Publications by authors named "Mélanie Legrand"

30 Publications

  • Page 1 of 1

The impact of the Fungus-Host-Microbiota interplay upon Candida albicans infections: current knowledge and new perspectives.

FEMS Microbiol Rev 2021 May;45(3)

NEXBIOME Therapeutics, 22 allée Alan Turing, 63000 Clermont-Ferrand, France.

Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.
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http://dx.doi.org/10.1093/femsre/fuaa060DOI Listing
May 2021

Factors that influence bidirectional long-tract homozygosis due to double-strand break repair in Candida albicans.

Genetics 2021 May;218(1)

Institut Pasteur, INRA, Unité Biologie et Pathogénicité Fongiques, Paris, France.

Genomic rearrangements have been associated with the acquisition of adaptive phenotypes, allowing organisms to efficiently generate new favorable genetic combinations. The diploid genome of Candida albicans is highly plastic, displaying numerous genomic rearrangements that are often the by-product of the repair of DNA breaks. For example, DNA double-strand breaks (DSB) repair using homologous-recombination pathways are a major source of loss-of-heterozygosity (LOH), observed ubiquitously in both clinical and laboratory strains of C. albicans. Mechanisms such as break-induced replication (BIR) or mitotic crossover (MCO) can result in long tracts of LOH, spanning hundreds of kilobases until the telomere. Analysis of I-SceI-induced BIR/MCO tracts in C. albicans revealed that the homozygosis tracts can ascend several kilobases toward the centromere, displaying homozygosis from the break site toward the centromere. We sought to investigate the molecular mechanisms that could contribute to this phenotype by characterizing a series of C. albicans DNA repair mutants, including pol32-/-, msh2-/-, mph1-/-, and mus81-/-. The impact of deleting these genes on genome stability revealed functional differences between Saccharomyces cerevisiae (a model DNA repair organism) and C. albicans. In addition, we demonstrated that ascending LOH tracts toward the centromere are associated with intrinsic features of BIR and potentially involve the mismatch repair pathway which acts upon natural heterozygous positions. Overall, this mechanistic approach to study LOH deepens our limited characterization of DNA repair pathways in C. albicans and brings forth the notion that centromere proximal alleles from DNA break sites are not guarded from undergoing LOH.
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http://dx.doi.org/10.1093/genetics/iyab028DOI Listing
May 2021

Novel partners of USP6 gene in a spectrum of bone and soft tissue lesions.

Virchows Arch 2021 Feb 9. Epub 2021 Feb 9.

Service d'anatomie et cytologie pathologiques, CHRU de Tours, Avenue de la République, 37044 Cedex 9, Tours, France.

Nodular fasciitis, primary aneurysmal bone cyst, myositis ossificans, and their related lesions are benign tumors that share common histological features and a chromosomal rearrangement involving the ubiquitin-specific peptidase 6 (USP6) gene. The identification of an increasing number of new partners implicated in USP6 rearrangements demonstrates a complex tumorogenesis of this tumor spectrum. In this study on a series of 77 tumors (28 nodular fasciitis, 42 aneurysmal bone cysts, and 7 myositis ossificans) from the database of the French Sarcoma Group, we describe 7 new partners of the USP6 gene. For this purpose, rearrangements were first researched by multiplexed RT-qPCRs in the entire population. A targeted RNA sequencing was then used on samples selected according to a high USP6-transcription level expression estimated by RT-qPCR. Thanks to this multistep approach, besides the common USP6 fusions observed, we detected novel USP6 partners: PDLIM7 and MYL12A in nodular fasciitis and TPM4, DDX17, GTF2I, KLF3, and MEF2A in aneurysmal bone cysts. In order to try to bring to light the role played by the recently identified USP6 partners in this lesional spectrum, their functions are discussed. Taking into account that a traumatic participation has long been mentioned in the histogenesis of most of these lesions and because of their morphological resemblance to organizing granulation reparative tissue or callus, a focus is placed on their relationship with tissue remodeling and, to a lesser extent, with bone metabolism.
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http://dx.doi.org/10.1007/s00428-021-03047-zDOI Listing
February 2021

Bisphosphonates for the treatment of fibrous dysplasia of bone.

Bone 2021 02 2;143:115784. Epub 2020 Dec 2.

INSERM UMR 1033, Université de Lyon, Hôpital E Herriot, Lyon cedex 03, France.

Introduction: Fibrous dysplasia of bone (FD) is a rare congenital bone disease, due to a somatic mutation of GNAS. This mutation results in a defect of osteoblast differentiation and mineralization and also an increase in bone resorption by large active osteoclasts. Bone pain is present in half of patients and is the main determinant of quality of life of patients with FD. Bisphosphonates are known to reduce bone pain and reduce the risk of fracture in patients with bone metastases or Paget's disease. Bisphosphonates may have similar effects in FD. In this article, we have reviewed the therapeutic potential of bisphosphonates to reduce bone pain due to FD, improve bone strength and reduce the occurrence of fracture.

