Comprehensive understanding of an organism requires that we understand the contributions of most, if not all, of its genes. Classical genetic approaches to this issue have involved systematic deletion of each gene in the genome, with comprehensive sets of mutants available only for very-well-studied model organisms. We took a different approach, harnessing the power of in vivo transposition coupled with deep sequencing to identify >500,000 different mutations, one per cell, in the prevalent human fungal pathogen Candida albicans and to map their positions across the genome. The transposition approach is efficient and less labor-intensive than classic approaches. Here, we describe the production and analysis (aided by machine learning) of a large collection of mutants and the comprehensive identification of 1,610 C. albicans genes that are essential for growth under standard laboratory conditions. Among these C. albicans essential genes, we identify those that are also essential in two distantly related model yeasts as well as those that are conserved in all four major human fungal pathogens and that are not conserved in the human genome. This list of genes with functions important for the survival of the pathogen provides a good starting point for the development of new antifungal drugs, which are greatly needed because of the emergence of fungal pathogens with elevated resistance and/or tolerance of the currently limited set of available antifungal drugs.

HighlightsThe cell biology of Candida albicans is adapted both for life as a commensal and as a pathogen.C. albicans can either downregulate or upregulate virulence properties in the human host.This fungus modulates the activity of phagocytes to enable its own survival.Candida is metabolically flexible enabling it to survive in multiple niches in the host.

Aspergillus fumigatus causes invasive pulmonary disease in immunocompromised hosts and allergic asthma in atopic individuals. We studied the contribution of lung eosinophils to these fungal diseases. By in vivo intracellular cytokine staining and confocal microscopy, we observed that eosinophils act as local sources of IL-23 and IL-17. Remarkably, mice lacking eosinophils had a >95% reduction in the percentage of lung IL-23p19+ cells as well as markedly reduced IL-23 heterodimer in lung lavage fluid. Eosinophils killed A. fumigatus conidia in vivo. Eosinopenic mice had higher mortality rates, decreased recruitment of inflammatory monocytes, and decreased expansion of lung macrophages after challenge with conidia. All of these functions underscore a potential protective role for eosinophils in acute aspergillosis. Given the postulated role for IL-17 in asthma pathogenesis, we assessed whether eosinophils could act as sources of IL-23 and IL-17 in models where mice were sensitized to either A. fumigatus antigens or ovalbumin (OVA). We found IL-23p19+ IL-17AF+ eosinophils in both allergic models. Moreover, close to 95% of IL-23p19+ cells and >90% of IL-17AF+ cells were identified as eosinophils. These data establish a new paradigm in acute and allergic aspergillosis whereby eosinophils act not only as effector cells but also as immunomodulatory cells driving the IL-23/IL-17 axis and contributing to inflammatory cell recruitment.

Graphical abstract(a) Morphotypes of Candida albicans (blue sector, vegetative forms; pink sector, forms related to mating or changes in ploidy). Cell types shown include: budding yeast cells; elongated conjoined yeasts forming pseudohyphae; parallel-sided hyphae; chlamydospores formed from suspensor cells; enlarged goliath cells formed under zinc deprivation (unpublished study, image from Duncan Wilson); intestinal gut form cells (image from Suzanne Noble); mating competence defined by whitegrey-opaque cell transitions (images from Guanghua Huang); elongated chemotactic shmoo-mating projections leading to tetraploid zygote formation (images from David Soll and Karla Daniels); trimera formed by unequal chromosome segregation under antifungal exposure (image from Judith Berman). (b) Superficial yeast colonization and invasion of the chicken chorioallantoic membrane by C. albicans hyphal cells. AbstractCandida albicans is normally a harmless commensal of human beings, but it can cause superficial infections of the mucosa (oral/vaginal thrush) in healthy individuals and (rarely) infections of the skin or nails. It can also become invasive, causing life-threatening systemic and bloodstream infections in immunocompromised hosts, where the mortality rate can be as high as 50 %. It is the most common cause of serious fungal infection and is a common cause of nosocomial infections in hospitals. Some strains have been recognized that are resistant to azoles or echinocandins, which are the first-line antifungals for treatment of C. albicans infections.

Background: GPI anchor is essential for virulence of C. albicans. Little is known about its GPI biosynthetic pathway. We explore roles of two GPI-N-acetylglucosaminyltransferase subunits catalyzing the first step. Results: Subunits GPI2 and GPI19 are negatively co-regulated, affecting Ras1 activity and ERG11 levels, respectively. Conclusion: GPI2/GPI19 levels affect morphogenesis and ergosterol biosynthesis. Significance: C. albicans can be targeted by modulation of cross-talk among major pathways.