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1. A gain-of-function mutation in zinc cluster transcription factor Rob1 drives Candida albicans adaptive growth in the cystic fibrosis lung environment.

Author

Mayssa Gnaien, Corinne Maufrais, Yasmine Rebai, Aicha Kallel, Laurence Ma, Samia Hamouda, Fatma Khalsi, Khaoula Meftah, Hanen Smaoui, Monia Khemiri, Sondes Hadj Fredj, Sophie Bachellier-Bassi, Imène Najjar, Taieb Messaoud, Khadija Boussetta, Kalthoum Kallel, Helmi Mardassi, Christophe d'Enfert, Marie-Elisabeth Bougnoux, and Sadri Znaidi

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

Candida albicans chronically colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens (e.g. Pseudomonas aeruginosa) and contributes to disease severity. We hypothesize that C. albicans undergoes specific adaptation mechanisms that explain its persistence in the CF lung environment. To identify the underlying genetic and phenotypic determinants, we serially recovered 146 C. albicans clinical isolates over a period of 30 months from the sputum of 25 antifungal-naive CF patients. Multilocus sequence typing analyses revealed that most patients were individually colonized with genetically close strains, facilitating comparative analyses between serial isolates. We strikingly observed differential ability to filament and form monospecies and dual-species biofilms with P. aeruginosa among 18 serial isolates sharing the same diploid sequence type, recovered within one year from a pediatric patient. Whole genome sequencing revealed that their genomes were highly heterozygous and similar to each other, displaying a highly clonal subpopulation structure. Data mining identified 34 non-synonymous heterozygous SNPs in 19 open reading frames differentiating the hyperfilamentous and strong biofilm-former strains from the remaining isolates. Among these, we detected a glycine-to-glutamate substitution at position 299 (G299E) in the deduced amino acid sequence of the zinc cluster transcription factor ROB1 (ROB1G299E), encoding a major regulator of filamentous growth and biofilm formation. Introduction of the G299E heterozygous mutation in a co-isolated weak biofilm-former CF strain was sufficient to confer hyperfilamentous growth, increased expression of hyphal-specific genes, increased monospecies biofilm formation and increased survival in dual-species biofilms formed with P. aeruginosa, indicating that ROB1G299E is a gain-of-function mutation. Disruption of ROB1 in a hyperfilamentous isolate carrying the ROB1G299E allele abolished hyperfilamentation and biofilm formation. Our study links a single heterozygous mutation to the ability of C. albicans to better survive during the interaction with other CF-associated microbes and illuminates how adaptive traits emerge in microbial pathogens to persistently colonize and/or infect the CF-patient airways.

Published
2024
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2. Inflammatory Bowel Disease Increases the Severity of Myocardial Infarction after Acute Ischemia–Reperfusion Injury in Mice

Author

Wael Mami, Soumaya Znaidi-Marzouki, Raoudha Doghri, Melika Ben Ahmed, Sadri Znaidi, and Erij Messadi

Subjects
myocardial infarction, ischemia–reperfusion injury, inflammatory bowel disease, cytokines, animal model, translational medicine, Biology (General), QH301-705.5
Abstract

(1) Background: Increased risk of myocardial infarction (MI) has been linked to several inflammatory conditions, including inflammatory bowel disease (IBD). However, the relationship between IBD and MI remains unclear. Here, we implemented an original mouse model combining IBD and MI to determine IBD’s impact on MI severity and the link between the two diseases. (2) Methods: An IBD model was established by dextran sulfate sodium (DSS) administration in drinking water, alone or with oral C. albicans (Ca) gavage. IBD severity was assessed by clinical/histological scores and intestinal/systemic inflammatory biomarker measurement. Mice were subjected to myocardial ischemia–reperfusion (IR), and MI severity was assessed by quantifying infarct size (IS) and serum cardiac troponin I (cTnI) levels. (3) Results: IBD mice exhibited elevated fecal lipocalin 2 (Lcn2) and IL-6 levels. DSS mice exhibited almost two-fold increase in IS compared to controls, with serum cTnI levels strongly correlated with IS. Ca inoculation tended to worsen DSS-induced systemic inflammation and IR injury, an observation which is not statistically significant. (4) Conclusions: This is the first proof-of-concept study demonstrating the impact of IBD on MI severity and suggesting mechanistic aspects involved in the IBD–MI connection. Our findings could pave the way for MI therapeutic approaches based on identified IBD-induced inflammatory mediators.

Published
2023
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3. Escherichia coli Nissle 1917 Antagonizes Candida albicans Growth and Protects Intestinal Cells from C. albicans-Mediated Damage

Author

Yasmine Rebai, Lysett Wagner, Mayssa Gnaien, Merle L. Hammer, Mario Kapitan, Maria Joanna Niemiec, Wael Mami, Amor Mosbah, Erij Messadi, Helmi Mardassi, Slavena Vylkova, Ilse D. Jacobsen, and Sadri Znaidi

Subjects
antifungal, Escherichia coli, Nissle 1917, probiotics, Candida albicans, polymicrobial interactions, Biology (General), QH301-705.5
Abstract

Candida albicans is a pathobiont of the gastrointestinal tract. It can contribute to the diversity of the gut microbiome without causing harmful effects. When the immune system is compromised, C. albicans can damage intestinal cells and cause invasive disease. We hypothesize that a therapeutic approach against C. albicans infections can rely on the antimicrobial properties of probiotic bacteria. We investigated the impact of the probiotic strain Escherichia coli Nissle 1917 (EcN) on C. albicans growth and its ability to cause damage to intestinal cells. In co-culture kinetic assays, C. albicans abundance gradually decreased over time compared with C. albicans abundance in the absence of EcN. Quantification of C. albicans survival suggests that EcN exerts a fungicidal activity. Cell-free supernatants (CFS) collected from C. albicans-EcN co-culture mildly altered C. albicans growth, suggesting the involvement of an EcN-released compound. Using a model of co-culture in the presence of human intestinal epithelial cells, we further show that EcN prevents C. albicans from damaging enterocytes both distantly and through direct contact. Consistently, both C. albicans’s filamentous growth and microcolony formation were altered by EcN. Taken together, our study proposes that probiotic-strain EcN can be exploited for future therapeutic approaches against C. albicans infections.

Published
2023
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4. Functional Portrait of Irf1 (Orf19.217), a Regulator of Morphogenesis and Iron Homeostasis in Candida albicans

Author

Lasse van Wijlick, Sadri Znaidi, Arturo Hernández-Cervantes, Virginia Basso, Sophie Bachellier-Bassi, and Christophe d’Enfert

Subjects
Candida albicans, iron homeostasis, trace metals, RNA-Seq, ChIP-Seq, Orf19.217/Irf1, Microbiology, QR1-502
Abstract

The alternate growth of Candida albicans between a unicellular yeast form and a multicellular hyphal form is crucial for its ability to cause disease. Interestingly, both morphological forms support distinct functions during proliferation in the human host. We previously identified ORF19.217 (C2_08890W_A), encoding a zinc-finger transcription factor of the C2H2 family, in a systematic screen of genes whose overexpression contributes to C. albicans’ morphological changes. Conditional overexpression of ORF19.217 with the strong tetracycline-inducible promoter (PTET) resulted in a hyperfilamentous phenotype. We examined growth of the orf19.217 knockout-mutant in different hypha-inducing conditions and found that the mutant still formed hyphae under standard hypha-inducing conditions. To further investigate the function of Orf19.217 in C. albicans, we combined genome-wide expression (RNA-Seq) and location (ChIP-Seq) analyses. We found that Orf19.217 is involved in regulatory processes comprising hyphal morphogenesis and iron acquisition. Comparative analysis with existing C. albicans hyphal transcriptomes indicates that Orf19.217-mediated filamentation is distinct from a true hyphal program. Further, the orf19.217 knockout-mutant did not show increased sensitivity to iron deprivation, but ORF19.217 overexpression was able to rescue the growth of a hap5-mutant, defective in a subunit of the CCAAT-complex, which is essential for iron acquisition. This suggested that Orf19.217 is involved in regulation of iron acquisition genes during iron deprivation and acts in a parallel pathway to the established CCAAT-complex. Interestingly, the orf19.217-mutant turned out to be defective in its ability to form filaments under iron-deficiency. Taken together our findings propose that the transcription factor Orf19.217 stimulates expression of the hyphal regulators EFG1 and BRG1 to promote filamentous growth under iron deprivation conditions, allowing the fungus to escape these iron-depleted conditions. The transcription factor therefore appears to be particularly important for adaptation of C. albicans to diverse environmental conditions in the human host. In regard to the newly identified functions, we have given the regulator the name Irf1, Iron-dependent Regulator of Filamentation.