Material And Methods: We have reviewed 234 articles examining the effect of bisphosphonates on FD/McCune Albright Syndrome with no date limit, in PubMed and selected the articles with highest quality of methodology.

Results: Pamidronate therapy significantly decreased bone pain and bone resorption (urinary NTX, urinary and serum CTX). Pamidronate may improve radiological lesions of FD patients (filling of osteolytic lesion and/or cortical thickening). This data with intravenous pamidronate, however, has been obtained from observational studies and no randomized controlled trial is available. Randomized placebo-controlled trials of oral bisphosphonates (alendronate or risedronate) have failed to demonstrate a significant decrease in bone pain over placebo. Several studies including one randomized controlled trial have shown an increase in bone mineral density (BMD) at FD sites with oral and intravenous bisphosphonate treatment. No effect on occurrence of fracture has been reported.

Conclusion: In conclusion, intravenous bisphosphonates may be proposed to treat persistent, moderate to severe bone pain of FD, e.g., according to the guidelines from the FD/MAS International Consortium. Oral bisphosphonates should not be used in this indication.
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http://dx.doi.org/10.1016/j.bone.2020.115784DOI Listing
February 2021

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

The impact of the Fungus-Host-Microbiota interplay upon Candida albicans infections: current knowledge and new perspectives.

FEMS Microbiol Rev 2021 May;45(3)

NEXBIOME Therapeutics, 22 allée Alan Turing, 63000 Clermont-Ferrand, France.

Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.
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http://dx.doi.org/10.1093/femsre/fuaa060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100220PMC
May 2021

Imminent fracture risk.

Joint Bone Spine 2021 05 7;88(3):105105. Epub 2020 Nov 7.

Department of Rheumatology, Edouard Herriot University Hospital, 5, Place d'Arsonval, 69003 Lyon, France; INSERM UMR 1033, Université de Lyon, Lyon, France.

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http://dx.doi.org/10.1016/j.jbspin.2020.105105DOI Listing
May 2021

Use of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes in Candida albicans.

mSphere 2020 09 2;5(5). Epub 2020 Sep 2.

Institut Pasteur, INRA, Unité Biologie et Pathogénicité Fongiques, Paris, France

Most of our knowledge relating to molecular mechanisms of human fungal pathogenesis in relies on reverse genetics approaches, requiring strain engineering. DNA-mediated transformation of has been described as highly mutagenic, potentially accentuated by the organism's genome plasticity, including the acquisition of genomic rearrangements, notably upon exposure to stress. The advent of CRISPR-Cas9 has vastly accelerated the process of genetically modifying strains, especially in diploid (such as ) and polyploid organisms. The effects of unleashing this nuclease within the genome of are unknown, although several studies in other organisms report Cas9-associated toxicity and off-target DNA breaks. Upon the construction of a strain collection, we took the opportunity to compare strains which were constructed using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation strategies, by quantifying and describing transformation-induced loss-of-heterozygosity and hyperploidy events. Our analysis of 57 strains highlights the mutagenic effects of transformation in , regardless of the transformation protocol, but also underscores interesting differences in terms of genomic changes between strains obtained using different transformation protocols. Indeed, although strains constructed using the CRISPR-Cas9-free transformation method display numerous concomitant genomic changes randomly distributed throughout their genomes, the use of CRISPR-Cas9 leads to a reduced overall number of genome changes, particularly hyperploidies. Overall, in addition to facilitating strain construction by reducing the number of transformation steps, the CRISPR-Cas9-dependent transformation strategy in appears to limit transformation-associated genome changes. Genome editing is essential to nearly all research studies aimed at gaining insight into the molecular mechanisms underlying various biological processes, including those in the opportunistic pathogen The adaptation of the CRISPR-Cas9 system greatly facilitates genome engineering in many organisms. However, our understanding of the effects of CRISPR-Cas9 technology on the biology of is limited. In this study, we sought to compare the extents of transformation-induced genomic changes within strains engineered using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation methods. CRISPR-Cas9-dependent transformation allows one to simultaneously target both homologs and, importantly, appears less mutagenic in , since strains engineered using CRISPR-Cas9 display an overall decrease in concomitant genomic changes.
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http://dx.doi.org/10.1128/mSphere.00620-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471004PMC
September 2020

Multiple Erosions and Crusts on Legs in a Returning Traveller: A Quiz.

Acta Derm Venereol 2020 08 25;100(15):adv00253. Epub 2020 Aug 25.

Department of Dermatology, University Hospital Center of Tours, 37044 Tours Ceddex 9, France.

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http://dx.doi.org/10.2340/00015555-3604DOI Listing
August 2020

A Signature of Circulating miRNAs Associated With Fibrous Dysplasia of Bone: the mirDys Study.

J Bone Miner Res 2020 10 21;35(10):1881-1892. Epub 2020 Jul 21.

Department of Rheumatology, Edouard Herriot University Hospital, Lyon, France.

