Integrated post-genomic cell wall analysis reveals floating biofilm formation associated with high expression of flocculins in the pathogen Pichia kudriavzevii.

The pathogenic yeast Pichia kudriavzevii, previously known as Candida krusei, is more distantly related to Candida albicans than clinically relevant CTG-clade Candida species. Its cell wall, a dynamic organelle that is the first point of interaction between pathogen and host, is relatively understudied, and its wall proteome remains unidentified to date. Here, we present an integrated study of the cell wall in P. kudriavzevii. Our comparative genomic studies and experimental data indicate that the general structure of the cell wall in P. kudriavzevii is similar to Saccharomyces cerevisiae and C. albicans and is comprised of ß-1,3-glucan, ß-1,6-glucan, chitin, and mannoproteins. However, some pronounced differences with C. albicans walls were observed, for instance, higher mannan and protein levels and altered protein mannosylation patterns. Further, despite absence of proteins with high sequence similarity to Candida adhesins, protein structure modeling identified eleven proteins related to flocculins/adhesins in S. cerevisiae or C. albicans. To obtain a proteomic comparison of biofilm and planktonic cells, P. kudriavzevii cells were grown to exponential phase and in static 24-h cultures. Interestingly, the 24-h static cultures of P. kudriavzevii yielded formation of floating biofilm (flor) rather than adherence to polystyrene at the bottom. The proteomic analysis of both conditions identified a total of 33 cell wall proteins. In line with a possible role in flor formation, increased abundance of flocculins, in particular Flo110, was observed in the floating biofilm compared to exponential cells. This study is the first to provide a detailed description of the cell wall in P. kudriavzevii including its cell wall proteome, and paves the way for further investigations on the importance of flor formation and flocculins in the pathogenesis of P. kudriavzevii.

Characterization of Awp14, A Novel Cluster III Adhesin Identified in a High Biofilm-Forming Candida glabrata Isolate.

Candida glabrata is among the most prevalent causes of candidiasis. Unlike Candida albicans, it is not capable of changing morphology between yeast and hyphal forms but instead has developed other virulence factors. An important feature is its unprecedented large repertoire of predicted cell wall adhesins, which are thought to enable adherence to a variety of surfaces under different conditions. Here, we analyzed the wall proteome of PEU1221, a high biofilm-forming clinical strain isolated from an infected central venous catheter, under biofilm-forming conditions. This isolate shows increased incorporation of putative adhesins, including eight proteins that were not detected in walls of reference strain ATCC 2001, and of which Epa22, Awp14, and Awp2e were identified for the first time. The proteomics data suggest that cluster III adhesin Awp14 is relatively abundant in PEU1221. Phenotypic studies with awp14Δ deletion mutants showed that Awp14 is not responsible for the high biofilm formation of PEU1221 onto polystyrene. However, awp14Δ mutant cells in PEU1221 background showed a slightly diminished binding to chitin and seemed to sediment slightly slower than the parental strain suggesting implication in fungal cell-cell interactions. By structural modeling, we further demonstrate similarity between the ligand-binding domains of cluster III adhesin Awp14 and those of cluster V and VI adhesins. In conclusion, our work confirms the increased incorporation of putative adhesins, such as Awp14, in high biofilm-forming isolates, and contributes to decipher the precise role of these proteins in the establishment of C. glabrata infections.

Host Age and Denture Wearing Jointly Contribute to Oral Colonization with Intrinsically Azole-Resistant Yeasts in the Elderly.

