PLB-985 Neutrophil-Like Cells as a Model To Study Aspergillus fumigatus Pathogenesis

Polymorphonuclear leukocytes are an important defense against human fungal pathogens, yet our model systems to study this group of cells remain very limited in scope. In this study, we established that differentiated PLB-985 cells can serve as a model to recapitulate several important aspects of human polymorphonuclear leukocyte interactions with the important human fungal pathogen Aspergillus fumigatus .

Chromosome-Level Genome Assembly of a Human Fungal Pathogen Reveals Synteny among Geographically Distinct Species

Histoplasma species are dimorphic fungi causing significant morbidity and mortality worldwide. These fungi grow as mold in the soil and as budding yeast within the human host. Histoplasma can be isolated from soil in diverse regions, including North America, South America, Africa, and Europe.

Methods for Manipulating Cryptococcus Spores

Spores are essential for the long-term survival of many diverse organisms, due to their roles in reproduction and stress resistance. In the environmental human fungal pathogen, Cryptococcus, basidiospores are robust cells with the ability to cause disease in animal models of infection. Here we describe methods for producing and purifying Cryptococcus basidiospores in quantities sufficient for large-scale analyses. The production of high numbers of pure spores has facilitated the development of new assays, including quantitative germination assays, and enabled transcriptomic, proteomic, and virulence studies, leading to discoveries of behaviors and properties unique to spores and spore-mediated disease.

A J Domain Protein Functions as a Histone Chaperone to Maintain Genome Integrity and the Response to DNA Damage in a Human Fungal Pathogen

DNA replication, gene expression, and genomic repair all require precise coordination of the many proteins that interact with DNA. This includes the histones as well as their chaperones.

CO2 accelerates Candida albicans biofilm formation and reveals novel approaches to their inhibition on airway management devices

C. albicans is the predominant human fungal pathogen worldwide and frequently colonises medical devices, such as voice prosthesis, as a biofilm. It is a dimorphic yeast that can switch between yeast and hyphal forms in response to environmental cues, a property that is essential during biofilm establishment and maturation. One such cue is elevation of CO2 levels, as observed in exhaled breath for example. However, despite the clear medical relevance the effects of high CO2 levels on C. albicans biofilm growth has not been investigated to date. Here, we show that 5% CO2 significantly enhances each stage of the C. albicans biofilm forming process; from attachment through maturation to dispersion, via stimulation of the Ras/cAMP/PKA signalling pathway. Transcriptome analysis of biofilm formation under elevated CO2 conditions revealed the activation of key biofilm formation pathways governed by the central biofilm regulators Efg1, Brg1, Bcr1 and Ndt80. Biofilms grown in under elevated CO2 conditions also exhibit increases in azole resistance, tolerance to nutritional immunity and enhanced glucose uptake capabilities. We thus characterise the mechanisms by which elevated CO2 promote C. albicans biofilm formation. We also investigate the possibility of re-purposing drugs that can target the CO2 activated metabolic enhancements observed in C. albicans biofilms. Using this approach we can significantly reduce multi-species biofilm formation in a high CO2 environment and demonstrate a significant extension of the lifespan of voice prostheses in a patient trial. Our research demonstrates a bench to bedside approach to tackle Candida albicans biofilm formation.

Tolerance to fluconazole in Candida albicans is regulated by temperature and aneuploidy

Candida albicans is a prevalent human fungal pathogen. Azoles are the most widely used antifungal drugs. Drug tolerance in bacteria is well defined and thoroughly studied, but in fungi, the definition of drug tolerance and the mechanism that drive it are not well understood. Here, we found that a large proportion of clinical isolates were intrinsically tolerant to fluconazole, and/or could be induced by high temperature (37°C) to become tolerant (conditionally tolerant). When treated with inhibitory doses of fluconazole, non-tolerant strains became tolerant by forming aneuploids involving different chromosomes, with chromosome R duplication as the most recurrent mechanism. Tolerance determines the ability to grow in the presence of fluconazole and other azoles, in a manner independent of the MIC. Both temperature conditional tolerance and the associated aneuploidy were sensitive to FK506, an inhibitor of calcineurin. Intrinsic and conditional tolerance were also abolished by deletions of genes encoding the calcineurin (CMP1 and CNB1). However, the dependence of tolerance on calcineurin could be bypassed by a different aneuploid chromosome. Thus, fluconazole tolerance in C. albicans is regulated by temperature and by aneuploidy and is dependent upon aneuploidy, but this dependence can be bypassed by an additional aneuploidy.