Fibrous dysplasia (FD) is a rare bone disease caused by activating mutations of GNAS encoding the Gsα protein, enhancing cyclic adenosine monophosphate (cAMP) production by overstimulation of adenylyl cyclase and impairing osteoblastic differentiation. The clinical presentation ranges from asymptomatic to polyostotic forms with severe disability, explained by the mosaic distribution of the GNAS mutation. Physicians have to deal with the gap of knowledge in FD pathogenesis, the absence of prognostic markers and the lack of specific treatment. The identification of specific biomarkers for FD is an important step to improve the clinical and therapeutic approaches. An epigenetic regulation driven by microRNAs (miRNAs), known as promising biomarkers in bone disease, could be involved in FD. We have sought circulating miRNAs that are differentially expressed in FD patients compared to controls and would reflect dysregulations of osteogenesis-related genes and bone disorder. The global miRNA profiling was performed using Next Generation Sequencing in patient serum collected from a discovery cohort of 20 patients (10 polyostotic and 10 monostotic) and 10 controls. From these, we selected 19 miRNAs for a miRNA validation phase from serum of 82 patients and 82 controls, using real-time qPCR. Discovery screening identified 111 miRNAs differentially expressed in patient serum, after adjusting for the false discovery rate (FDR). Among the 82 patients, 55% were polyostotic, and 73% were women with a mean age of 42 years. Six miRNAs (miR-25-3p, miR-93-5p, miR-182-5p, miR-324-5p, miR-363-3p, and miR-451a) were significantly overexpressed in serum, with FDR <0.05. The expression level of these six miRNAs was not associated with the FD severity. In conclusion, we identified a signature of circulating miRNAs associated with FD. These miRNAs are potential negative regulators of gene expression in bone cell progenitors, suggesting their activity in FD by interfering with osteoblastic and osteoclastic differentiation to impair bone mineralization and remodeling processes. © 2020 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.4111DOI Listing
October 2020

Umbilicated Papules on the Face of an Adult Patient: A Quiz.

Acta Derm Venereol 2020 Jul 28;100(14):adv00213. Epub 2020 Jul 28.

Department of Dermatology, Centre Hospitalier Regional Universitaire de Tours, FR-37044 Tours, France.

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http://dx.doi.org/10.2340/00015555-3528DOI Listing
July 2020

Candida albicans: An Emerging Yeast Model to Study Eukaryotic Genome Plasticity.

Trends Genet 2019 04 28;35(4):292-307. Epub 2019 Feb 28.

Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India. Electronic address:

Saccharomyces cerevisiae and Schizosaccharomyces pombe have served as uncontested unicellular model organisms, as major discoveries made in the field of genome biology using yeast genetics have proved to be relevant from yeast to humans. The yeast Candida albicans has attracted much attention because of its ability to switch between a harmless commensal and a dreaded human pathogen. C. albicans bears unique features regarding its life cycle, genome structure, and dynamics, and their links to cell biology and adaptation to environmental challenges. Examples include a unique reproduction cycle with haploid, diploid, and tetraploid forms; a distinctive organisation of chromosome hallmarks; a highly dynamic genome, with extensive karyotypic variations, including aneuploidies, isochromosome formation, and loss-of-heterozygosity; and distinctive links between the response to DNA alterations and cell morphology. These features have made C. albicans emerge as a new and attractive unicellular model to study genome biology and dynamics in eukaryotes.
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http://dx.doi.org/10.1016/j.tig.2019.01.005DOI Listing
April 2019

Identification of Recessive Lethal Alleles in the Diploid Genome of a Candida albicans Laboratory Strain Unveils a Potential Role of Repetitive Sequences in Buffering Their Deleterious Impact.

mSphere 2019 02 13;4(1). Epub 2019 Feb 13.

Institut Pasteur, INRA, Unité Biologie et Pathogénicité Fongiques, Paris, France

The heterozygous diploid genome of is highly plastic, with frequent loss of heterozygosity (LOH) events. In the SC5314 laboratory strain, while LOH events are ubiquitous, a chromosome homozygosis bias is observed for certain chromosomes, whereby only one of the two homologs can occur in the homozygous state. This suggests the occurrence of recessive lethal allele(s) (RLA) preventing large-scale LOH events on these chromosomes from being stably maintained. To verify the presence of an RLA on chromosome 7 (Chr7), we utilized a system that allows (i) DNA double-strand break (DSB) induction on Chr7 by the I-I endonuclease and (ii) detection of the resulting long-range homozygosis. I-I successfully induced a DNA DSB on both Chr7 homologs, generally repaired by gene conversion. Notably, cells homozygous for the right arm of Chr7B were not recovered, confirming the presence of RLA(s) in this region. Genome data mining for RLA candidates identified a premature nonsense-generating single nucleotide polymorphism (SNP) within the HapB allele of C7_03400c whose ortholog encodes the essential Mtr4 RNA helicase. Complementation with a wild-type copy of rescued cells homozygous for the right arm of Chr7B, demonstrating that the RLA is responsible for the Chr7 homozygosis bias in strain SC5314. Furthermore, we observed that the major repeat sequences (MRS) on Chr7 acted as hot spots for interhomolog recombination. Such recombination events provide with increased opportunities to survive DNA DSBs whose repair can lead to homozygosis of recessive lethal or deleterious alleles. This might explain the maintenance of MRS in this species. is a major fungal pathogen, whose mode of reproduction is mainly clonal. Its genome is highly tolerant to rearrangements, in particular loss of heterozygosity events, known to unmask recessive lethal and deleterious alleles in heterozygous diploid organisms such as By combining a site-specific DSB-inducing system and mining genome sequencing data of 182  isolates, we were able to ascribe the chromosome 7 homozygosis bias of the laboratory strain SC5314 to an heterozygous SNP introducing a premature STOP codon in the gene. We have also proposed genome-wide candidates for new recessive lethal alleles. We additionally observed that the major repeat sequences (MRS) on chromosome 7 acted as hot spots for interhomolog recombination. Maintaining MRS in could favor haplotype exchange, of vital importance to LOH events, leading to homozygosis of recessive lethal or deleterious alleles that inevitably accumulate upon clonality.
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http://dx.doi.org/10.1128/mSphere.00709-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374597PMC
February 2019