In elderly patients, several morbidities or medical treatments predisposing for fungal infections occur at a higher frequency, leading to high mortality and morbidity in this vulnerable patient group. Often, this is linked to an innately azole-resistant yeast species such as Candida glabrata or C. krusei. Additionally, host age per se and the wearing of dentures have been determined to influence the mix of colonizing species and, consequently, the species distribution of invasive fungal infections. Since both old age and the wearing of dentures are two tightly connected parameters, it is still unclear which of them is the main contributor. Here, we performed a cross-sectional study on a cohort (N = 274) derived from three groups of healthy elderly, diseased elderly, and healthy young controls. With increasing host age, the frequency of oral colonization by a non-albicans Candida species, mainly by C. glabrata, also increased, and the wearing of dentures predisposed for colonization by C. glabrata irrespectively of host age. Physically diseased hosts, on the other hand, were more frequently orally colonized by C. albicans than by other yeasts. For both C. albicans and C. glabrata, isolates from the oral cavity did not generally display an elevated biofilm formation capacity. In conclusion, intrinsically azole-drug-resistant, non-albicans Candida yeasts are more frequent in the oral cavities of the elderly, and fungal cells not contained in biofilms may predispose for subsequent systemic infection with these organisms. This warrants further exploration of diagnostic procedures, e.g., before undergoing elective abdominal surgery or when using indwelling devices on this patient group.

Mass Spectrometry-Based Proteomic and Immunoproteomic Analyses of the Candida albicans Hyphal Secretome Reveal Diagnostic Biomarker Candidates for Invasive Candidiasis.

Invasive candidiasis (IC) is associated with high morbidity and mortality in hospitalized patients if not diagnosed early. Long-term use of central venous catheters is a predisposing factor for IC. Hyphal forms of Candida albicans (the major etiological agent of IC) are related to invasion of host tissues. The secreted proteins of hyphae are involved in virulence, host interaction, immune response, and immune evasion. To identify IC diagnostic biomarker candidates, we characterized the C. albicans hyphal secretome by gel-free proteomic analysis, and further assessed the antibody-reactivity patterns to this subproteome in serum pools from 12 patients with non-catheter-associated IC (ncIC), 11 patients with catheter-associated IC (cIC), and 11 non-IC patients. We identified 301 secreted hyphal proteins stratified to stem from the extracellular region, cell wall, cell surface, or intracellular compartments. ncIC and cIC patients had higher antibody levels to the hyphal secretome than non-IC patients. Seven secreted hyphal proteins were identified to be immunogenic (Bgl2, Eno1, Pgk1, Glx3, Sap5, Pra1 and Tdh3). Antibody-reactivity patterns to Bgl2, Eno1, Pgk1 and Glx3 discriminated IC patients from non-IC patients, while those to Sap5, Pra1 and Tdh3 differentiated between cIC and non-IC patients. These proteins may be useful for development of future IC diagnostic tests.

High Biofilm Formation of Non-Smooth Candida parapsilosis Correlates with Increased Incorporation of GPI-Modified Wall Adhesins.

Candida parapsilosis is among the most frequent causes of candidiasis. Clinical isolates of this species show large variations in colony morphotype, ranging from round and smooth to a variety of non-smooth irregular colony shapes. A non-smooth appearance is related to increased formation of pseudohyphae, higher capacity to form biofilms on abiotic surfaces, and invading agar. Here, we present a comprehensive study of the cell wall proteome of C. parapsilosis reference strain CDC317 and seven clinical isolates under planktonic and sessile conditions. This analysis resulted in the identification of 40 wall proteins, most of them homologs of known Candida albicans cell wall proteins, such as Gas, Crh, Bgl2, Cht2, Ecm33, Sap, Sod, Plb, Pir, Pga30, Pga59, and adhesin family members. Comparative analysis of exponentially growing and stationary phase planktonic cultures of CDC317 at 30 °C and 37 °C revealed only minor variations. However, comparison of smooth isolates to non-smooth isolates with high biofilm formation capacity showed an increase in abundance and diversity of putative wall adhesins from Als, Iff/Hyr, and Hwp families in the latter. This difference depended more strongly on strain phenotype than on the growth conditions, as it was observed in planktonic as well as biofilm cells. Thus, in the set of isolates analyzed, the high biofilm formation capacity of non-smooth C. parapsilosis isolates with elongated cellular phenotypes correlates with the increased surface expression of putative wall adhesins in accordance with their proposed cellular function.

Candida parapsilosis Colony Morphotype Forecasts Biofilm Formation of Clinical Isolates.