Published
2022
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5. A conserved regulator controls asexual sporulation in the fungal pathogen Candida albicans

Author

Arturo Hernández-Cervantes, Sadri Znaidi, Lasse van Wijlick, Iryna Denega, Virginia Basso, Jeanne Ropars, Natacha Sertour, Derek Sullivan, Gary Moran, Louise Basmaciyan, Fabienne Bon, Frédéric Dalle, Marie-Elisabeth Bougnoux, Teun Boekhout, Ying Yang, Zongwei Li, Sophie Bachellier-Bassi, and Christophe d’Enfert

Subjects
Science
Abstract

Transcription factor Rme1 regulates meiosis and pseudohyphal growth in baker’s yeast, but its role in the meiosis-defective pathogen Candida albicans is unclear. Here, Hernández-Cervantes et al. show that Rme1 activates the expression of genes required for formation of asexual spores in Candida species.

Published
2020
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6. Identification and Characterization of Mediators of Fluconazole Tolerance in Candida albicans

Author

Eric Delarze, Ludivine Brandt, Emilie Trachsel, Marion Patxot, Claire Pralong, Fabio Maranzano, Murielle Chauvel, Mélanie Legrand, Sadri Znaidi, Marie-Elisabeth Bougnoux, Christophe d’Enfert, and Dominique Sanglard

Subjects
Candida, fluconazole, drug resistance, drug tolerance, calcineurin, Microbiology, QR1-502
Abstract

Candida albicans is an important human pathogen and a major concern in intensive care units around the world. C. albicans 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 C. albicans 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 C. albicans 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 C. albicans by an overexpression strategy focusing on 572 C. albicans genes. This strategy led to the identification of two transcription factors, CRZ1 and GZF3. CRZ1 is a C2H2-type transcription factor that is part of the calcineurin-dependent pathway in C. albicans, while GZF3 is a GATA-type transcription factor of unknown function in C. albicans. Overexpression of each gene resulted in an increase of FLC tolerance, however, only the deletion of CRZ1 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.

Published
2020
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7. mSphere of Influence: Decoding Transcriptional Regulatory Networks To Illuminate the Mechanisms of Microbial Pathogenicity

Author

Sadri Znaidi

Subjects
Candida albicans, genomics, host-pathogen interactions, iron metabolism, transcription factors, Microbiology, QR1-502
Abstract

ABSTRACT Sadri Znaidi works in the field of molecular mycology with a focus on functional genomics in Candida albicans. In this mSphere of Influence article, he reflects on how the paper “An iron homeostasis regulatory circuit with reciprocal roles in Candida albicans commensalism and pathogenesis” by Chen et al. (C. Chen, K. Pande, S. D. French, B. B. Tuch, and S. M. Noble, Cell Host Microbe 10:118–135, 2011, https://doi.org/10.1016/j.chom.2011.07.005) made an impact on his research on how transcriptional regulatory networks function to control C. albicans’ ability to efficiently interact with the host environment.

Published
2020
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8. A conserved fungal hub protein involved in adhesion and drug resistance in the human pathogen Candida albicans

Author

Hélène Martin-Yken, Tina Bedekovic, Alexandra C. Brand, Mathias L. Richard, Sadri Znaidi, Christophe d'Enfert, and Etienne Dague

Subjects
Cytology, QH573-671
Abstract

Drug resistance and cellular adhesion are two key elements of both dissemination and prevalence of the human fungal pathogen Candida albicans. Smi1 belongs to a family of hub proteins conserved among the fungal kingdom whose functions in cellular signaling affect morphogenesis, cell wall synthesis and stress resistance. The data presented here indicate that C. albicans SMI1 is a functional homolog of Saccharomyces cerevisiae KNR4 and is involved in the regulation of cell wall synthesis. Expression of SMI1 in S. cerevisiae knr4Δ null mutants rescued their sensitivity to caspofungin and to heat stress. Deletion of SMI1 in C. albicans resulted in sensitivity to the cell-wall-perturbing compounds Calcofluor White and Caspofungin. Analysis of wild-type and mutant cells by Atomic Force Microscopy showed that the Young’s Modulus (stiffness) of the cell wall was reduced by 85% upon deletion of SMI1, while cell surface adhesion measured by Force Spectroscopy showed that the surface expression of adhesive molecules was also reduced in the mutant. Over-expression of SMI1, on the contrary, increased cell surface adhesion by 6-fold vs the control strain. Finally, Smi1-GFP localized as cytoplasmic patches and concentrated spots at the sites of new cell wall synthesis including the tips of growing hyphae, consistent with a role in cell wall regulation. Thus, Smi1 function appears to be conserved across fungi, including the yeast S. cerevisiae, the yeast and hyphal forms of C. albicans and the filamentous fungus Neurospora crassa. Keywords: Fungal cell wall, Adhesion, Caspofungin, Candida albicans, Atomic Force Microscopy

Published
2018
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9. High Frequency of Enterocytozoon bieneusi Genotype WL12 Occurrence among Immunocompromised Patients with Intestinal Microsporidiosis

Author

Mariem Messaoud, Salma Abbes, Mayssa Gnaien, Yasmine Rebai, Aicha Kallel, Sana Jemel, Ghaya Cherif, Mohamed Amine Skhairia, Sonia Marouen, Najla Fakhfekh, Helmi Mardassi, Slaheddine Belhadj, Sadri Znaidi, and Kalthoum Kallel

Subjects
Enterocytozoon bieneusi, Encephalitozoon spp, immunocompromised patients, HIV, emerging infectious diseases, genotype WL12, Biology (General), QH301-705.5
Abstract

Microsporidiosis is an emerging opportunistic infection causing severe digestive disorders in immunocompromised patients. The aim of this study was to investigate the prevalence of intestinal microsporidia carriage among immunocompromised patients hospitalized at a major hospital complex in the Tunis capital area, Tunisia (North Africa), and perform molecular epidemiology and population structure analyses of Enterocytozoon bieneusi, which is an emerging fungal pathogen. We screened 250 stool samples for the presence of intestinal microsporidia from 171 patients, including 81 organ transplant recipients, 73 Human Immunodeficiency Virus (HIV)-positive patients, and 17 patients with unspecified immunodeficiency. Using a nested PCR-based diagnostic approach for the detection of E. bieneusi and Encephalitozoon spp., we identified 18 microsporidia-positive patients out of 171 (10.5%), among which 17 were infected with E. bieneusi. Microsporidia-positive cases displayed chronic diarrhea (17 out of 18), which was associated more with HIV rather than with immunosuppression other than HIV (12 out of 73 versus 6 out of 98, respectively, p = 0.02) and correlated with extended hospital stays compared to microsporidia-negative cases (60 versus 19 days on average, respectively; p = 0.001). Strikingly, internal transcribed spacer (ITS)-based genotyping of E. bieneusi strains revealed high-frequency occurrence of ITS sequences that were identical (n = 10) or similar (with one single polymorphic site, n = 3) to rare genotype WL12. Minimum-spanning tree analyses segregated the 17 E. bieneusi infection cases into four distinct genotypic clusters and confirmed the high prevalence of genotype WL12 in our patient population. Phylogenetic analyses allowed the mapping of all 17 E. bieneusi strains to zoonotic group 1 (subgroups 1a and 1b/1c), indicating loose host specificity and raising public health concern. Our study suggests a probable common source of E. bieneusi genotype WL12 transmission and prompts the implementation of a wider epidemiological investigation.