[Microcystic serous cystadenoma: An uncommon neoplasm of pancreas. Report of two cases].

Ann Pathol 2019 Aug 31;39(4):292-296. Epub 2019 Jan 31.

Service d'anatomie et cytologie pathologiques, hôpital de la Source, BP 86709, 45067 Orléans cedex 2, France.

Microcystic variant of serous cystadenoma of the pancreas is a rare neoplasm; essentially located in the body or tail of the pancreas and associated with the von Hippel-Lindau. Often, patients are asymptomatic and the neoplasm is incidentally discovered. Usually radiographic manifestations are characteristic. Histopathological examination revealed uniform clear cuboidal cells; they can be confused with other clear cell neoplasms like renal cell carcinomas, well-differentiated neuroendocrine tumors and solid pseudopapillary tumors of the pancreas. Immunohistochemistry can be help to establish the diagnosis and to remove differential diagnosis. Serous cystadenoma is a benign neoplasm whose prognosis is excellent. We herein report two cases of microcystic serous cystadenomas of the pancreas diagnosed in two asymptomatic women and review analysis in the literature to remind the main features of this lesion and the main differential diagnosis.
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http://dx.doi.org/10.1016/j.annpat.2018.12.007DOI Listing
August 2019

Generating genomic platforms to study Candida albicans pathogenesis.

Nucleic Acids Res 2018 08;46(14):6935-6949

Fungal Biology and Pathogenicity Unit, Department of Mycology, Institut Pasteur, INRA, Paris 75015, France.

The advent of the genomic era has made elucidating gene function on a 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 toward 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 5099 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.
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http://dx.doi.org/10.1093/nar/gky594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101633PMC
August 2018

Analysis of Repair Mechanisms following an Induced Double-Strand Break Uncovers Recessive Deleterious Alleles in the Candida albicans Diploid Genome.

mBio 2016 10 11;7(5). Epub 2016 Oct 11.

Institut Pasteur, INRA, Unité Biologie et Pathogénicité Fongiques, Paris, France

The diploid genome of the yeast Candida albicans is highly plastic, exhibiting frequent loss-of-heterozygosity (LOH) events. To provide a deeper understanding of the mechanisms leading to LOH, we investigated the repair of a unique DNA double-strand break (DSB) in the laboratory C. albicans SC5314 strain using the I-SceI meganuclease. Upon I-SceI induction, we detected a strong increase in the frequency of LOH events at an I-SceI target locus positioned on chromosome 4 (Chr4), including events spreading from this locus to the proximal telomere. Characterization of the repair events by single nucleotide polymorphism (SNP) typing and whole-genome sequencing revealed a predominance of gene conversions, but we also observed mitotic crossover or break-induced replication events, as well as combinations of independent events. Importantly, progeny that had undergone homozygosis of part or all of Chr4 haplotype B (Chr4B) were inviable. Mining of genome sequencing data for 155 C. albicans isolates allowed the identification of a recessive lethal allele in the GPI16 gene on Chr4B unique to C. albicans strain SC5314 which is responsible for this inviability. Additional recessive lethal or deleterious alleles were identified in the genomes of strain SC5314 and two clinical isolates. Our results demonstrate that recessive lethal alleles in the genomes of C. albicans isolates prevent the occurrence of specific extended LOH events. While these and other recessive lethal and deleterious alleles are likely to accumulate in C. albicans due to clonal reproduction, their occurrence may in turn promote the maintenance of corresponding nondeleterious alleles and, consequently, heterozygosity in the C. albicans species.

Importance: Recessive lethal alleles impose significant constraints on the biology of diploid organisms. Using a combination of an I-SceI meganuclease-mediated DNA DSB, a fluorescence-activated cell sorter (FACS)-optimized reporter of LOH, and a compendium of 155 genome sequences, we were able to unmask and identify recessive lethal and deleterious alleles in isolates of Candida albicans, a diploid yeast and the major fungal pathogen of humans. Accumulation of recessive deleterious mutations upon clonal reproduction of C. albicans could contribute to the maintenance of heterozygosity despite the high frequency of LOH events in this species.
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http://dx.doi.org/10.1128/mBio.01109-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061868PMC
October 2016

A FACS-optimized screen identifies regulators of genome stability in Candida albicans.

