Sébastien C. Ortiz

Background Pulmonary infections caused by Aspergillus fumigatus are a global health concern. In the lungs, A. fumigatus employs a variety of virulence and adaptation mechanisms that allows it to survive and evade local immune responses. A. fumigatus protein kinases co-ordinately regulate adaptation to the lung environment and antifungal agents. However, to improve our understanding of the role of these fungal protein kinases in subverting the antifungal responses exhibited by specific host cells, in vitro infection models should be developed. Here, we describe a functional genomics approach to evaluate the role of A. fumigatus protein kinases upon infection of airway epithelial cells, leveraging the use of our existing library of A. fumigatus deletion mutants for kinase functions and bar-seq. Methods We infected A549 alveolar epithelial cells with a pool of 111 A. fumigatus barcoded null mutant strains in genes encoding protein kinases and used bar-seq to identify which of these are involved in fungal survival upon A549 infection. To assess the optimal method for understanding host-pathogen interactions, three approaches aimed at removing extracellular conidia were evaluated: pharmacological treatment with the antifungal nystatin, washes of unbound conidia, and differential fluorescent staining of extracellular and intracellular conidia followed by cell sorting. The identified candidates were further validated using targeted approaches. Results Our data revealed new roles for A. fumigatus protein kinases during infection of airway epithelial cells. Modification of the experimental setup allowed us to differentiate between the genes associated with uptake and those required to withstand other stresses imposed by exposure to mammalian cells. Conclusions This protocol provides a new platform to ascribe pathogenicity-associated functions to A. fumigatus genes and allows prioritization for downstream experiments in mice, potentially reducing the number of studies needed in live animals. This is particularly pertinent, as genome-wide barcoded collections with thousands of strains have become available.

Background Pulmonary infections caused by Aspergillus fumigatus are a global health concern. In the lungs, A. fumigatus employs a variety of virulence and adaptation mechanisms that allows it to survive and evade local immune responses. A. fumigatus protein kinases co-ordinately regulate adaptation to the lung environment and antifungal agents. However, to improve our understanding of the role of these fungal protein kinases in subverting the antifungal responses exhibited by specific host cells, in vitro infection models should be developed. Here, we describe a functional genomics approach to evaluate the role of A. fumigatus protein kinases upon infection of airway epithelial cells, leveraging the use of our existing library of A. fumigatus deletion mutants for kinase functions and bar-seq. Methods We infected A549 alveolar epithelial cells with a pool of 111 A. fumigatus barcoded null mutant strains in genes encoding protein kinases and used bar-seq to identify which of these are involved in fungal survival upon A549 infection. To assess the optimal method for understanding host-pathogen interactions, three approaches aimed at removing extracellular conidia were evaluated: pharmacological treatment with the antifungal nystatin, washes of unbound conidia, and differential fluorescent staining of extracellular and intracellular conidia followed by cell sorting. The identified candidates were further validated using targeted approaches. Results Our data revealed new roles for A. fumigatus protein kinases during infection of airway epithelial cells. Modification of the experimental setup allowed us to differentiate between the genes associated with uptake and those required to withstand other stresses imposed by exposure to mammalian cells. Conclusions This protocol provides a new platform to ascribe pathogenicity-associated functions to A. fumigatus genes and allows prioritization for downstream experiments in mice, potentially reducing the number of studies needed in live animals. This is particularly pertinent, as genome-wide barcoded collections with thousands of strains have become available.

Human fungal pathogens, including Cryptococcus neoformans, cause 1.5 million annual deaths. Cryptococcus causes disease when it disseminates out of the lung and into the brain, which can occur years after initial exposure (latency) via mechanisms that remain unknown. Spores of Cryptococcus display distinct surface epitopes, host interactions, and disease kinetics to the vegetatively growing yeast morphotype, yet they remain understudied, likely contributing to our lack of understanding of pathogenesis. One of the first barriers spores encounter is non-professional phagocytic a irway e pithelial c ells (AECs). Here, we demonstrate that Cryptococcus spores are preferentially internalized by AECs both in vitro and in vivo . Once inside, spores can germinate, subsequently replicate, persist, and/or escape. This ability to enter AECs correlates with a preferential ability of spores to cross AEC barriers. Together, our work indicates that AECs internalize Cryptococcus spores and may serve as a previously ignored intracellular host niche, providing alternative hypotheses for both Cryptococcus dissemination and latency. IMPORTANCE: Fungal spores are a dormant, stress-resistant, and relatively understudied cell type and are presumed infectious cell types in cryptococcal disease. Cryptococcus spores have been shown to display distinct disease kinetics to the vegetative yeast morphotype and are significantly better at disseminating out of the host lung. While the molecular mechanisms by which spores disseminate out of the lung have yet to be identified, their preferential ability to get inside host cells likely enables their dissemination. Here, we show that spores, unlike yeast, readily get taken up by non-professional phagocytic cells, airway epithelial cells, both in vitro and in vivo, and once inside can germinate, replicate, escape, and/or persist. These results provide a previously unexplored host cell type that Cryptococcus can inhabit, likely affecting disease kinetics and could be a critical interaction in understanding both extrapulmonary dissemination and latency.