Published
2021
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10. Mechanisms Underlying the Delayed Activation of the Cap1 Transcription Factor in Candida albicans following Combinatorial Oxidative and Cationic Stress Important for Phagocytic Potency

Author

Iaroslava Kos, Miranda J. Patterson, Sadri Znaidi, Despoina Kaloriti, Alessandra da Silva Dantas, Carmen M. Herrero-de-Dios, Christophe d’Enfert, Alistair J. P. Brown, and Janet Quinn

Subjects
Microbiology, QR1-502
Abstract

ABSTRACT Following phagocytosis, microbes are exposed to an array of antimicrobial weapons that include reactive oxygen species (ROS) and cationic fluxes. This is significant as combinations of oxidative and cationic stresses are much more potent than the corresponding single stresses, triggering the synergistic killing of the fungal pathogen Candida albicans by “stress pathway interference.” Previously we demonstrated that combinatorial oxidative plus cationic stress triggers a dramatic increase in intracellular ROS levels compared to oxidative stress alone. Here we show that activation of Cap1, the major regulator of antioxidant gene expression in C. albicans, is significantly delayed in response to combinatorial stress treatments and to high levels of H2O2. Cap1 is normally oxidized in response to H2O2; this masks the nuclear export sequence, resulting in the rapid nuclear accumulation of Cap1 and the induction of Cap1-dependent genes. Here we demonstrate that following exposure of cells to combinatorial stress or to high levels of H2O2, Cap1 becomes trapped in a partially oxidized form, Cap1OX-1. Notably, Cap1-dependent gene expression is not induced when Cap1 is in this partially oxidized form. However, while Cap1OX-1 readily accumulates in the nucleus and binds to target genes following high-H2O2 stress, the nuclear accumulation of Cap1OX-1 following combinatorial H2O2 and NaCl stress is delayed due to a cationic stress-enhanced interaction with the Crm1 nuclear export factor. These findings define novel mechanisms that delay activation of the Cap1 transcription factor, thus preventing the rapid activation of the stress responses vital for the survival of C. albicans within the host. IMPORTANCE Combinatorial stress-mediated synergistic killing represents a new unchartered area in the field of stress signaling. This phenomenon contrasts starkly with “stress cross-protection,” where exposure to one stress protects against subsequent exposure to a different stress. Previously we demonstrated that the pathogen Candida albicans is acutely sensitive to combinations of cationic and oxidative stresses, because the induction of H2O2-responsive genes is blocked in the presence of cationic stress. We reveal that this is due to novel mechanisms that delay activation of the Cap1 AP-1-like transcription factor, the major regulator of the H2O2-induced regulon. Cap1 becomes trapped in a partially oxidized form following simultaneous exposure to oxidative and cationic stresses. In addition, cationic stress promotes the interaction of Cap1 with the Crm1 nuclear export factor, thus inhibiting its nuclear accumulation. These mechanisms probably explain the potency of neutrophils, which employ multiple stresses to kill fungal pathogens.

Published
2016
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11. Targeted changes of the cell wall proteome influence Candida albicans ability to form single- and multi-strain biofilms.

Author

Vitor Cabral, Sadri Znaidi, Louise A Walker, Hélène Martin-Yken, Etienne Dague, Mélanie Legrand, Keunsook Lee, Murielle Chauvel, Arnaud Firon, Tristan Rossignol, Mathias L Richard, Carol A Munro, Sophie Bachellier-Bassi, and Christophe d'Enfert

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

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.

Published
2014
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12. High-throughput functional profiling of the human fungal pathogen Candida albicans genome

Author

Murielle Chauvel, Sophie Bachellier-Bassi, Anne-Marie Guérout, Keunsook K. Lee, Corinne Maufrais, Emmanuelle Permal, Juliana Pipoli Da Fonseca, Sadri Znaidi, Didier Mazel, Carol A. Munro, Christophe d’Enfert, Melanie Legrand, Biologie et Pathogénicité fongiques - Fungal Biology and Pathogenicity (BPF), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Plasticité du Génome Bactérien - Bacterial Genome Plasticity (PGB), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK., NGeneBio [Seoul, Corée du sud], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Biomics (plateforme technologique), Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP), This work was financially supported by a Biomedical Resources grant from the Wellcome Trust (The Candida albicans ORFeome project, 088858/Z/09/Z to CAM and CD), the European Commission (FINSysB, PITN-GA-2008-214004 to CD), and the Agence Nationale de la Recherche (KANJI, ANR-08-MIE-033-01 to CD, CANDICOL, ANR-10-01 to CD). CAM would like to acknowledge support from a Royal Society Leverhulme Trust Senior Research Fellowship (SRF∖R1∖180115). We acknowledge support from the French Government’s Investissement d’Avenir program (Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases, ANR-10-LABX-62-IBEID)., ANR-08-MIEN-0033,KANJI,Colonisation et invasion de la muqueuse digestive par le champignon pathogène de l'homme Candida albicans(2008), ANR-10-PATH-0008,CANDICOL(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), and European Project: 214004,PEOPLE,FP7-PEOPLE-2007-1-1-ITN,FINSYSB(2008)

Subjects
[SDV]Life Sciences [q-bio], Candida albicans, overexpression mutants, ORFeome, General Medicine, Large-scale systematic studies, Bar-seq, Molecular Biology, Microbiology, Candida albicans ORFeome Overexpression mutants Bar-seq Large-scale systematic studies, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology
Abstract

International audience; Candida albicans is a major fungal pathogen of humans. Although its genome has been sequenced more than two decades ago, there are still over 4300 uncharacterized C. albicans genes. We previously generated an ORFeome as well as a collection of destination vectors to facilitate overexpression of C. albicans ORFs. Here, we report the construction of ~2500 overexpression mutants and their evaluation by in vitro spotting on rich medium and in a liquid pool experiment in rich medium, allowing the identification of genes whose overexpression has a fitness cost. The candidates were further validated at the individual strain level. This new resource allows large-scale screens in different growth conditions to be performed routinely. Altogether, based on the concept of identifying functionally related genes by cluster analysis, the availability of this overexpression mutant collection will facilitate the character- ization of gene functions in C. albicans.

Published
2023
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13. Genotypic and phenotypic portrait of Candida albicans clinical isolates colonizing the airways of patients with cystic fibrosis

Author

Mayssa Gnaien, Aicha Kallel, Fatma Khalsi, Samia Hamouda, Hanen Smaoui, Monia Khemiri, Sondes Hadj Fredj, Taieb Messaoud, Khadija Boussetta, Kalthoum Kallel, Christophe d'Enfert, Marie-Elisabeth Bougnoux, Helmi Mardassi, and Sadri Znaidi

Subjects
General Materials Science
Abstract

Candida albicans colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, and contributes to disease severity. We serially recovered 160 C. albicans clinical isolates over a period of 30 months from the sputum of 23 pediatric and 2 adult antifungal-naive CF patients at Children’s Hospital Tunis and characterized the genotype and phenotype of a subset of strains using multilocus sequence typing (MLST) and growth assays on multiple stress-, filamentous growth- and biofilm-inducing media. Out of 16 patients regularly sampled for at least 9 months, 8 and 4 were chronically and transiently colonized with C. albicans, respectively. MLST analyses of 56 strains originating from 15 patients indicated that each patient was colonized with a single strain, while 8 patients (53%) carried isolates from clade 4 known to be enriched with strains from Middle East-Africa. A subset of these isolates with the same sequence type and colonizing 3 unrelated patients displayed altered susceptibility to cell wall-perturbing agents, suggesting changes in cell wall structure/function during growth in the CF lung. We also observed differential ability to filament and/or form biofilms in a set of identical isolates from clade 10 sampled over a period of 9 months in a pediatric CF patient, suggesting alterations in phenotypes associated with virulence. Our findings will rely on future whole-genome sequencing analyses to identify polymorphisms that could explain the emergence of new traits in C. albicans strains thriving in the CF host environment.

Published
2021
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14. Ethylzingerone, a Novel Compound with Antifungal Activity

Author

Rosemary Ann Barnes, Laurence Decourty, Sylvie Cupferman, Florence Menard-Szczebara, Christophe d'Enfert, Cosmin Saveanu, Maria Dalko-Sciba, Jean-Yves Maillard, Sadri Znaidi, Rebecca Wesgate, Murielle Chauvel, Tristan Rossignol, Biologie et Pathogénicité fongiques, Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), Laboratoire de Microbiologie Moléculaire, Vaccinologie et Développement Biotechnologique (LR11IPT01), Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Cardiff University, Génétique des Interactions macromoléculaires, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), L’Oréal Research and Innovation, University Hospital of Wales [Cardiff, UK], Cardiff University (Cardiff University), L'Oreal Group, L'Oreal, France, ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-08-JCJC-0019,GENO-GIM,Obtention d'une carte d'interactions génétiques à l'échelle génomique chez la levure(2008), Biologie et Pathogénicité fongiques (BPF), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP), Génétique des Interactions macromoléculaires / Genetics of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and University Hospital of Wales (UHW)

Subjects
Antifungal Agents, [SDV]Life Sciences [q-bio], Saccharomyces cerevisiae, HEPB, Microbiology, ethylzingerone, 03 medical and health sciences, chemistry.chemical_compound, hydroxyethoxyphenyl butanone, Candida albicans, Aromatic amino acids, Pharmacology (medical), Mechanisms of Action: Physiological Effects, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, Amino acid synthesis, 030304 developmental biology, Pharmacology, chemistry.chemical_classification, 0303 health sciences, Candida glabrata, biology, cosmetics, 030306 microbiology, Antimicrobial, biology.organism_classification, Corpus albicans, 3. Good health, Triclosan, Infectious Diseases, chemistry, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, antifungal, mechanism of action
Abstract