Eukaryot Cell 2015 Mar 16;14(3):311-22. Epub 2015 Jan 16.

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

Loss of heterozygosity (LOH) plays important roles in genome dynamics, notably, during tumorigenesis. In the fungal pathogen Candida albicans, LOH contributes to the acquisition of antifungal resistance. In order to investigate the mechanisms that regulate LOH in C. albicans, we have established a novel method combining an artificial heterozygous locus harboring the blue fluorescent protein and green fluorescent protein markers and flow cytometry to detect LOH events at the single-cell level. Using this fluorescence-based method, we have confirmed that elevated temperature, treatment with methyl methanesulfonate, and inactivation of the Mec1 DNA damage checkpoint kinase triggered an increase in the frequency of LOH. Taking advantage of this system, we have searched for C. albicans genes whose overexpression triggered an increase in LOH and identified four candidates, some of which are known regulators of genome dynamics with human homologues contributing to cancer progression. Hence, the approach presented here will allow the implementation of new screens to identify genes that are important for genome stability in C. albicans and more generally in eukaryotic cells.
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http://dx.doi.org/10.1128/EC.00286-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4346560PMC
March 2015

Targeted changes of the cell wall proteome influence Candida albicans ability to form single- and multi-strain biofilms.

PLoS Pathog 2014 Dec 11;10(12):e1004542. Epub 2014 Dec 11.

Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Département Génomes et Génétique, Paris, France; INRA, USC2019, Paris, France.

Biofilm formation is an important virulence trait of the pathogenic yeast Candida albicans. We have combined gene overexpression, strain barcoding and microarray profiling to screen a library of 531 C. albicans conditional overexpression strains (∼10% of the genome) for genes affecting biofilm development in mixed-population experiments. The overexpression of 16 genes increased strain occupancy within a multi-strain biofilm, whereas overexpression of 4 genes decreased it. The set of 16 genes was significantly enriched for those encoding predicted glycosylphosphatidylinositol (GPI)-modified proteins, namely Ihd1/Pga36, Phr2, Pga15, Pga19, Pga22, Pga32, Pga37, Pga42 and Pga59; eight of which have been classified as pathogen-specific. Validation experiments using either individually- or competitively-grown overexpression strains revealed that the contribution of these genes to biofilm formation was variable and stage-specific. Deeper functional analysis of PGA59 and PGA22 at a single-cell resolution using atomic force microscopy showed that overexpression of either gene increased C. albicans ability to adhere to an abiotic substrate. However, unlike PGA59, PGA22 overexpression led to cell cluster formation that resulted in increased sensitivity to shear forces and decreased ability to form a single-strain biofilm. Within the multi-strain environment provided by the PGA22-non overexpressing cells, PGA22-overexpressing cells were protected from shear forces and fitter for biofilm development. Ultrastructural analysis, genome-wide transcript profiling and phenotypic analyses in a heterologous context suggested that PGA22 affects cell adherence through alteration of cell wall structure and/or function. Taken together, our findings reveal that several novel predicted GPI-modified proteins contribute to the cooperative behaviour between biofilm cells and are important participants during C. albicans biofilm formation. Moreover, they illustrate the power of using signature tagging in conjunction with gene overexpression for the identification of novel genes involved in processes pertaining to C. albicans virulence.
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http://dx.doi.org/10.1371/journal.ppat.1004542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263760PMC
December 2014

Distinct and redundant roles of exonucleases in Cryptococcus neoformans: implications for virulence and mating.

Fungal Genet Biol 2014 Dec 28;73:20-8. Epub 2014 Sep 28.

Institut Pasteur, Unité Biologie et Pathogénicité Fongiques - INRA USC2019, 75015 Paris, France. Electronic address:

Opportunistic pathogens like Cryptococcus neoformans are constantly exposed to changing environments, in their natural habitat as well as when encountering a human host. This requires a coordinated program to regulate gene expression that can act at the levels of mRNA synthesis and also mRNA degradation. Here, we find that deletion of the gene encoding the major cytoplasmic 5'→3' exonuclease Xrn1p in C. neoformans has important consequences for virulence associated phenotypes such as growth at 37 °C, capsule and melanin. In an invertebrate model of cryptococcosis the alteration of these virulence properties corresponds to avirulence of the xrn1Δ mutant strains. Additionally, deletion of XRN1 impairs uni- and bisexual mating. On a molecular level, the absence of XRN1 is associated with the upregulation of other major exonuclease encoding genes (i.e. XRN2 and RRP44). Using inducible alleles of RRP44 and XRN2, we show that artificial overexpression of these genes alters LAC1 gene expression and mating. Our data thus suggest the existence of a complex interdependent regulation of exonuclease encoding genes that impact upon virulence and mating in C. neoformans.
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http://dx.doi.org/10.1016/j.fgb.2014.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382001PMC
December 2014

A study of the DNA damage checkpoint in Candida albicans: uncoupling of the functions of Rad53 in DNA repair, cell cycle regulation and genotoxic stress-induced polarized growth.

