The NDR/LATS Kinase Cbk1 Controls the Activity of the Transcriptional Regulator Bcr1 during Biofilm Formation in Candida albicans

In nature, many microorganisms form specialized complex, multicellular, surface-attached communities called biofilms. These communities play critical roles in microbial pathogenesis. The fungal pathogen Candida albicans is associated with catheter-based infections due to its ability to establish biofilms. The transcription factor Bcr1 is a master regulator of C. albicans biofilm development, although the full extent of its regulation remains unknown. Here, we report that Bcr1 is a phosphoprotein that physically interacts with the NDR kinase Cbk1 and undergoes Cbk1-dependent phosphorylation. Mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to alanine markedly impaired Bcr1 function during biofilm formation and virulence in a mouse model of disseminated candidiasis. Cells lacking Cbk1, or any of its upstream activators, also had reduced biofilm development. Notably, mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to glutamate in cbk1Δ cells upregulated the transcription of Bcr1-dependent genes and partially rescued the biofilm defects of a cbk1Δ strain. Therefore, our data uncovered a novel role of the NDR/LATS kinase Cbk1 in the regulation of biofilm development through the control of Bcr1

Systematic Gene Overexpression in Candida albicans identifies a Regulator of Early Adaptation to the Mammalian Gut

We are grateful to members of the genomics core facility (PF2, Génopole) for the availability of the microarray scanner and the Alain Jacquier’s lab for making the GenePix software available. We are grateful to Drs. Suzanne Noble and Aaron Mitchell for providing C. albicans mutant collections. We thank all members of the Fungal Biology & Pathogenicity Unit, particularly Drs. Anne Neville and Adeline Feri for their numerous insights during the course of this project. 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.; 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.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Candida albicans biofilms are generally devoid of persister cells

International audienceCandida albicans is known for its ability to form biofilms - communities of microorganisms embedded in an extracellular matrix developing on different surfaces. Biofilms are highly tolerant to antifungal therapy. This phenomenon has been partially explained by the appearance of so-called persister cells, phenotypic variants of wild-type cells, capable of surviving very high concentrations of antimicrobial agents. Persister cells in C. albicans were found exceptionally in biofilms while none were detected in planktonic cultures of this fungus. Yet, this topic remains controversial as others could not observe persister cells in biofilms formed by the C. albicans SC5314 laboratory strain. Due to ambiguous data in the literature, this work aimed to re-evaluate the presence of persister cells in C. albicans biofilms. We demonstrated that isolation of C. albicans "persister cells" as described previously was likely to be the result of survival of biofilm cells that were not reached by the antifungal. We tested biofilms of SC5314 and its derivatives, as well as 95 clinical isolates, using an improved protocol, demonstrating that persister cells are not a characteristic trait of C. albicans biofilms. Although some clinical isolates are able to yield survivors upon the antifungal treatment of biofilms, this phenomenon is rather stochastic and inconsistent

Bending stiffness of Candida albicans hyphae as a proxy of cell wall properties

International audienceThe cell wall is a key component of fungi. It constitutes a highly regulated viscoelastic shell which counteracts internal cell turgor pressure. Its mechanical properties thus contribute to define cell morphology. Measurements of the elastic moduli of the fungal cell wall have been carried out in many species including Candida albicans, a major human opportunistic pathogen. They mainly relied on atomic force microscopy, and mostly considered the yeast form. We developed a parallelized pressure-actuated microfluidic device to measure the bending stiffness of hyphae. We found that the cell wall stiffness lies in the MPa range. We then used three different ways to disrupt cell wall physiology: inhibition of beta-glucan synthesis, a key component of the inner cell wall; application of a hyperosmotic shock triggering a sudden decrease of the hyphal diameter; deletion of two genes encoding GPI-modified cell wall proteins resulting in reduced cell wall thickness. The bending stiffness values were affected to different extents by these environmental stresses or genetic modifications. Overall, our results support the elastic nature of the cell wall and its ability to remodel at the scale of the entire hypha over minutes

Cell cycle-dependent kinetochore localization of condensin complex in Saccharomyces cerevisiae.