International audience; Preservatives increase the shelf life of cosmetic products by preventing growth of contaminating microbes, including bacteria and fungi. In recent years, the Scientific Committee on Consumer Safety (SCCS) has recommended the ban or restricted use of a number of preservatives due to safety concerns. Here, we characterize the antifungal activity of ethylzingerone (hydroxyethoxyphenyl butanone [HEPB]), an SCCS-approved new preservative for use in rinse-off, oral care, and leave-on cosmetic products. We show that HEPB significantly inhibits growth of Candida albicans, Candida giabrata, and Saccharomyces cerevisiae, acting fungicidally against C. albicans. Using transcript profiling experiments, we found that the C. albicans transcriptome responded to HEPB exposure by increasing the expression of genes involved in amino acid biosynthesis while activating pathways involved in chemical detoxification/oxidative stress response. Comparative analyses revealed that C. albicans phenotypic and transcriptomic responses to HEPB treatment were distinguishable from those of two widely used preservatives, triclosan and methylparaben. Chemogenomic analyses, using a barcoded S. cerevisiae nonessential mutant library, revealed that HEPB antifungal activity strongly interfered with the biosynthesis of aromatic amino acids. The trpl LI mutants in S. cerevisiae and C. albicans were particularly sensitive to HEPB treatment, a phenotype rescued by exogenous addition of tryptophan to the growth medium, providing a direct link between HEPB mode of action and tryptophan availability. Collectively, our study sheds light on the antifungal activity of HEPB, a new molecule with safe properties for use as a preservative in the cosmetic industry, and exemplifies the powerful use of functional genomics to illuminate the mode of action of antimicrobial agents.

Published
2021
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15. High Frequency of Enterocytozoon bieneusi Genotype WL12 Occurrence among Immunocompromised Patients with Intestinal Microsporidiosis

Author

Ghaya Cherif, A. Kallel, Mariem Messaoud, Slaheddine Belhadj, Najla Fakhfekh, Helmi Mardassi, Mayssa Gnaien, Sana Jemel, Sonia Marouen, Sadri Znaidi, Kalthoum Kallel, Mohamed Amine Skhairia, Yasmine Rebai, Salma Abbes, Hôpital La Rabta [Tunis], Laboratoire de Microbiologie Moléculaire, Vaccinologie et Développement Biotechnologique (LR11IPT01), Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Université de Tunis El Manar (UTM), Biologie et Pathogénicité fongiques - Fungal Biology and Pathogenicity (BPF), Institut Pasteur [Paris] (IP)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Financial support to K.K. (Research Unit # UR17SP03) was obtained from the Tunisian Ministry of Health and the Ministry of Higher Education & Scientific Research. S.Z. is an Institut Pasteur International Network Affiliate Program Fellow. S.Z. was supported by grants/funds from the Laboratoire de Microbiologie Moléculaire, Vaccinologie et Développement Biotechnologique (LR16IPT01, Institut Pasteur de Tunis, Tunis-Belvédère, Tunisia), the Unité Biologie & Pathogénicité Fongiques (Institut Pasteur, Paris, France), the Institut Pasteur de Tunis Programmes Collaboratifs Internes (Grants # PCI22 and PCI32), the Ministry of Higher Education and Scientific Research (Young Investigator Grant Award # 19PEJC06-02) and the Institut Pasteur Division of International Affairs, Paris, France (Institut Pasteur International Network Affiliate Program Fellowship).

Subjects
Microbiology (medical), Encephalitozoon spp, Enterocytozoon bieneusi, Opportunistic infection, [SDV]Life Sciences [q-bio], Plant Science, Microsporidiosis, Article, emerging infectious diseases, 03 medical and health sciences, fluids and secretions, Genotype, molecular diagnosis, parasitic diseases, medicine, Genotyping, lcsh:QH301-705.5, Ecology, Evolution, Behavior and Systematics, Immunodeficiency, 030304 developmental biology, 0303 health sciences, biology, Molecular epidemiology, 030306 microbiology, Transmission (medicine), fungi, virus diseases, HIV, biology.organism_classification, medicine.disease, genotype WL12, Virology, lcsh:Biology (General), immunocompromised patients
Abstract

Microsporidiosis is an emerging opportunistic infection causing severe digestive disorders in immunocompromised patients. The aim of this study was to investigate the prevalence of intestinal microsporidia carriage among immunocompromised patients hospitalized at a major hospital complex in the Tunis capital area, Tunisia (North Africa), and perform molecular epidemiology and population structure analyses of Enterocytozoon bieneusi, which is an emerging fungal pathogen. We screened 250 stool samples for the presence of intestinal microsporidia from 171 patients, including 81 organ transplant recipients, 73 Human Immunodeficiency Virus (HIV)-positive patients, and 17 patients with unspecified immunodeficiency. Using a nested PCR-based diagnostic approach for the detection of E. bieneusi and Encephalitozoon spp., we identified 18 microsporidia-positive patients out of 171 (10.5%), among which 17 were infected with E. bieneusi. Microsporidia-positive cases displayed chronic diarrhea (17 out of 18), which was associated more with HIV rather than with immunosuppression other than HIV (12 out of 73 versus 6 out of 98, respectively, p = 0.02) and correlated with extended hospital stays compared to microsporidia-negative cases (60 versus 19 days on average, respectively, p = 0.001). Strikingly, internal transcribed spacer (ITS)-based genotyping of E. bieneusi strains revealed high-frequency occurrence of ITS sequences that were identical (n = 10) or similar (with one single polymorphic site, n = 3) to rare genotype WL12. Minimum-spanning tree analyses segregated the 17 E. bieneusi infection cases into four distinct genotypic clusters and confirmed the high prevalence of genotype WL12 in our patient population. Phylogenetic analyses allowed the mapping of all 17 E. bieneusi strains to zoonotic group 1 (subgroups 1a and 1b/1c), indicating loose host specificity and raising public health concern. Our study suggests a probable common source of E. bieneusi genotype WL12 transmission and prompts the implementation of a wider epidemiological investigation.

Published
2021
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17. Pathway Maps of Orphan and Complex Diseases Using an Integrative Computational Approach

Author

Sadri Znaidi, Houcemeddine Othman, Soumaya Kouidhi, Kais Ghedira, Sghaier Haïtham, Sonia Kechaou, Yosr Hamdi, and Imen Rabhi

Subjects
Article Subject, Apoptosis pathways, Gene regulatory network, Apoptosis, Computational biology, Disease, Orphan diseases, General Biochemistry, Genetics and Molecular Biology, Rare Diseases, Neoplasms, Databases, Genetic, Medicine, Animals, Humans, Computer Simulation, Gene Regulatory Networks, Protein Interaction Maps, General Immunology and Microbiology, business.industry, Computational Biology, General Medicine, B cell receptor signaling, Patient diagnosis, Mutation, High incidence, P53 signaling, business, Algorithms, Research Article, Signal Transduction
Abstract

Orphan diseases (ODs) are progressive genetic disorders, which affect a small number of people. The principal fundamental aspects related to these diseases include insufficient knowledge of mechanisms involved in the physiopathology necessary to access correct diagnosis and to develop appropriate healthcare. Unlike ODs, complex diseases (CDs) have been widely studied due to their high incidence and prevalence allowing to understand the underlying mechanisms controlling their physiopathology. Few studies have focused on the relationship between ODs and CDs to identify potential shared pathways and related molecular mechanisms which would allow improving disease diagnosis, prognosis, and treatment. We have performed a computational approach to studying CDs and ODs relationships through (1) connecting diseases to genes based on genes-diseases associations from public databases, (2) connecting ODs and CDs through binary associations based on common associated genes, and (3) linking ODs and CDs to common enriched pathways. Among the most shared significant pathways between ODs and CDs, we found pathways in cancer, p53 signaling, mismatch repair, mTOR signaling, B cell receptor signaling, and apoptosis pathways. Our findings represent a reliable resource that will contribute to identify the relationships between drugs and disease-pathway networks, enabling to optimise patient diagnosis and disease treatment.

