Guilhem Janbon

Candida albicans Biofilms: a Developmental State Associated With Specific and Stable Gene Expression Patterns

ABSTRACT Like many bacteria, yeast species can form biofilms on several surfaces. Candida albicans colonizes the surfaces of catheters, prostheses, and epithelia, forming biofilms that are extremely resistant to antifungal drugs. We have used transcript profiling to investigate the specific properties of C. albicans biofilms. Biofilm and planktonic cultures produced under different conditions of nutrient flow, aerobiosis, or glucose concentration were compared by overall gene expression correlation. Correlation was much higher between biofilms than planktonic populations irrespective of the growth conditions, indicating that biofilm populations formed in different environments display very similar and specific transcript profiles. A first cluster of 325 differentially expressed genes was identified. In agreement with the overrepresentation of amino acid biosynthesis genes in this cluster, Gcn4p, a regulator of amino acid metabolism, was shown to be required for normal biofilm growth. To identify biofilm-related genes that are independent of mycelial development, we studied the transcriptome of biofilms produced by a wild-type, hypha-producing strain and a cph1/cph1 efg1/efg1 strain defective for hypha production. This analysis identified a cluster of 317 genes expressed independently of hypha formation, whereas 86 genes were dependent on mycelial development. Both sets revealed the activation of the sulfur-amino acid biosynthesis pathway as a feature of C. albicans biofilms.

A Yeast under Cover: the Capsule of Cryptococcus neoformans

Few fungi are pathogenic to humans. Of these, Cryptococcus neoformans has emerged as an important cause of mortality in immunocompromised patients, especially those with AIDS. As a result, extensive research efforts have addressed the pathogenesis and virulence of this organism. C. neoformans is a basidiomycetous fungus that is ubiquitous in the environment, where it is found in soil, in association with certain trees, and in bird guano (16). Because of its ubiquity, it has been suggested that most people are exposed to C. neoformans early in life (41). The fungus is heterothallic, with mating types MATa and MAT. Asexual reproduction takes place either by budding or, in the case of MAT cells, by haploid fruiting in response to nutrient deprivation or exposure to the mating pheromone a factor (106). Sexual reproduction occurs when cells of opposite mating types come together to form a heterokaryon that ultimately leads to the production of basidia and basidiospores (64). Desiccated cells and the spores formed by haploid fruiting or sexual reproduction have all been suggested to serve as infective particles, which must be less than 2 m in diameter to penetrate the lung parenchyma (44, 86). Infection occurs when the fungal particles are inhaled and enter the alveolar space. In most immunocompetent individuals, this infection is either cleared or remains dormant until an immune imbalance leads to further development. In the setting of compromised immune function, however, the fungus disseminates, with particular tropism for the central nervous system. In severe cases, cryptococcal infection progresses to a meningoencephalitis that is fatal if left untreated. C. neoformans virulence is mediated predominantly by a polysaccharide capsule that surrounds its cell wall and has multiple effects on the host immune system. This structure provides a physical barrier that interferes with normal phagocytosis and clearance by the immune system. Capsule components inhibit the production of proinflammatory cytokines, deplete complement components (by efficiently binding them), and reduce leukocyte migration to sites of inflammation (11). The capsule also constitutes the major diagnostic feature of cryptococcosis, because its components can be detected in the bloodstream and it can be visualized with light microscopy by using India ink staining. The capsule excludes the ink particles and forms characteristic halos (Fig. 1A) whose diameters are often several times that of the cell. The elaborate structure of the capsule may also be appreciated by electron microscopy (Fig. 1B and C). Given the importance of the capsule in cryptococcal disease, tremendous effort has been applied in recent years to understanding its biology. This review focuses on the resulting advances in our understanding of the structure and synthesis of the capsular components, the incorporation of these components into the existing capsular network, the association between the capsule and the cell wall, and the regulation of capsule growth.

Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

Cryptococcus neoformans Senses CO2 through the Carbonic Anhydrase Can2 and the Adenylyl Cyclase Cac1

ABSTRACT Cryptococcus neoformans, a fungal pathogen of humans, causes fatal meningitis in immunocompromised patients. Its virulence is mainly determined by the elaboration of a polysaccharide capsule surrounding its cell wall. During its life, C. neoformans is confronted with and responds to dramatic variations in CO2 concentrations; one important morphological change triggered by the shift from its natural habitat (0.033% CO2) to infected hosts (5% CO2) is the induction of capsule biosynthesis. In cells, CO2 is hydrated to bicarbonate in a spontaneous reaction that is accelerated by carbonic anhydrases. Here we show that C. neoformans contains two β-class carbonic anhydrases, Can1 and Can2. We further demonstrate that CAN2, but not CAN1, is abundantly expressed and essential for the growth of C. neoformans in its natural environment, where CO2 concentrations are limiting. Structural studies reveal that Can2 forms a homodimer in solution. Our data reveal Can2 to be the main carbonic anhydrase and suggest a physiological role for bicarbonate during C. neoformans growth. Bicarbonate directly activates the C. neoformans Cac1 adenylyl cyclase required for capsule synthesis. We show that this specific activation is optimal at physiological pH.

Cas1p is a membrane protein necessary for the O‐acetylation of the Cryptococcus neoformans capsular polysaccharide

The capsule is certainly the most obvious virulence factor for Cryptococcus neoformans. The main capsule constituents are glucuronoxylomannans (GXM). Several studies have focused on the structure and chemistry of the GXM component of the capsule, yet little is known about the genetic basis of the capsule construction. Using a monoclonal antibody specific to a sugar epitope, we isolated a capsule‐structure mutant strain and cloned by complementation a gene named CAS1 that codes for a putative membrane protein. Although no sequence homology was found with any known protein in the different databases, protein analysis using the Propsearch software classified Cas1p as a putative glycosyltransferase. Cas1p is a well‐conserved evolutionary protein, as we identified one orthologue in the human genome, one in the drosophila genome and four in the plant Arabidopsis thaliana genome. Analysis of the capsule structure after CAS1 deletion showed that it is required for GXM O‐acetylation.

The Yak1p kinase controls expression of adhesins and biofilm formation in Candida glabrata in a Sir4p‐dependent pathway

Biofilm is the predominant type of microbial development in natural environments, and potentially represents a major form of resistance or source of recurrence during host infection. Although a large number of studies have focussed on the genetics of bacterial biofilm formation, very little is known about the genes involved in this type of growth in fungi. A genetic screen for Candida glabrata Biofilm mutants was performed using a 96‐well plate model of biofilm formation. Study of the isolated mutant strains allowed the identification of four genes involved in biofilm formation (RIF1, SIR4, EPA6 and YAK1). Epa6p is a newly identified adhesin required for biofilm formation in this pathogenic yeast. EPA6 and its close paralogue EPA7 are located in subtelomeric regions and their transcription is regulated by Sir4p and Rif1p, two proteins involved in subtelomeric silencing. Biofilm growth conditions induce the transcription of EPA6 and EPA7: this is dependent on the presence of an intact subtelomeric silencing machinery and is independent of the Mpk1p signalling pathway. Finally, the kinase Yak1p is required for expression of both adhesin genes and acts through a subtelomeric silencing machinery‐dependent pathway.

Cryptococcus neoformans and Cryptococcus gattii, the Etiologic Agents of Cryptococcosis

Cryptococcus neoformans and Cryptococcus gattii are the two etiologic agents of cryptococcosis. They belong to the phylum Basidiomycota and can be readily distinguished from other pathogenic yeasts such as Candida by the presence of a polysaccharide capsule, formation of melanin, and urease activity, which all function as virulence determinants. Infection proceeds via inhalation and subsequent dissemination to the central nervous system to cause meningoencephalitis. The most common risk for cryptococcosis caused by C. neoformans is AIDS, whereas infections caused by C. gattii are more often reported in immunocompetent patients with undefined risk than in the immunocompromised. There have been many chapters, reviews, and books written on C. neoformans. The topics we focus on in this article include species description, pathogenesis, life cycle, capsule, and stress response, which serve to highlight the specializations in virulence that have occurred in this unique encapsulated melanin-forming yeast that causes global deaths estimated at more than 600,000 annually.