International audienceIn budding yeast mitosis is endonuclear and associated with a very limited condensation of the chromosomes. Despite this partial chromosomal condensation, condensin is conserved and essential for the Saccharomyces cerevisiae mitotic cycle. Here, we investigate the localization of condensin during the mitotic cycle. In addition to a constitutive association with rDNA, we have discovered that condensin is localized to the kinetochore in a cell cycle-dependent manner. Shortly after duplication of the spindle pole body, the yeast equivalent of the centrosome, we observed a local enrichment of condensin colocalizing with kinetochore components. This specific association is consistent with mutant phenotypes of chromosome loss and defective sister chromatid separation at anaphase. During a short period of the cell cycle, we observed, at the single cell level, a spatial proximity of condensin and a cohesin rosette, without colocalization. Furthermore, using a genetic screen we demonstrated that condensin localization at kinetochores is specifically impaired in a mutant for ulp2/smt4, an abundant SUMO protease. In conclusion, during chromosome segregation, we established a SUMO-dependent cell cycle-specific condensin concentration colocalizing with kinetochores

Involvement of amyloid proteins in the formation of biofilms in the pathogenic yeast Candida albicans

International audienceCandida species represent a major fungal threat for human health. Within the Candida genus, the yeast Candida albicans is the most frequently incriminated species during episodes of candidiasis or candidemia. Biofilm formation is used by C. albicans to produce a microbial community that is important in an infectious context. The cell wall, the most superficial cellular compartment, is of paramount importance regarding the establishment of biofilms. C. albicans cell wall contains proteins with amyloid properties that are necessary for biofilm formation due to their adhesion properties. This review focuses on these amyloid proteins during biofilm formation in the yeast C. albicans

A protocol for ultrastructural study of Candida albicans biofilm using transmission electron microscopy

International audienceThis protocol describes how to analyze C. albicans biofilm using transmission electron microscopy. We present two approaches to observe the ultrastructure of fungal cells within unperturbed biofilms, as well as an immunogold labeling procedure. This approach maintains the architecture of the fungal biofilm close to its native state by growing C. albicans biofilm on a plastic surface. After the freeze substitution procedure, classical transmission electron microscopy or electron tomography will allow the ultrastructural analysis of the microbial community

Transcript profiling reveals the role of PDB1, a subunit of the pyruvate dehydrogenase complex, in Candida albicans biofilm formation

International audienceCandida albicans, the most prevalent fungal pathogen in the human microbiota can form biofilms on implanted medical devices. These biofilms are tolerant to conventional antifungal drugs and the host immune system as compared to the free-floating planktonic cells. Several in vitro models of biofilm formation have been used to determine the C. albicans biofilm-forming process, regulatory networks, and their properties. Here, we performed a genome-wide transcript profiling with C. albicans cells grown in YPD medium both in planktonic and biofilm condition. Transcript profiling of YPD-grown biofilms was further compared with published Spider medium-grown biofilm transcriptome data. This comparative analysis highlighted the differentially expressed genes and the pathways altered during biofilm formation. In addition, we demonstrated that overexpression of the PDB1 gene encoding a subunit of the pyruvate dehydrogenase resulted in defective biofilm formation. Altogether, this comparative analysis of transcript profiles from two different studies provides a robust reading on biofilm-altered genes and pathways during C. albicans biofilm development

Regulators of commensal and pathogenic life-styles of an opportunistic fungus -Candida albicans

International audienceThe yeast Candida albicans is primarily a commensal of humans that colonizes the mucosal surfaces of the gastrointestinal and genital tracts. Yet, C. albicans can under certain circumstances undergo a shift from commensalism to pathogenicity. This transition is governed by fungal factors such as morphological transitions, environmental cues for instance relationships with gut microbiota and the host immune system. C. albicans utilizes distinct sets of regulatory programs to colonize or infect its host and to evade the host defense systems. Moreover, an orchestrated iron acquisition mechanism operates to adapt to specific niches with variable iron availability. Studies on regulatory networks and morphogenesis of these two distinct modes of C. albicans growth, suggest that both yeast and hyphal forms exist in both growth patterns and the regulatory circuits are inter‐connected. Here, we summarize current knowledge about C. albicans commensal‐to‐pathogen shift, its regulatory elements and their contribution to human disease

Overexpression approaches to advance understanding of Candida albicans

International audienceCandida albicans is an opportunistic fungal pathogen that is responsible for infections linked to high mortality. Loss-of-function approaches, taking advantage of gene knockouts or inducible down-regulation, have been successfully used in this species in order to understand gene function. However, overexpression of a gene provides an alternative, powerful tool to elucidate gene function and identify novel phenotypes. Notably, overexpression can identify pathway components that might remain undetected using loss-of-function approaches. Several repressible or inducible promoters have been developed which allow to shut off or turn on the expression of a gene in C. albicans upon growth in the presence of a repressor or inducer. In this review, we summarize recent overexpression approaches used to study different aspects of C. albicans biology, including morphogenesis, biofilm formation, drug tolerance, and commensalism