Published
2020

18. Identification and Characterization of Mediators of Fluconazole Tolerance in Candida albicans

Author

Fabio Maranzano, Claire Pralong, Dominique Sanglard, Mélanie Legrand, Murielle Chauvel, Eric Delarze, Sadri Znaidi, Marion Patxot, Emilie Trachsel, Christophe d'Enfert, Ludivine Brandt, Marie-Elisabeth Bougnoux, Lausanne University Hospital, Université de Lausanne = University of Lausanne (UNIL), Biologie et Pathogénicité fongiques (BPF), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP), This work was supported by Swiss National Research Foundation grants 31003A_146936 and 31003A_172958 to DS. The authors thank Françoise Ischer and Danielle Brandalise for excellent technical assistance and M. Arendrup for generous gift of C. albicans isolates. The content of this manuscript has been published as part of the thesis of ED (Delarze, 2019). SZ was the recipient of post-doctoral fellowships from the European Commission (FINSysB, PITN-GA-2008-214004), the Agence Nationale de la Recherche (KANJI, ANR-08-MIE-033-01) and the French Government’s Investissement d’Avenir program (Institut de Recherche Technologique BIOASTER, ANR-10-AIRT-03). Cd’E is supported by the French Government’s Investissement d’Avenir program (Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases, ANR-10-LABX-62-IBEID)., ANR-08-MIEN-0033,KANJI,Colonisation et invasion de la muqueuse digestive par le champignon pathogène de l'homme Candida albicans(2008), ANR-10-AIRT-0003,BIOASTER,BIOASTER(2010), and ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010)

Subjects
Microbiology (medical), [SDV]Life Sciences [q-bio], lcsh:QR1-502, Human pathogen, Drug resistance, Microbiology, Candida, calcineurin, drug resistance, drug tolerance, fluconazole, lcsh:Microbiology, 03 medical and health sciences, Drug tolerance, Intensive care, hemic and lymphatic diseases, medicine, Candida albicans, Gene, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, 030304 developmental biology, 0303 health sciences, biology, 030306 microbiology, biology.organism_classification, Corpus albicans, 3. Good health, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Fluconazole, medicine.drug
Abstract

International audience; Candida albicans is an important human pathogen and a major concern in intensive care units around the world. C. albicans 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 C. albicans 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 C. albicans 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 C. albicans by an overexpression strategy focusing on 572 C. albicans genes. This strategy led to the identification of two transcription factors, CRZ1 and GZF3. CRZ1 is a C2H2-type transcription factor that is part of the calcineurin-dependent pathway in C. albicans, while GZF3 is a GATA-type transcription factor of unknown function in C. albicans. Overexpression of each gene resulted in an increase of FLC tolerance, however, only the deletion of CRZ1 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.

Published
2020
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19. A conserved regulator controls asexual sporulation in the fungal pathogen Candida albicans

Author

Marie-Elisabeth Bougnoux, Natacha Sertour, Jeanne Ropars, Zongwei Li, Gary P. Moran, Sophie Bachellier-Bassi, Virginia Basso, Arturo Hernández-Cervantes, Fabienne Bon, Christophe d'Enfert, Ying Yang, Lasse van Wijlick, Frédéric Dalle, Iryna Denega, Teun Boekhout, Louise Basmaciyan, Derek J. Sullivan, Sadri Znaidi, Biologie et Pathogénicité fongiques - Fungal Biology and Pathogenicity (BPF), Institut Pasteur [Paris] (IP)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Laboratoire de Microbiologie Moléculaire, Vaccinologie et Développement Biotechnologique (LR11IPT01), Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Université de Tunis El Manar (UTM), Université Sorbonne Paris Cité (USPC), Trinity College Dublin, Procédés Alimentaires et Microbiologiques [Dijon] (PAM), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université Bourgogne Franche-Comté [COMUE] (UBFC), Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon), Unité de Parasitologie-Mycologie, Service de Microbiologie [Hôpital Necker-Enfants-Malades, Paris], Assistance Publique - Hôpitaux de Paris, Westerdijk Fungal Biodiversity Institute [Utrecht] (WI), Royal Netherlands Academy of Arts and Sciences (KNAW), Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam [Amsterdam] (UvA), Beijing Institute of Radiation medicine, Institute for Disease Control & Prevention [Beijing], This research was supported by grants from the Agence Nationale de la Recherche (KANJI, ANR‐08‐MIE‐033‐01 to C.d.E. and F.D., CANDIHUB, ANR‐14‐CE‐0018 to C.d.E., IBEID, ANR-10-LABX-62-IBEID to C.d.E.), the European Commission (FINSysB, PITN‐GA‐2008‐214004 to C.d.E.) and the French Government’s Investissement d’Avenir program (Institut de Recherche Technologique BIOASTER, ANR‐10‐AIRT‐03 to C.d.E. and F.D.). S.Z. is an Institut Pasteur International Network Affiliate Program Fellow. S.Z., L.v.W. and A.H.C. were the recipients of post‐doctoral fellowships from the European Commission (FINSysB, PITN‐GA‐2008‐214004 to SZ), the Agence Nationale de la Recherche (KANJI, ANR‐08‐MIE‐033‐01 to S.Z., ERA‐Net Infect‐ERA, FUNCOMPATH, ANR‐14‐IFEC‐0004 to A.H.C., CANDIHUB, ANR‐14‐CE‐0018 to L.v.W.), the French Government’s Investissement d’Avenir program (Institut de Recherche Technologique BIOASTER, ANR‐10‐AIRT‐03 to S.Z. and A.H.C.) from the National Council of Science and Technology, Mexico to A.H.C. V.B. was supported by a grant from the Pasteur-Paris University (PPU) International PhD program and the 'Fondation Daniel et Nina Carasso', ID belongs to the Pasteur-Paris University (PPU) International PhD program, which has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 665807, and from the Institut Carnot Pasteur Microbes & Santé., ANR-08-MIEN-0033,KANJI,Colonisation et invasion de la muqueuse digestive par le champignon pathogène de l'homme Candida albicans(2008), ANR-14-CE14-0018,CANDIHUB,Réponse au stress et virulence chez les champignons pathogènes de l'homme : une approche comparative systémique des réseaux de régulation chez les espèces du genre Candida(2014), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-AIRT-0003,BIOASTER,BIOASTER(2010), ANR-14-IFEC-0004,FunComPath,From fungal commensalism to pathogenicity:dissection of the colonization-to-infection shift of Candida albicans(2014), European Project: 214004,PEOPLE,FP7-PEOPLE-2007-1-1-ITN,FINSYSB(2008), European Project: 665807,H2020,H2020-MSCA-COFUND-2014,PASTEURDOC(2015), Westerdijk Fungal Biodiversity Institute - Yeast Research, Westerdijk Fungal Biodiversity Institute, Institut Pasteur [Paris]-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Westerdijk Fungal Biodiversity Insitute [Utrecht] (WI), and Evolutionary and Population Biology (IBED, FNWI)

Subjects
0301 basic medicine, Hyphal growth, Spores, Science, Spores, Fungal/genetics, 030106 microbiology, Saccharomyces cerevisiae, Candida albicans/classification, [SDV.BDLR.RA]Life Sciences [q-bio]/Reproductive Biology/Asexual reproduction, General Physics and Astronomy, Asexual sporulation, General Biochemistry, Genetics and Molecular Biology, Article, Fungal Proteins, 03 medical and health sciences, Mice, Fungal biology, Pseudohyphal growth, Fungal genetics, Gene Expression Regulation, Fungal, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Candida albicans, Animals, Cellular microbiology, Inbred BALB C, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, Mice, Inbred BALB C, Multidisciplinary, Fungal/genetics, biology, Gene Expression Profiling, Candidemia, General Chemistry, Spores, Fungal, biology.organism_classification, Corpus albicans, Cell biology, Fungal Proteins/genetics, 030104 developmental biology, Fungal, Gene Expression Regulation, Candidemia/microbiology, Female, Gene Expression Profiling/methods, Pathogens, Chromatin immunoprecipitation
Abstract

Transcription factor Rme1 is conserved among ascomycetes and regulates meiosis and pseudohyphal growth in Saccharomyces cerevisiae. The genome of the meiosis-defective pathogen Candida albicans encodes an Rme1 homolog that is part of a transcriptional circuitry controlling hyphal growth. Here, we use chromatin immunoprecipitation and genome-wide expression analyses to study a possible role of Rme1 in C. albicans morphogenesis. We find that Rme1 binds upstream and activates the expression of genes that are upregulated during chlamydosporulation, an asexual process leading to formation of large, spherical, thick-walled cells during nutrient starvation. RME1 deletion abolishes chlamydosporulation in three Candida species, whereas its overexpression bypasses the requirement for chlamydosporulation cues and regulators. RME1 expression levels correlate with chlamydosporulation efficiency across clinical isolates. Interestingly, RME1 displays a biphasic pattern of expression, with a first phase independent of Rme1 function and dependent on chlamydospore-inducing cues, and a second phase dependent on Rme1 function and independent of chlamydospore-inducing cues. Our results indicate that Rme1 plays a central role in chlamydospore development in Candida species., Transcription factor Rme1 regulates meiosis and pseudohyphal growth in baker’s yeast, but its role in the meiosis-defective pathogen Candida albicans is unclear. Here, Hernández-Cervantes et al. show that Rme1 activates the expression of genes required for formation of asexual spores in Candida species.