Mol Microbiol 2014 Feb 25;91(3):452-71. Epub 2013 Dec 25.

Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Département Génomes et Génétique, 25, rue du Docteur Roux, F-75015, Paris, France; INRA USC2019, 25, rue du Docteur Roux, F-75015, Paris, France; Univ. Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, rue du Docteur Roux, F-75015, Paris, France.

In response to genotoxic stress (GS), Candida albicans can undergo polarized growth and massive genome rearrangements including loss-of-heterozygosity (LOH) events. We evaluated the contribution of the CaRad53p and CaDun1p kinases of the DNA damage checkpoint (DDCP) in these processes. Characterization of C. albicans rad53ΔΔ and dun1ΔΔ mutants revealed that the two kinases were involved in the maintenance of heterozygosity. SNP-RFLP typing and whole-genome sequencing of rad53ΔΔ isolates having undergone a LOH revealed that, according to the chromosome on which LOH had occurred, these were predominantly due to break-induced replication/mitotic cross-over or chromosome loss. Loss of CaRAD53 also resulted in frequent aneuploidies. Deletion of CaDUN1 led to an increase in recombination-dependent LOH but did not trigger aneuploidies. It also increased GS sensitivity but did not impair GS-induced polarized growth contrary to CaRAD53 deletion. Characterization of CaRad53p site-directed mutants demonstrated that its kinase activity and N-terminal phosphorylation sites were crucial for its function in the resistance to GS, maintenance of heterozygosity, cell cycle regulation and polarized growth. Moreover, using phosphomimic mutants, we revealed an uncoupling of the functions of CaRad53p in these different processes, thus providing a novel understanding of how the DDCP may regulate downstream events in response to GS.
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http://dx.doi.org/10.1111/mmi.12471DOI Listing
February 2014

A versatile overexpression strategy in the pathogenic yeast Candida albicans: identification of regulators of morphogenesis and fitness.

PLoS One 2012 25;7(9):e45912. Epub 2012 Sep 25.

Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Département Génomes et Génétique, Paris, France.

Candida albicans is the most frequently encountered human fungal pathogen, causing both superficial infections and life-threatening systemic diseases. Functional genomic studies performed in this organism have mainly used knock-out mutants and extensive collections of overexpression mutants are still lacking. Here, we report the development of a first generation C. albicans ORFeome, the improvement of overexpression systems and the construction of two new libraries of C. albicans strains overexpressing genes for components of signaling networks, in particular protein kinases, protein phosphatases and transcription factors. As a proof of concept, we screened these collections for genes whose overexpression impacts morphogenesis or growth rates in C. albicans. Our screens identified genes previously described for their role in these biological processes, demonstrating the functionality of our strategy, as well as genes that have not been previously associated to these processes. This article emphasizes the potential of systematic overexpression strategies to improve our knowledge of regulatory networks in C. albicans. The C. albicans plasmid and strain collections described here are available at the Fungal Genetics Stock Center. Their extension to a genome-wide scale will represent important resources for the C. albicans community.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045912PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457969PMC
May 2013

Modular gene over-expression strategies for Candida albicans.

Methods Mol Biol 2012 ;845:227-44

Département Génomes et Génétique, Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Paris, France.

Over-expression is a valid functional genomics approach to characterise genes of unknown function on a genome-wide scale. Strains are engineered to over-express a specific gene and the resulting gain-of-function phenotype assessed. Here, we describe the strategy we are adopting to synthesise a Candida albicans ORFeome collection and the options available to create over-expressing strains from this collection.
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http://dx.doi.org/10.1007/978-1-61779-539-8_15DOI Listing
June 2012

The contribution of the S-phase checkpoint genes MEC1 and SGS1 to genome stability maintenance in Candida albicans.

Fungal Genet Biol 2011 Aug 13;48(8):823-30. Epub 2011 Apr 13.

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis MN 55455, USA.

Genome rearrangements, a common feature of Candida albicans isolates, are often associated with the acquisition of antifungal drug resistance. In Saccharomyces cerevisiae, perturbations in the S-phase checkpoints result in the same sort of Gross Chromosomal Rearrangements (GCRs) observed in C. albicans. Several proteins are involved in the S. cerevisiae cell cycle checkpoints, including Mec1p, a protein kinase of the PIKK (phosphatidyl inositol 3-kinase-like kinase) family and the central player in the DNA damage checkpoint. Sgs1p, the ortholog of BLM, the Bloom's syndrome gene, is a RecQ-related DNA helicase; cells from BLM patients are characterized by an increase in genome instability. Yeast strains bearing deletions in MEC1 or SGS1 are viable (in contrast to the inviability seen with loss of MEC1 in S. cerevisiae) but the different deletion mutants have significantly different phenotypes. The mec1Δ/Δ colonies have a wild-type colony morphology, while the sgs1Δ/Δ mutants are slow-growing, producing wrinkled colonies with pseudohyphal-like cells. The mec1Δ/Δ mutants are only sensitive to ethylmethane sulfonate (EMS), methylmethane sulfonate (MMS), and hydroxyurea (HU) but the sgs1Δ/Δ mutants exhibit a high sensitivity to all DNA-damaging agents tested. In an assay for chromosome 1 integrity, the mec1Δ/Δ mutants exhibit an increase in genome instability; no change was observed in the sgs1Δ/Δ mutants. Finally, loss of MEC1 does not affect sensitivity to the antifungal drug fluconazole, while loss of SGS1 leads to an increased susceptibility to fluconazole. Neither deletion elevated the level of antifungal drug resistance acquisition.
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http://dx.doi.org/10.1016/j.fgb.2011.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126902PMC
August 2011

