in Neurospora crassa. Kozak-controlled regulation is correlated with insertions in translational initiation factors in fidelity-determining regions that contact the initiator tRNA. Thus, start codon context is a signal that programs the expression and structures of proteins in fungi.

genomics↗

bioRxiv2018

Generating genomic platforms to study Candida albicans pathogenesis

The advent of the genomic era has made elucidating gene function at large scale a pressing challenge. ORFeome collections, whereby almost all ORFs of a given species are cloned and can be subsequently leveraged in multiple functional genomic approaches, represent valuable resources towards this endeavor. Here we provide novel, genome-scale tools for the study of Candida albicans, a commensal yeast that is also responsible for frequent superficial and disseminated infections in humans. We have generated an ORFeome collection composed of 5,102 ORFs cloned in a Gateway donor vector, representing 83% of the currently annotated coding sequences of C. albicans. Sequencing data of the cloned ORFs are available in the CandidaOrfDB database at http://candidaorfeome.eu. We also engineered 49 expression vectors with a choice of promoters, tags, and selection markers and demonstrated their applicability to the study of target ORFs transferred from the C. albicans ORFeome. In addition, the use of the ORFeome in the detection of protein-protein interaction was demonstrated. Mating-compatible strains as well as Gateway-compatible two-hybrid vectors were engineered, validated and used in a proof of concept experiment. These unique and valuable resources should greatly facilitate future functional studies in C. albicans and the elucidation of mechanisms that underlie its pathogenicity.

genomics↗