Published
2020
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20. The two-component response regulator Skn7 belongs to a network of transcription factors regulating morphogenesis inCandida albicansand independently limits morphogenesis-induced ROS accumulation

Author

Sophie Bachellier-Bassi, Virginia Basso, Sadri Znaidi, Franziska Schoenherr, Salomé LeibundGut-Landmann, Christophe d'Enfert, Vitor Cabral, and Valentine Lagage

Subjects
0301 basic medicine, Regulation of gene expression, 030106 microbiology, Regulator, Morphogenesis, Fungal genetics, Biology, biology.organism_classification, Microbiology, Cell biology, 03 medical and health sciences, Response regulator, Transcriptional regulation, Candida albicans, Molecular Biology, Intracellular
Abstract

Skn7 is a conserved fungal heat shock factor-type transcriptional regulator. It participates in maintaining cell wall integrity and regulates the osmotic/oxidative stress response (OSR) in S. cerevisiae, where it is part of a two-component signal transduction system. Here, we comprehensively address the function of Skn7 in the human fungal pathogen Candida albicans. We provide evidence reinforcing functional divergence, with loss of the cell wall/osmotic stress-protective roles and acquisition of the ability to regulate morphogenesis on solid medium. Mapping of the Skn7 transcriptional circuitry, through combination of genome-wide expression and location technologies, pointed to a dual regulatory role encompassing OSR and filamentous growth. Genetic interaction analyses revealed close functional interactions between Skn7 and master regulators of morphogenesis, including Efg1, Cph1 and Ume6. Intracellular biochemical assays revealed that Skn7 is crucial for limiting the accumulation of reactive oxygen species (ROS) in filament-inducing conditions on solid medium. Interestingly, functional domain mapping using site-directed mutagenesis allowed decoupling of Skn7 function in morphogenesis from protection against intracellular ROS. Our work identifies Skn7 as an integral part of the transcriptional circuitry controlling C. albicans filamentous growth and illuminates how C. albicans relies on an evolutionarily-conserved regulator to protect itself from intracellular ROS during morphological development. This article is protected by copyright. All rights reserved.

Published
2017
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21. From Genes to Networks: The Regulatory Circuitry Controlling Candida albicans Morphogenesis

Author

Virginia, Basso, Christophe, d'Enfert, Sadri, Znaidi, and Sophie, Bachellier-Bassi

Subjects
Virulence, Candida albicans, Morphogenesis, Animals, Humans, Signal Transduction, Transcription Factors
Abstract

Candida albicans is a commensal yeast of most healthy individuals, but also one of the most prevalent human fungal pathogens. During adaptation to the mammalian host, C. albicans encounters different niches where it is exposed to several types of stress, including oxidative, nitrosative (e.g., immune system), osmotic (e.g., kidney and oral cavity) stresses and pH variation (e.g., gastrointestinal (GI) tract and vagina). C. albicans has developed the capacity to respond to the environmental changes by modifying its morphology, which comprises the yeast-to-hypha transition, white-opaque switching, and chlamydospore formation. The yeast-to-hypha transition has been very well characterized and was shown to be modulated by several external stimuli that mimic the host environment. For instance, temperature above 37 ℃, serum, alkaline pH, and CO

Published
2018

22. Erratum: Generating genomic platforms to study Candida albicans pathogenesis

Author

Mélanie, Legrand, Sophie, Bachellier-Bassi, Keunsook K, Lee, Yogesh, Chaudhari, Hélène, Tournu, Laurence, Arbogast, Hélène, Boyer, Murielle, Chauvel, Vitor, Cabral, Corinne, Maufrais, Audrey, Nesseir, Irena, Maslanka, Emmanuelle, Permal, Tristan, Rossignol, Louise A, Walker, Ute, Zeidler, Sadri, Znaidi, Floris, Schoeters, Charlotte, Majgier, Renaud A, Julien, Laurence, Ma, Magali, Tichit, Christiane, Bouchier, Patrick, Van Dijck, Carol A, Munro, and Christophe, d'Enfert

Subjects
NAR Breakthrough Article
Abstract

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.

Published
2018

23. Systematic gene overexpression in Candida albicans identifies a regulator of early adaptation to the mammalian gut

Author

Thierry Jouault, Frédéric Dalle, Sophie Bachellier-Bassi, Ralitsa Atanassova, Frédéric Vincent, Valérie Gouyer, Christophe d'Enfert, Carol A. Munro, Natacha Sertour, Jean-Luc Desseyn, Sadri Znaidi, Arturo Hernández-Cervantes, Marie-Elisabeth Bougnoux, Lasse van Wijlick, Université de Tunis El Manar (UTM), Biologie et Pathogénicité fongiques, Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), Laboratoire de Microbiologie Moléculaire, Vaccinologie et Développement Biotechnologique (LR11IPT01), Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Lille Inflammation Research International Center - U 995 (LIRIC), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Agroécologie [Dijon], Université de Bourgogne (UB)-Institut National de la Recherche Agronomique (INRA)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, University of Aberdeen, Laboratoire de parasitologie mycologie (CHU de Dijon), Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon), CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), This work has been supported by grants from the Agence Nationale de la Recherche (KANJI, ANR‐08‐MIE‐033‐01 to C. d'E. and F. D., ERA‐Net Infect‐ERA, FUNCOMPATH, ANR‐14‐IFEC‐0004, and CANDIHUB, ANR‐14‐CE‐0018 to C. d'E.), the French Government's Investissement d'Avenir program (Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases, ANR‐10‐LABX‐62‐IBEID to C. d'E. and Institut de Recherche Technologique BIOASTER, ANR‐10‐AIRT‐03 to C. d'E., F. D., and T. J.), the European Commission (FinSysB, PITN‐GA‐2008‐214004 to C. d'E.), and the Wellcome Trust (The Candida albicans ORFeome project, WT088858MA to C. d'E. and C. M.). C. M. acknowledges support from the Medical Research Council, UK (New Investigator Award, G0400284), the MRC Centre for Medical Mycology (MR/N006364/1), and the University of Aberdeen. S. Z. is an Institut Pasteur International Network Affiliate Program Fellow. S. Z., L. v. W., and A. H. C. were the recipients of post‐doctoral fellowships from the European Commission (FINSysB, PITN‐GA‐2008‐214004 to S. Z.), the Agence Nationale de la Recherche (KANJI, ANR‐08‐MIE‐033‐01 to S. Z., ERA‐Net Infect‐ERA, FUNCOMPATH, ANR‐14‐IFEC‐0004 to A. H. C., CANDIHUB, ANR‐14‐CE‐0018 to L. v. W), and the French Government's Investissement d'Avenir program (Institut de Recherche Technologique BIOASTER, ANR‐10‐AIRT‐03 to S. Z. and A. H. C.)., ANR-08-MIEN-0033,KANJI,Colonisation et invasion de la muqueuse digestive par le champignon pathogène de l'homme Candida albicans(2008), ANR-14-IFEC-0004,FunComPath,From fungal commensalism to pathogenicity:dissection of the colonization-to-infection shift of Candida albicans(2014), ANR-14-CE14-0018,CANDIHUB,Réponse au stress et virulence chez les champignons pathogènes de l'homme : une approche comparative systémique des réseaux de régulation chez les espèces du genre Candida(2014), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-AIRT-0003,BIOASTER,BIOASTER(2010), European Project: 214004,PEOPLE,FP7-PEOPLE-2007-1-1-ITN,FINSYSB(2008), Université Tunis El Manar (UTM), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris], Lille Inflammation Research International Center (LIRIC), Institut National de la Recherche Agronomique (INRA)-Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université Bourgogne Franche-Comté [COMUE] (UBFC), KANJI, ANR-08-MIE-033-01,KANJI, ANR-08-MIE-033-01,KANJI, ANR-08-MIE-033-01, ANR-14-CE14-0018,CANDIHUB,Réponse au stress et virulence chez les champignons pathogènes de l’homme : une approche comparative systémique des réseaux de régulation chez les espèces du genre Candida(2014), ANR-10-LABX-62-IBEID,IBEID,Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases'(2010), ANR-10-AIRT-0003,BIOASTER,Investissement d'Avenir BIOASTER(2012), Biologie et Pathogénicité fongiques (BPF), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP), Lille Inflammation Research International Center - U 995 [LIRIC], Ben Hassine, AbdelHakim, Maladies Infectieuses et environnement - Colonisation et invasion de la muqueuse digestive par le champignon pathogène de l'homme Candida albicans - - KANJI2008 - ANR-08-MIEN-0033 - MIE - VALID, Recherche transnationale sur les maladies infectieuses humaines - From fungal commensalism to pathogenicity:dissection of the colonization-to-infection shift of Candida albicans - - FunComPath2014 - ANR-14-IFEC-0004 - IFEC - VALID, Appel à projets générique - Réponse au stress et virulence chez les champignons pathogènes de l'homme : une approche comparative systémique des réseaux de régulation chez les espèces du genre Candida - - CANDIHUB2014 - ANR-14-CE14-0018 - Appel à projets générique - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Instituts de recherche technologique - BIOASTER - - BIOASTER2010 - ANR-10-AIRT-0003 - AIRT - VALID, and Pathogenomics and Systems Biology of Fungal Infections - An Integrative Approach - FINSYSB - - PEOPLE2008-10-01 - 2012-09-30 - 214004 - VALID

