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

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.

Use of the Fluorescent Dye Thioflavin T to Track Amyloid Structures in the Pathogenic Yeast Candida albicans.

The human pathogenic yeast Candida albicans can attach to epithelial cells or indwelling medical devices to form biofilms. These microbial communities are highly problematic in the clinic as they reduce both sensitivity to antifungal drugs and detection of fungi by the immune system. Amyloid structures are highly organized quaternary structures that play a critical role in biofilm establishment by allowing fungal cells to adhere to each other. Thus, fungal amyloids are exciting targets to develop new antifungal strategies. Thioflavin T is a specific fluorescent dye widely used to study amyloid properties of target proteins in vitro (spectrophotometry) and in vivo (epifluorescence/confocal microscopy). Notably, thioflavin T has been used to demonstrate the ability of Als5, a C. albicans adhesin, to form an amyloid fiber upon adhesion. We have developed a pipeline that allows us to study amyloid properties of target proteins using thioflavin T staining in vitro and in vivo, as well as in intact fungal biofilms. In brief, we used thioflavin T to sequentially stain (i) amyloid peptides, (ii) recombinant proteins, (iii) fungal cells treated or not with amyloid peptides, (iv) fungal amyloids enriched by cell fractionation, and (v) intact biofilms of C. albicans. Contrary to other methods, our pipeline gives a complete picture of the amyloid behavior of target proteins, from in vitro analysis to intact fungal biofilms. Using this pipeline will allow an assessment of the relevance of the in vitro results in cells and the impact of amyloids on the development and/or maintenance of fungal biofilm. Key features • Study of amyloid properties of fungal proteins. • Visualization of the subcellular localization of fungal amyloid material using epifluorescence or confocal microscopy. • Unraveling of the amyloid properties of target proteins and their physiological meaning for biofilm formation. • Observation of the presence of amyloid structures with live-cell imaging on intact fungal biofilm using confocal microscopy.

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

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 characterization of gene functions in C. albicans.

The Pga59 cell wall protein is an amyloid forming protein involved in adhesion and biofilm establishment in the pathogenic yeast Candida albicans.

The human commensal fungus Candida albicans can attach to epithelia or indwelling medical devices and form biofilms, that are highly tolerant to antifungal drugs and can evade the immune response. The cell surface protein Pga59 has been shown to influence adhesion and biofilm formation. Here, we present evidence that Pga59 displays amyloid properties. Using electron microscopy, staining with an amyloid fibre-specific dye and X-ray diffraction experiments, we showed that the predicted amyloid-forming region of Pga59 is sufficient to build up an amyloid fibre in vitro and that recombinant Pga59 can also adopt a cross-ß amyloid fibre architecture. Further, mutations impairing Pga59 amyloid assembly led to diminished adhesion to substrates and reduced biofilm production. Immunogold labelling on amyloid structures extracted from C. albicans revealed that Pga59 is used by the fungal cell to assemble amyloids within the cell wall in response to adhesion. Altogether, our results suggest that Pga59 amyloid properties are used by the fungal cell to mediate cell-substrate interactions and biofilm formation.

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

Candida 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.

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

The 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.

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

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 (P TET ) 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.

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

This 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.

Spatiotemporal dynamics of calcium signals during neutrophil cluster formation.

Neutrophils form cellular clusters or swarms in response to injury or pathogen intrusion. Yet, intracellular signaling events favoring this coordinated response remain to be fully characterized. Here, we show that calcium signals play a critical role during mouse neutrophil clustering around particles of zymosan, a structural fungal component. Pioneer neutrophils recognizing zymosan or live Candida albicans displayed elevated calcium levels. Subsequently, a transient wave of calcium signals in neighboring cells was observed followed by the attraction of neutrophils that exhibited more persistent calcium signals as they reached zymosan particles. Calcium signals promoted LTB4 production while the blocking of extracellular calcium entry or LTB4 signaling abrogated cluster formation. Finally, using optogenetics to manipulate calcium influx in primary neutrophils, we show that calcium signals could initiate recruitment of neighboring neutrophils in an LTB4-dependent manner. Thus, sustained calcium responses at the center of the cluster are necessary and sufficient for the generation of chemoattractive gradients that attract neutrophils in a self-reinforcing process.