Genetic analysis of sirtuin deacetylases in hyphal growth of Candida albicans

Candida albicans is a major human fungal pathogen that encounters varied host environments during infection. In response to environmental cues, C. albicans switches between ovoid yeast and elongated hyphal growth forms, and this morphological plasticity contributes to virulence. Environmental changes that alter the cell’s metabolic state could be sensed by sirtuins, which are NAD+-dependent deacetylases. Here we studied the roles of three sirtuin deacetylases, Sir2, Hst1, and Hst2, in hyphal growth of C. albicans. We made single, double, and triple sirtuin knockout strains and tested their ability to switch from yeast to hyphae. We found that true hyphae formation was significantly reduced by the deletion of SIR2 but not HST1 or HST2. Moreover, the expression of hyphal-specific genes HWP1, ALS3, and ECE1 decreased in the sir2Δ/Δ mutant compared to wild-type. This regulation of hyphae formation was dependent on the deacetylase activity of Sir2, as a point mutant lacking deacetylase activity had a similar defect in hyphae formation as the sir2Δ/Δ mutant. Finally, we found that Sir2 and Hst1 were localized to the nucleus, with Sir2 specifically focused in the nucleolus. This nuclear localization suggests a role for Sir2 and Hst1 in regulating gene expression. In contrast, Hst2 was localized to the cytoplasm. In conclusion, our results suggest that Sir2 plays a critical and non-redundant role in hyphal growth of C. albicans.

A GDPase/UDPase Bifunctional Enzyme From Candida Albicans: Purification and Biochemical Characterization

The most frequently isolated human fungal pathogen is Candida albicans which is responsible for about 50% of all Candida infections. In healthy individuals, this organism resides as a part of the normal microbiota in equilibrium with the host. However, under certain conditions, particularly in immunocompromised patients, this opportunistic pathogen adheres to host cells causing serious systemic infections. Thus, much effort has been dedicated to the study of its physiology with emphasis on factors associated to pathogenicity. A representative analysis deals with the mechanisms of glycoprotein assembly as many cell surface antigens and other macromolecules that modulate the immune system fall within this chemical category. In this regard, studies of the terminal protein glycosylation stage which occurs in Golgi vesicles has led to the identification of nucleotidases that convert glycosyltransferase-generated dinucleotides into the corresponding mononucleotides, thus playing a double function: their activity prevent inhibition of further glycosyl transfer by the accumulation of dinucleotides and the resulting mononucleotides are exchanged by specific membrane transporters for equimolecular amounts of sugar donors from the cytosol. Here, using a simple protocol for protein separation we isolated a bifunctional nucleotidase from C. albicans active on GDP and UDP that was characterized in terms of its molecular mass, response to bivalent ions and other factors, substrate specificity and affinity. Results are discussed in terms of the similarities and differences of this nucleotidase with similar counterparts from other organisms thus contributing to the knowledge of a bifunctional diphosphatase not described before in C. albicans.

Microdomain protein Nce102 is a local sensor of plasma membrane sphingolipid balance

Sphingolipids are essential building blocks of eukaryotic membranes and important signalling molecules, tightly regulated in response to environmental and physiological inputs. Mechanism of sphingolipid level perception at the plasma membrane remains unclear. In Saccharomyces cerevisiae, Nce102 protein has been proposed to function as sphingolipid sensor as it changes its plasma membrane distribution in response to sphingolipid biosynthesis inhibition. We show that Nce102 redistributes specifically in regions of increased sphingolipid demand, e.g., membranes of nascent buds. Furthermore, we report that production of Nce102 increases following sphingolipid biosynthesis inhibition and Nce102 is internalized when excess sphingolipid precursors are supplied. This suggests that the total amount of Nce102 in the plasma membrane is a measure of the current need for sphingolipids, whereas its local distribution marks sites of high sphingolipid demand. Physiological role of Nce102 in regulation of sphingolipid synthesis is demonstrated by mass spectrometry analysis showing reduced levels of complex sphingolipids and long-chain bases in nce102? deletion mutant. Nce102 behaves analogously in human fungal pathogen Candida albicans, suggesting a conserved principle of local sphingolipid control across species.

Host Lung Environment Limits Aspergillus fumigatus Germination through an SskA-Dependent Signaling Response

Aspergillus fumigatus is an important human fungal pathogen particularly in immunocompromised individuals. Initiation of growth by A. fumigatus in the lung is important for its pathogenicity in murine models.