Subjects
0301 basic medicine, [SDV.MHEP.AHA] Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], 030106 microbiology, Immunology, Microbiology, Mannosyltransferases, Biological pathway, Transcriptome, Fungal Proteins, Mannans, 03 medical and health sciences, chemistry.chemical_compound, transcriptomics, regulatory networks, Cell Wall, Virology, Gene Expression Regulation, Fungal, Candida albicans, signature‐tagged overexpression, [SDV.MHEP.AHA]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Animals, Gene Regulatory Networks, Promoter Regions, Genetic, Gene, Transcription factor, Research Articles, Fungal protein, Mice, Inbred BALB C, CRZ2, chromatin immunoprecipitation‐on‐chip, biology, chromatin immunoprecipitation-on-chip, gastrointestinal colonisation, signature-tagged overexpression, Tunicamycin, Hydrogen-Ion Concentration, biology.organism_classification, Phenotype, Cell biology, Gastrointestinal Microbiome, Gastrointestinal Tract, chemistry, Female, Microorganisms, Genetically-Modified, Research Article
Abstract

International audience; Candida albicans is part of the human gastrointestinal (GI) microbiota. To better understand how C. albicans efficiently establishes GI colonisation, we competitively challenged growth of 572 signature-tagged strains (~10% genome coverage), each conditionally overexpressing a single gene, in the murine gut. We identified CRZ2, a transcription factor whose overexpression and deletion respectively increased and decreased early GI colonisation. Using clues from genome-wide expression and gene-set enrichment analyses, we found that the optimal activity of Crz2p occurs under hypoxia at 37°C, as evidenced by both phenotypic and transcriptomic analyses following CRZ2 genetic perturbation. Consistent with early colonisation of the GI tract, we show that CRZ2 overexpression confers resistance to acidic pH and bile salts, suggesting an adaptation to the upper sections of the gut. Genome-wide location analyses revealed that Crz2p directly modulates the expression of many mannosyltransferase- and cell-wall protein-encoding genes, suggesting a link with cell-wall function. We show that CRZ2 overexpression alters cell-wall phosphomannan abundance and increases sensitivity to tunicamycin, suggesting a role in protein glycosylation. Our study reflects the powerful use of gene overexpression as a complementary approach to gene deletion to identify relevant biological pathways involved in C. albicans interaction with the host environment.

Published
2018
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24. Generating genomic platforms to studyCandida albicanspathogenesis

Author

Corinne Maufrais, Sadri Znaidi, Irena Maslanka, Charlotte Majgier, Renaud A. Julien, Carol A. Munro, Louise A. Walker, Laurence Arbogast, Sophie Bachellier-Bassi, Yogesh Chaudhari, Ute Zeidler, Laurence Ma, Keunsook K. Lee, Murielle Chauvel, Tristan Rossignol, Floris Schoeters, Christophe d'Enfert, Christiane Bouchier, Audrey Nesseir, Emmanuelle Permal, Hélène Tournu, Magali Tichit, Vitor Cabral, Mélanie Legrand, Patrick Van Dijck, Helene Boyer, Biologie et Pathogénicité fongiques, Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), University of Aberdeen, Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Modul-Bio, Biomics, Centre de Recherche et Innovation Technologique (CITECH), Institut Pasteur [Paris]-Institut Pasteur [Paris], Wellcome Trust [088858/Z/09/Z to C.A.M., C.D.], European Commission (FINSysB) [PITN-GA-2008-214004 to C.D.] Agence Nationale de la Recherche [KANJI, ANR-08-MIE-033-01 to C.D., CANDICOL, ANR-10-01 to C.D.], Belgian Science Policy Office, Interuniversity Attraction Poles Programme [IAP P7/28 to P.V.D.], Medical Research Council New Investigator Award [G0400284 to C.A.M.], MRC Centre for Medical Mycology and the University of Aberdeen [MR/M026663/1], FINSysB Consortium PhD Fellowship [PITN-GA-2008-214004 to V.C.], DIM-MalInf Région Ile-de-France PhD Fellowship (to A.N.), C. albicans ORFeome Project Post-doctoral Fellowship [WT088858MA to E.P.], NPARI Consortium Postdoctoral Fellowship [LSHE-CT-2006-037692 to T.R.], Programme Fungi from Institut Carnot-Pasteur Maladies Infectieuses Postdoctoral Fellowship (to U.Z.), FINSysB Post-doctoral Fellowship [PITN-GA-2008-214004 to S.Z.], KANJI consortia Post-doctoral Fellowship [ANR-08-MIE-033-01 to S.Z.], KU Leuven CREA Grant [2011-2013 to H.T.], France Génomique Consortium [ANR10-INBS-09-08], French Government’s Investissement d’Avenir program (Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases) [ANR-10-LABX-62-IBEID]. Funding for open access charge: Institutional (Institut Pasteur)., ANR-08-MIEN-0033,KANJI,Colonisation et invasion de la muqueuse digestive par le champignon pathogène de l'homme Candida albicans(2008), ANR-10-PATH-0008,CANDICOL(2010), ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 214004,PEOPLE,FP7-PEOPLE-2007-1-1-ITN,FINSYSB(2008), Institut Pasteur [Paris], Institut National de la Recherche Agronomique (INRA), Université Catholique de Louvain (UCL), Université Sorbonne Paris Cité (COMUE) (USPC), MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Université Paris Saclay (COmUE), Biologie et Pathogénicité fongiques (BPF), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut Pasteur [Paris] (IP), Bachellier- Bassi, Sophie, D'Enfert, Christophe, Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris], Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], ANR: ANR-10-INBS-09-08,The 'France Génomique' consortium, ANR-10-LABX-62-IBEID,IBEID,Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases'(2010), and Bachellier-Bassi, Sophie

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], 030106 microbiology, Genetic Vectors, Genomics, Computational biology, Genome, Pathogenesis, Open Reading Frames, 03 medical and health sciences, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Candida albicans, Protein Interaction Mapping, Genetics, Vector (molecular biology), ORFS, ORFeome, Gene, 030304 developmental biology, 0303 health sciences, biology, 030302 biochemistry & molecular biology, biology.organism_classification, Corpus albicans, Open reading frame, 030104 developmental biology, Databases, Nucleic Acid, Function (biology)
Abstract

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 ofCandida 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 ofC. albicans. Sequencing data of the cloned ORFs are available in the CandidaOrfDB database athttp://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 theC. albicansORFeome. 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 inC. albicansand the elucidation of mechanisms that underlie its pathogenicity.