ChIP-SICAP: A New Tool to Explore Gene-Regulatory Networks in Candida albicans and Other Yeasts.

Chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) is a powerful method to identify protein interactions, and has long been used to gain insights into regulatory networks in relevant fungal species as well as many other organisms. In this chapter, we discuss a similar technique called ChIP-SICAP (chromatin immunoprecipitation with selective isolation of chromatin-associated proteins) that overcomes many of the traditional limitations of ChIP-MS, and describe a protocol that allows ChIP-SICAP to be applied to Candida albicans and other yeasts. Notably, the technique design permits stringent washing to remove contaminating proteins and antibodies before subsequent mass spectrometry processing, allows for genome-wide mapping of the bait protein by ChIP-seq after ChIP-SICAP from the same sample through a DNA recovery process, and specifically purifies and identifies proteins associating with chromatin. In the future, ChIP-SICAP will provide the yeast genomics research community an additional method to explore the complex dynamics of the gene-regulatory networks modulating morphology, metabolism and response to stress.

Overexpression approaches to advance understanding of Candida albicans.

Candida 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.

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

The 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.

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

Candida 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 conserved regulator controls asexual sporulation in the fungal pathogen Candida albicans.

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.

The Candida albicans biofilm gene circuit modulated at the chromatin level by a recent molecular histone innovation.

Histone H3 and its variants regulate gene expression but the latter are absent in most ascomycetous fungi. Here, we report the identification of a variant histone H3, which we have designated H3VCTG because of its exclusive presence in the CTG clade of ascomycetes, including Candida albicans, a human pathogen. C. albicans grows both as single yeast cells and hyphal filaments in the planktonic mode of growth. It also forms a three-dimensional biofilm structure in the host as well as on human catheter materials under suitable conditions. H3VCTG null (hht1/hht1) cells of C. albicans are viable but produce more robust biofilms than wild-type cells in both in vitro and in vivo conditions. Indeed, a comparative transcriptome analysis of planktonic and biofilm cells reveals that the biofilm circuitry is significantly altered in H3VCTG null cells. H3VCTG binds more efficiently to the promoters of many biofilm-related genes in the planktonic cells than during biofilm growth, whereas the binding of the core canonical histone H3 on the corresponding promoters largely remains unchanged. Furthermore, biofilm defects associated with master regulators, namely, biofilm and cell wall regulator 1 (Bcr1), transposon enhancement control 1 (Tec1), and non-dityrosine 80 (Ndt80), are significantly rescued in cells lacking H3VCTG. The occupancy of the transcription factor Bcr1 at its cognate promoter binding sites was found to be enhanced in the absence of H3VCTG in the planktonic form of growth resulting in enhanced transcription of biofilm-specific genes. Further, we demonstrate that co-occurrence of valine and serine at the 31st and 32nd positions in H3VCTG, respectively, is essential for its function. Taken together, we show that even in a unicellular organism, differential gene expression patterns are modulated by the relative occupancy of the specific histone H3 type at the chromatin level.

Candida albicans Biofilms Are Generally Devoid of Persister Cells.

Candida albicans is known for its ability to form biofilms, which are 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 reevaluate the presence of persister cells in C. albicans biofilms. We demonstrated that the isolation of C. albicans "persister cells" as described previously was likely to be the result of the 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.

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

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 â„ƒ, serum, alkaline pH, and CO2 concentration are all reported to enhance filamentation. The transition is characterized by the activation of an intricate regulatory network of signaling pathways, involving many transcription factors. The regulatory pathways that control either the stress response or morphogenesis are required for full virulence and promote survival of C. albicans in the host. Many of these transcriptional circuitries have been characterized, highlighting the complexity and the interconnections between the different pathways. Here, we present the major signaling pathways and the main transcription factors involved in the yeast-to-hypha transition. Furthermore, we describe the role of heat shock transcription factors in the morphogenetic transition, providing an edifying example of the complex cross talk between pathways involved in morphogenesis and stress response.

Generating genomic platforms to study Candida albicans pathogenesis.

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.

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

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.