Isolation and characterization of capsule structure mutant strains of Cryptococcus neoformans

The capsule of Cryptococcus neoformans is the most obvious virulence factor of this pathogenic yeast. The main capsule constituents are glucuronoxylomannans (GXM). Although several studies have focused on GXM composition and structure, very little is known about their genetics. To elucidate the relationship between the capsule structure and the pathophysiology of the cryptococcosis, genetic screening for mutant strains producing a structurally modified capsule was set up. Using monoclonal antibodies specific for different capsule sugar epitopes, we isolated strains with different mutated capsule structures (Cas mutants). According to their reactivities with various monoclonal antibodies, the mutants were classified into six groups (Cas1 to Cas6). One Cas2 mutant was used to clone the corresponding gene by complementation. This gene (USX1) encodes the previously identified UDP‐xylose synthase. We demonstrated that it is necessary for both capsule xylosylation and C. neoformans virulence.

Systematic capsule gene disruption reveals the central role of galactose metabolism on Cryptococcus neoformans virulence.

The polysaccharidic capsule is the main virulence factor of Cryptococcus neoformans. It primarily comprised of two polysaccharides: glucuronoxylomannan (GXM, 88% of the capsule mass) and galactoxylomannan (GalXM, 7% of the capsule mass). We constructed a large collection of mutant strains in which genes potentially involved in capsule biosynthesis were deleted. We used a new post‐genomic approach to study the virulence of the strains. Primers specific for unique tags associated with the disruption cassette were used in a real‐time PCR virulence assay to measure the fungal burden of each strain in different organs of mice in multi‐infection experiments. With this very sensitive assay, we identified a putative UDP‐glucose epimerase (Uge1p) and a putative UDP‐galactose transporter (Ugt1p) essential for C. neoformans virulence. The uge1Δ and ugt1Δ strains are temperature sensitive and do not produce GalXM but synthesize a larger capsule. These mutant strains (GalXM negative, GXM positive) are not able to colonize the brain even at the first day of infection whereas GXM‐negative strains (GalXM positive) can still colonize the brain, although less efficiently than the wild‐type strain.

Identification of a New Family of Genes Involved in β-1,2-Mannosylation of Glycans in Pichia pastoris and Candida albicans

Structural studies of cell wall components of the pathogenic yeast Candida albicans have demonstrated the presence of β-1,2-linked oligomannosides in phosphopeptidomannan and phospholipomannan. During C. albicans infection, β-1,2-oligomannosides play an important role in host/pathogen interactions by acting as adhesins and by interfering with the host immune response. Despite the importance of β-1,2-oligomannosides, the genes responsible for their synthesis have not been identified. The main reason is that the reference species Saccharomyces cerevisiae does not synthesize β-linked mannoses. On the other hand, the presence of β-1,2-oligomannosides has been reported in the cell wall of the more genetically tractable C. albicans relative, P. pastoris. Here we present the identification, cloning, and characterization of a novel family of fungal genes involved in β-mannose transfer. Employing in silico analysis, we identified a family of four related new genes in P. pastoris and subsequently nine homologs in C. albicans. Biochemical, immunological, and structural analyses following deletion of four genes in P. pastoris and deletion of four genes acting specifically on C. albicans mannan demonstrated the involvement of these new genes in β-1,2-oligomannoside synthesis. Phenotypic characterization of the strains deleted in β-mannosyltransferase genes (BMTs) allowed us to describe the stepwise activity of Bmtps and acceptor specificity. For C. albicans, despite structural similarities between mannan and phospholipomannan, phospholipomannan β-mannosylation was not affected by any of the CaBMT1–4 deletions. Surprisingly, depletion in mannan major β-1,2-oligomannoside epitopes had little impact on cell wall surface β-1,2-oligomannoside antigenic expression.