Published
2018
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26. Targeted changes of the cell wall proteome influence [i]Candida albican[/i]s ability to form single- and multi-strain biofilms

Author

Louise A. Walker, Vitor Cabral, Keunsook K. Lee, Tristan Rossignol, Murielle Chauvel, Christophe d'Enfert, Mathias L. Richard, Etienne Dague, Arnaud Firon, Sadri Znaidi, Carol A. Munro, Mélanie Legrand, Hélène Martin-Yken, Sophie Bachellier-Bassi, Biologie et Pathogénicité fongiques, Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris], School of Medical Sciences, University of Aberdeen, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Équipe NanoBioSystèmes (LAAS-NBS), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, European Commission (EURESFUN) [LSHM-CT-2005-518199], European Commission (Galar Fungail 2) [MRTN-CT-2003-504148], European Commission (FINSysB) [PITN-GA-2008-214004], Agence Nationale de la Recherche (KANJI) [ANR-08-MIE-033-01], Wellcome Trust (The Candida albicans ORFeome project) [WT088858MA], Institut Pasteur (Bourse Roux), Medical Research Council [G0400284], Agence Nationale de la Recherche (AFMYST project) [ANR-11-JSV5-001-01, SD 30024331], ANR-08-MIEN-0033,KANJI,Colonisation et invasion de la muqueuse digestive par le champignon pathogène de l'homme Candida albicans(2008), ANR-11-JSV5-0001,AFMYST,Etude Biophysique, biochimique et biomoléculaire de la paroi des levures.(2011), Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, Université Paris-Sorbonne (UP4), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse 1 Capitole (UT1), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Biologie et Pathogénicité fongiques (BPF), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), Cabral, Vitor, and Znaidi, Sadri

Subjects
Genetic Screens, Proteome, [SDV]Life Sciences [q-bio], Gene Identification and Analysis, EFFICIENT, Yeast and Fungal Models, DEFECTIVE MUTANTS, biofilm, Transcriptome, INFECTION MODEL, DNA library construction, Cell Wall, Gene Expression Regulation, Fungal, Genomic Library Screening, Candida albicans, lcsh:QH301-705.5, protéome, GENE-EXPRESSION, biology, Ecology, Genomic Library Construction, Corpus albicans, Phenotype, Hyperexpression Techniques, Cellular Structures and Organelles, Shear Strength, VIRULENCE FACTORS, Research Article, lcsh:Immunologic diseases. Allergy, TRANSCRIPTIONAL REGULATOR, PROTEINS, Gene prediction, Immunology, Virulence, Library Screening, DNA construction, Microbiology, Microbial Ecology, MECHANISMS, Fungal Proteins, Cell Walls, Model Organisms, Virology, Cell Adhesion, Genetics, Gene Expression and Vector Techniques, Molecular Biology, Gene, Molecular Biology Assays and Analysis Techniques, Ecology and Environmental Sciences, Biofilm, Biology and Life Sciences, Cell Biology, biology.organism_classification, Molecular biology, Research and analysis methods, ONTOLOGY, Molecular biology techniques, lcsh:Biology (General), Biofilms, Parasitology, OVEREXPRESSION, lcsh:RC581-607
Abstract

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., Author Summary Candida albicans is the most prevalent human fungal pathogen. Its ability to cause disease relies, in part, on the formation of biofilms, a protective structure of highly adherent cells tolerant to antifungal agents and the host immune response. The biofilm is considered as a persistent root of infection, disseminating infectious cells to other locations. In this study, we performed large-scale phenotypic analyses aimed at identifying genes whose overexpression affects biofilm development in C. albicans. Our screen relied on a collection of 531 C. albicans strains, each conditionally overexpressing one given gene and carrying one specific molecular tag allowing the quantification of strain abundance in mixed-population experiments. Our results strikingly revealed the enrichment of strains overproducing poorly-characterized surface proteins called Pgas (Putative GPI-Anchored proteins), within a 531-strain-containing biofilm model. We show that these PGA genes differentially contribute to single-strain and multi-strain biofilm formation and are involved in specific stages of the biofilm developmental process. Taken together, our results reveal the importance of C. albicans cell surface proteins during biofilm formation and reflect the powerful use of strain barcoding in combination with gene overexpression to identify genes and/or pathways involved in processes pertaining to virulence of pathogenic microbes.

Published
2014
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27. A highly parallel conditional overexpression screen, in vivo, identifies a new regulator of Candida albicans commensalism

Author

Arturo Hernández Cervantes, Thierry Jouault, Natacha Sertour, Christophe d'Enfert, Marie-Elisabeth Bougnoux, Frédéric Vincent, Frédéric Dalle, Valérie Gouyer, Jean-Luc Desseyn, and Sadri Znaidi

Subjects
Zinc finger transcription factor, Mannosyltransferase, Infectious Diseases, biology, Activator (genetics), Gene expression, Transcriptional regulation, Repressor, Candida albicans, biology.organism_classification, Gene, Molecular biology
Abstract

Candida albicans is part of the human mycobiota, coexisting with the plethora of bacterial species within the gut. To better understand how C. albicans efficiently establishes gastrointestinal (GI)-tract colonization, we competitively challenged growth of 579 signature-tagged C. albicans strains (∼ 10% genome coverage), each conditionally overexpressing a single gene, in the gut of a mouse model of GI-tract colonization. Our screen identified CRZ2, a Candida -specific zinc finger transcription factor of the C2H2 family, whose overexpression increased C. albicans intestinal colonization. Using an ex vivo model of Candida -colon explant adhesion assay, we further showed that CRZ2 overexpression imparts increased adhesion of C. albicans to colonic tissue. Genome-wide expression and location experiments revealed that Crz2p directly modulates the expression of many C. albicans mannosyltransferase and cell-wall protein-encoding genes and has dual transcriptional regulatory functions, acting as both activator and repressor of gene expression. Gene-set enrichment analyses indicated that CRZ2 overexpression triggers a substantial reprogramming of amino acid and metal/ion homeostases, while respectively turning on and off the hypoxic and oxidative respiration transcriptional programs. Our study reflects the powerful use of gene overexpression as a complementary approach to gene deletion to identify relevant biological circuits involved in C. albicans commensalism and links transcriptional regulation of cell-surface proteins to C. albicans ability to thrive within the host.

Published
2015
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28. How Did Institut Pasteur’s NGS Core Facility, Biomics, Manage the Coronavirus Disease 2019 Crisis?

Author

Laurence Ma, Valérie Briolat, Imène Najjar, Marc Monot, Laure Lemée, Laurence Motreff, Pôle Biomics (C2RT), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], The Biomics platform is supported by France Génomique (ANR-10-INBS-09-09) and Infrastructures en Biologie, Santé et Agronomie (IBiSA). This work was supported by the 'URGENCE COVID-19' fundraising campaign of IP., We thank the other members of Biomics who made it possible for the activities of the platform to continue during lockdown: Julian Garneau, Elodie Turc, George Haustant, Zachary Allouche, Thomas Cockelear, Juliana Pipoli Da Fonseca, and Etienne Kornobis. We would particularly like to thank our collaborator from the IP of Tunis Sadri Znaidi and the C2RT Head of Operations Anna Kheres for taking the time to read the manuscript and for giving recommendations and advice. The authors declare no conflicts of interest., ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), and Institut Pasteur [Paris] (IP)-Institut Pasteur [Paris] (IP)

Subjects
Paris, MESH: Pandemics, Process management, Quality management, [SDV]Life Sciences [q-bio], coronavirus, Risk management information systems, Crisis management, Article, MESH: Communicable Disease Control, Business continuity, Added value, Humans, MESH: COVID-19, MESH: SARS-CoV-2, Pandemics, Molecular Biology, MESH: High-Throughput Nucleotide Sequencing, MESH: Humans, Emergency management, business.industry, SARS-CoV-2, MESH: Paris, COVID-19, High-Throughput Nucleotide Sequencing, Benchmarking, OMICS sequencing, 3. Good health, Core Facility, NGS, Communicable Disease Control, MESH: Laboratories, Business, Laboratories, management
Abstract

International audience; In 2020, research entities at the Institut Pasteur (IP) in Paris, as elsewhere around the world, were closed because of the coronavirus disease 2019 (COVID-19) pandemic. However, IP core facilities, laboratories, services, and departments working on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and priority projects were authorized to continue working both on site and remotely. Given the importance of its role in SARS-CoV-2 genome-sequencing initiatives, the IP Biomics core facility was fully functional during the first (i.e., March-June 2020) and second (i.e., November-December 2020) national lockdowns. We describe here how Biomics successfully implemented an emergency management plan to deal with this health crisis. We highlight the internal deployment of the institutional business continuity plan (BCP) through a series of actions. We also address the impact of the COVID-19 crisis on Biomics staff and collaborators. The added value of quality management and the limitations of risk management systems are discussed. Finally, we suggest that the Biomics infrastructure and the BCP described here could be used for benchmarking purposes, for other next-generation sequencing core facilities wishing to implement/improve their processes, and for future major crisis management.

Published
2021
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