Candida parapsilosis is a frequent cause of fungal bloodstream infections, especially in critically ill neonates or immunocompromised patients. Due to the formation of biofilms, the use of indwelling catheters and other medical devices increases the risk of infection and complicates treatment, as cells embedded in biofilms display reduced drug susceptibility. Therefore, biofilm formation may be a significant clinical parameter, guiding downstream therapeutic choices. Here, we phenotypically characterized 120 selected isolates out of a prospective collection of 215 clinical C. parapsilosis isolates, determining biofilm formation, major emerging colony morphotype, and antifungal drug susceptibility of the isolates and their biofilms. In our isolate set, increased biofilm formation capacity was independent of body site of isolation and not predictable using standard or modified European Committee on Antimicrobial Susceptibility Testing (EUCAST) drug susceptibility testing protocols. In contrast, biofilm formation was strongly correlated with the appearance of non-smooth colony morphotypes and invasiveness into agar plates. Our data suggest that the observation of non-smooth colony morphotypes in cultures of C. parapsilosis may help as an indicator to consider the initiation of anti-biofilm-active therapy, such as the switch from azole- to echinocandin- or polyene-based strategies, especially in case of infections by potent biofilm-forming strains.

Phenotypic Variability in a Coinfection With Three Independent Candida parapsilosis Lineages.

The human pathogenic yeast Candida parapsilosis has gained significant importance over the past decades as one of the principal causes of fungal bloodstream infections. Isolates of C. parapsilosis are known to be able to switch between several different colony morphologies in vitro, which are correlated with different cell shapes, altered cell surface properties, and thus different capacities to form biofilms on indwelling medical devices. In a set of six clinical specimens from a single surgery patient yielding stable smooth- as well as crepe-morphology isolates, we investigated the differences between five of them on a phenotypic and genomic level. In contrast to the initial assumption that they were switched forms of a clonal strain, karyotyping and genome sequencing showed that the patient was colonized by at least three distinct linages. Statistical analysis placed these groups distantly across the population of C. parapsilosis. Interestingly the single blood culture isolate was of smooth morphology and matched with an isolate from the patient's nose of similar morphology. Strong variation between the isolates was seen in adhesin-encoding genes, where repeat regions showed significant variation in length and repeat-numbers, most strikingly in HWP1 of the smooth isolates. Although no differences in drug susceptibility were evident, the high phylogenetic distance separating the individual strains highlights the need for testing of multiple colonies in routine practice. The absence of biofilm formation in the blood stream isolate indicates a lack of respective adhesins in the cell wall, in turn pointing toward lack of adhesion as a positively contributing factor for dissemination.

Molecular Typing of Candida glabrata.

The yeast Candida glabrata has emerged, second only to Candida albicans, to be one of the most frequently isolated fungi in clinical specimen from human. Its frequent resistance towards azole antifungal drugs and the high capacity to form biofilms on indwelling catheters of individual isolates render it an often difficult to treat pathogen. Hence, there is a notably increasing scientific and clinical interest in this species. This has led to the development of a variety of molecular tools for genetic modification, strain collections, and last but not least different approaches to analyse the population structure among isolates of different geographical and clinical contexts. Often, these are used to study correlations (or the absence thereof) with different pathogenicity, virulence, or drug resistance traits. Three molecular methods have been used to type within the C. glabrata population on a genetic level by multiple studies: multi-locus sequence typing, microsatellite length polymorphisms, and clustering of whole-genome sequencing data, and these are subject of this review.

Genome Comparisons of Candida glabrata Serial Clinical Isolates Reveal Patterns of Genetic Variation in Infecting Clonal Populations.