Analysis of base excision and nucleotide excision repair in Candida albicans.

Microbiology (Reading) 2008 Aug;154(Pt 8):2446-2456

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.

Candida albicans, clinically the most important human fungal pathogen, rapidly develops resistance to antifungal drugs. The acquisition of resistance has been linked to various types of genome changes. As part of an ongoing study of this problem, we investigated mutation, genome stability and drug resistance acquisition in C. albicans strains with deletions in the base excision repair (BER) genes NTG1, APN1 and OGG1, and in the nucleotide excision repair (NER) genes RAD2 and RAD10. The BER mutants did not exhibit any change in their susceptibility to DNA-damaging agents, but the NER mutants were extremely sensitive to UV-induced DNA damage. We did not observe any significant change in mutation, genome stability and antifungal drug sensitivity in the mutant strains we tested. However, we detected a number of intriguing phenotypic differences between strains bearing deletions in equivalent C. albicans and Saccharomyces cerevisiae BER and NER genes, which may be related to differences in the life cycles of these two fungi.
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http://dx.doi.org/10.1099/mic.0.2008/017616-0DOI Listing
August 2008

Haplotype mapping of a diploid non-meiotic organism using existing and induced aneuploidies.

PLoS Genet 2008 Jan 20;4(1):e1. Epub 2007 Nov 20.

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America.

Haplotype maps (HapMaps) reveal underlying sequence variation and facilitate the study of recombination and genetic diversity. In general, HapMaps are produced by analysis of Single-Nucleotide Polymorphism (SNP) segregation in large numbers of meiotic progeny. Candida albicans, the most common human fungal pathogen, is an obligate diploid that does not appear to undergo meiosis. Thus, standard methods for haplotype mapping cannot be used. We exploited naturally occurring aneuploid strains to determine the haplotypes of the eight chromosome pairs in the C. albicans laboratory strain SC5314 and in a clinical isolate. Comparison of the maps revealed that the clinical strain had undergone a significant amount of genome rearrangement, consisting primarily of crossover or gene conversion recombination events. SNP map haplotyping revealed that insertion and activation of the UAU1 cassette in essential and non-essential genes can result in whole chromosome aneuploidy. UAU1 is often used to construct homozygous deletions of targeted genes in C. albicans; the exact mechanism (trisomy followed by chromosome loss versus gene conversion) has not been determined. UAU1 insertion into the essential ORC1 gene resulted in a large proportion of trisomic strains, while gene conversion events predominated when UAU1 was inserted into the non-essential LRO1 gene. Therefore, induced aneuploidies can be used to generate HapMaps, which are essential for analyzing genome alterations and mitotic recombination events in this clonal organism.
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http://dx.doi.org/10.1371/journal.pgen.0040001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2174976PMC
January 2008

Role of DNA mismatch repair and double-strand break repair in genome stability and antifungal drug resistance in Candida albicans.

Eukaryot Cell 2007 Dec 26;6(12):2194-205. Epub 2007 Oct 26.

Department of Genetics, Cell Biology, and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455, USA.

Drug resistance has become a major problem in the treatment of Candida albicans infections. Genome changes, such as aneuploidy, translocations, loss of heterozygosity, or point mutations, are often observed in clinical isolates that have become resistant to antifungal drugs. To determine whether these types of alterations result when DNA repair pathways are eliminated, we constructed yeast strains bearing deletions in six genes involved in mismatch repair (MSH2 and PMS1) or double-strand break repair (MRE11, RAD50, RAD52, and YKU80). We show that the mre11Delta/mre11Delta, rad50Delta/rad50Delta, and rad52Delta/rad52Delta mutants are slow growing and exhibit a wrinkly colony phenotype and that cultures of these mutants contain abundant elongated pseudohypha-like cells. These same mutants are susceptible to hydrogen peroxide, tetrabutyl hydrogen peroxide, UV radiation, camptothecin, ethylmethane sulfonate, and methylmethane sulfonate. The msh2Delta/msh2Delta, pms1Delta/pms1Delta, and yku80Delta/yku80Delta mutants exhibit none of these phenotypes. We observed an increase in genome instability in mre11Delta/mre11Delta and rad50Delta/rad50Delta mutants by using a GAL1/URA3 marker system to monitor the integrity of chromosome 1. We investigated the acquisition of drug resistance in the DNA repair mutants and found that deletion of mre11Delta/mre11Delta, rad50Delta/rad50Delta, or rad52Delta/rad52Delta leads to an increased susceptibility to fluconazole. Interestingly, we also observed an elevated frequency of appearance of drug-resistant colonies for both msh2Delta/msh2Delta and pms1Delta/pms1Delta (MMR mutants) and rad50Delta/rad50Delta (DSBR mutant). Our data demonstrate that defects in double-strand break repair lead to an increase in genome instability, while drug resistance arises more rapidly in C. albicans strains lacking mismatch repair proteins or proteins central to double-strand break repair.
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http://dx.doi.org/10.1128/EC.00299-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168241PMC
December 2007