Candida glabrata is an opportunistic fungal pathogen that currently ranks as the second most common cause of candidiasis. Although the mechanisms underlying virulence and drug resistance in C. glabrata are now starting to be elucidated, we still lack a good understanding of how this yeast adapts during the course of an infection. Outstanding questions are whether the observed genomic plasticity of C. glabrata plays a role during infection, or what levels of genetic variation exist within an infecting clonal population. To shed light onto the genomic variation within infecting C. glabrata populations, we compared the genomes of 11 pairs and one trio of serial clinical isolates, each obtained from a single patient. Our results provide a catalog of genetic variations existing within clonal infecting isolates, and reveal an enrichment of non-synonymous changes in genes encoding cell-wall proteins. Genetic variation and the presence of non-synonymous mutations and copy number variations accumulated within the host, suggest that clonal populations entail a non-negligible level of genetic variation that may reflect selection processes that occur within the human body. As we show here, these genomic changes can underlie phenotypic differences in traits that are relevant for infection.

Patterns of Genomic Variation in the Opportunistic Pathogen Candida glabrata Suggest the Existence of Mating and a Secondary Association with Humans.

Candida glabrata is an opportunistic fungal pathogen that ranks as the second most common cause of systemic candidiasis. Despite its genus name, this yeast is more closely related to the model yeast Saccharomyces cerevisiae than to other Candida pathogens, and hence its ability to infect humans is thought to have emerged independently. Moreover, C. glabrata has all the necessary genes to undergo a sexual cycle but is considered an asexual organism due to the lack of direct evidence of sexual reproduction. To reconstruct the recent evolution of this pathogen and find footprints of sexual reproduction, we assessed genomic and phenotypic variation across 33 globally distributed C. glabrata isolates. We cataloged extensive copy-number variation, which particularly affects genes encoding cell-wall-associated proteins, including adhesins. The observed level of genetic variation in C. glabrata is significantly higher than that found in Candida albicans. This variation is structured into seven deeply divergent clades, which show recent geographical dispersion and large within-clade genomic and phenotypic differences. We show compelling evidence of recent admixture between differentiated lineages and of purifying selection on mating genes, which provides the first evidence for the existence of an active sexual cycle in this yeast. Altogether, our data point to a recent global spread of previously genetically isolated populations and suggest that humans are only a secondary niche for this yeast.

Identification of Secreted Candida Proteins Using Mass Spectrometry.

Analysis of fungal secretomes using mass spectrometry is a useful technique in cell biology. Knowledge of the secretome of a human fungal pathogen may yield important information of host-pathogen interactions and may be useful for identifying vaccines candidates or diagnostic markers for antifungal strategies. In this chapter, with a main focus on sample preparation aspects, we describe the methodology that we apply for gel-independent batch identification and quantification of proteins that are secreted during growth in liquid cultures. Using these techniques with Candida and other yeast species, the majority of the identified proteins are classical secretory proteins and cell wall proteins containing N-terminal signal peptides for secretion, although dependent on sample preparation quality and the mass spectrometric analysis also usually, a number of nonsecretory proteins are identified.

Proteomic analysis of hyperadhesive Candida glabrata clinical isolates reveals a core wall proteome and differential incorporation of adhesins.

Attachment to human host tissues or abiotic medical devices is a key step in the development of infections by Candida glabrata. The genome of this pathogenic yeast codes for a large number of adhesins, but proteomic work using reference strains has shown incorporation of only few adhesins in the cell wall. By making inventories of the wall proteomes of hyperadhesive clinical isolates and reference strain CBS138 using mass spectrometry, we describe the cell wall proteome of C. glabrata and tested the hypothesis that hyperadhesive isolates display differential incorporation of adhesins. Two clinical strains (PEU382 and PEU427) were selected, which both were hyperadhesive to polystyrene and showed high surface hydrophobicity. Cell wall proteome analysis under biofilm-forming conditions identified a core proteome of about 20 proteins present in all C. glabrata strains. In addition, 12 adhesin-like wall proteins were identified in the hyperadherent strains, including six novel adhesins (Awp8-13) of which only Awp12 was also present in CBS138. We conclude that the hyperadhesive capacity of these two clinical C. glabrata isolates is correlated with increased and differential incorporation of cell wall adhesins. Future studies should elucidate the role of the identified proteins in the establishment of C. glabrata infections.