Homozygosity at the MTL locus in clinical strains of Candida albicans: karyotypic rearrangements and tetraploid formation.

Mol Microbiol 2004 Jun;52(5):1451-62

Department of Genetics, Cell Biology, and Development, University of Minnesota, 321 Church St. SE, Minneapolis, MN 55455, USA.

One hundred and twenty Candida albicans clinical isolates from the late 1980s and early 1990s were examined for homozygosity at the MTL locus. Of these, 108 were heterozygous (MTLa/MTLalpha), whereas seven were MTLa and five were MTLalpha. Five of the homozygous isolates were able to switch to the opaque cell morphology, while opaque cells were not detectable among the remaining seven. Nevertheless, all but one of the isolates homozygous at the MTL locus were shown to mate and to yield cells containing markers from both parents; the non-mater was found to have a frameshift in the MTLalpha1 gene. In contrast to Saccharomyces cerevisiae, C. albicans homozygotes with no active MTL allele failed to mate rather than mating as a cells. There was no correlation between homozygosity and fluconazole resistance, mating and fluconazole resistance or switching and fluconazole resistance, in part because most of the strains were isolated before the widespread use of this antifungal agent, and only three were in fact drug resistant. Ten of the 12 homozygotes had rearranged karyotypes involving one or more homologue of chromosomes 4, 5, 6 and 7. We suggest that karyotypic rearrangement, drug resistance and homozygosity come about as the result of induction of hyper-recombination during the infection process; hence, they tend to occur together, but each is the independent result of the same event. Furthermore, as clinical strains can mate and form tetraploids, mating and marker exchange are likely to be a significant part of the life cycle of C. albicans in vivo.
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http://dx.doi.org/10.1111/j.1365-2958.2004.04068.xDOI Listing
June 2004

MFalpha1, the gene encoding the alpha mating pheromone of Candida albicans.

Eukaryot Cell 2003 Dec;2(6):1350-60

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455, USA.

Candida albicans, the single most frequently isolated human fungal pathogen, was thought to be asexual until the recent discovery of the mating-type-like locus (MTL). Homozygous MTL strains were constructed and shown to mate. Furthermore, it has been demonstrated that opaque-phase cells are more efficient in mating than white-phase cells. The similarity of the genes involved in the mating pathway in Saccharomyces cerevisiae and C. albicans includes at least one gene (KEX2) that is involved in the processing of the alpha mating pheromone in the two yeasts. Taking into account this similarity, we searched the C. albicans genome for sequences that would encode the alpha pheromone gene. Here we report the isolation and characterization of the gene MFalpha1, which codes for the precursor of the alpha mating pheromone in C. albicans. Two active alpha-peptides, 13 and 14 amino acids long, would be generated after the precursor molecule is processed in C. albicans. To examine the role of this gene in mating, we constructed an mfalpha1 null mutant of C. albicans. The mfalpha1 null mutant fails to mate as MTLalpha, while MTLa mfalpha1 cells are still mating competent. Experiments performed with the synthetic alpha-peptides show that they are capable of inducing growth arrest, as demonstrated by halo tests, and also induce shmooing in MTLa cells of C. albicans. These peptides are also able to complement the mating defect of an MTLalpha kex2 mutant strain when added exogenously, thereby confirming their roles as alpha mating pheromones.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC326654PMC
http://dx.doi.org/10.1128/EC.2.6.1350-1360.2003DOI Listing
December 2003

Many of the genes required for mating in Saccharomyces cerevisiae are also required for mating in Candida albicans.

Mol Microbiol 2002 Dec;46(5):1345-51

Department of Genetics, Cell Biology, and Development, University of Minnesota, St. Paul, MN 55108, USA.

Candida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type (MAT) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae. We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTLa and MTL(alpha) strains, whereas disruptions in STE20 had no effect. A disruption in KEX2, a gene involved in processing the S. cerevisiae pheromone Mf(alpha), prevented mating in MTL(alpha) but not MTLa cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTLa but not in MTL(alpha) cells. Disruption of two cell wall genes, ALS1 and INT1, had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1. The results reveal that these two diverged yeasts show a surprising similarity in their mating processes.
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http://dx.doi.org/10.1046/j.1365-2958.2002.03263.xDOI Listing
December 2002