The human lung is confronted daily with thousands of microbial invaders reaching the lower respiratory tract. An efficient response by the resident type 1 and type 2 alveolar epithelial cells (AECs) and alveolar macrophages (AMs) cells during the early hours of innate immunity is a prerequisite to maintain a non-inflammatory state, but foremost to rapidly remove harmful substances. One such human-pathogenic invader is the opportunistic fungus Aspergillus fumigatus. If the spherical conidia are not cleared in time, they swell reaching approximately twice of their initial size and germinate to develop hyphae around six hours post-infection. This process of morphological change is crucial as it enables the pathogen to invade the alveolar epithelium and to reach the bloodstream, but also makes it conspicuous for the immune system. During this process, conidia are first in contact with AECs then with migrating AMs, both attempting to internalize and clear the fungus. However, the relative contribution of AMs and AECs to uptake of A. fumigatus remains an open question, especially the capabilities of the barely investigated type 1 AECs. In this study, we present a bottom-up modeling approach to incorporate experimental data on the dynamic increase of the conidial diameter and A. fumigatus uptake by AECs and AMs in a hybrid agent-based model (hABM) for the to-scale simulation of virtual infection scenarios in the human alveolus. By screening a wide range of parameters, we found that type 1 AECs, which cover approximately 95% of the alveolar surface, are likely to have a greater impact on uptake than type 2 AECs. Moreover, the majority of infection scenarios across the regime of tested parameters were cleared through uptake by AMs, whereas the contribution to conidial uptake by AECs was observed to be limited, indicating that their crucial support might mostly consist in mediating chemokine secretion for AM recruitment. Regardless, as the first host cell being confronted with A. fumigatus conidia, our results evidence the large potential impact of type 1 AECs antimicrobial activities, underlining the requirement of increasing experimental efforts on this alveolar constituent.

SUMMARY: Spores are primary infectious propagules for the majority of human fungal pathogens; however, relatively little is known about their fundamental biology. One strategy to address this deficiency has been to develop the basidiospores of Cryptococcus into a model for pathogenic spore biology. Here, we provide an update on the state of the field with a comprehensive review of the data generated from the study of Cryptococcus basidiospores from their formation (sporulation) and differentiation (germination) to their roles in pathogenesis. Importantly, we provide support for the presence of basidiospores in nature, define the key characteristics that distinguish basidiospores from yeast cells, and clarify their likely roles as infectious particles. This review is intended to demonstrate the importance of basidiospores in the field of Cryptococcus research and provide a solid foundation from which researchers who wish to study sexual spores in any fungal system can launch their studies.

Human fungal pathogens, including Cryptococcus neoformans, cause 1.5 million annual deaths. Cryptococcus causes disease when it disseminates out of the lung and into the brain which can occur years after initial exposure (latency) via mechanisms that remain unknown. Spores of Cryptococcus display distinct surface epitopes, host-interactions, and disease kinetics to the vegetatively growing yeast morphotype, yet they remain understudied likely contributing to our lack of understanding of pathogenesis. One of the first barriers spores encounter are non-professional phagocytic A irway E pithelial C ells (AECs). Here, we demonstrated that Cryptococcus spores preferentially invade AECs both in vitro and in vivo . Once inside spores can germinate, subsequently replicate, persist and/or escape. This ability to enter AECs correlates with a preferential ability of spore to cross AEC barriers. Together our work indicates that AECs can be invaded by Cryptococcus spores and may serve as a previously ignored intracellular host niche, providing alternative hypothesis for both Cryptococcus dissemination and latency.

Spores are critical morphotypes required for long-term survival of most fungi. Under the right environmental conditions, spores can escape dormancy and differentiate into vegetatively growing cells through the process of germination. For fatal human fungal pathogens like Cryptococcus , germination is the key differentiation process required for spores to initiate vegetative growth and ultimately cause disease; however, relatively little is known about the molecular mechanisms that control germination. To this end, we performed an extensive characterization of Cryptococcus spore germination through the morphological assessment of germinating spores, the inhibition of key eukaryotic processes, and the detailed quantification of fungal spore germination kinetics under numerous nutrient conditions. We identified temporal associations between molecular and morphological events and determined that carbon metabolism pathways (both glycolysis and oxidative phosphorylation) were required from the beginning of germination. We further determined that carbon sources are primarily used as fuel rather than as simply triggers of germination ‘commitment,’ and identified spore-specific carbon source utilization that is absent in yeast. Finally, we discovered the first spore-specific enzyme, Nth2, a trehalase that is required for germination when trehalose is the primary available carbon source. Together this work provides an extensive characterization of Cryptococcus spore germination and suggests that germination is more than simply a ‘modified cell cycle’ but is rather a highly adapted differentiation process.

Aspergillus fumigatus spores initiate more than 3,000,000 chronic and 300,000 invasive diseases annually, worldwide. Depending on the immune status of the host, inhalation of these spores can lead to a broad spectrum of disease, including invasive aspergillosis, which carries a 50% mortality rate overall; however, this mortality rate increases substantially if the infection is caused by azole-resistant strains or diagnosis is delayed or missed. Increasing resistance to existing antifungal treatments is becoming a major concern; for example, resistance to azoles (the first-line available oral drug against Aspergillus species) has risen by 40% since 2006. Despite high morbidity and mortality, the lack of an in-depth understanding of A. fumigatus pathogenesis and host response has hampered the development of novel therapeutic strategies for the clinical management of fungal infections. Recent advances in sample preparation, infection models and imaging techniques applied in vivo have addressed important gaps in fungal research, whilst questioning existing paradigms. This review highlights the successes and further potential of these recent technologies in understanding the host–pathogen interactions that lead to aspergillosis.

In studying the development of tolerance to common hospital cleaners (Oxivir^® and CaviCide™) in clinical isolate stocks of the emerging, multidrug-resistant yeast pathogen Candida auris, we selected for a cleaner-tolerant subpopulation of a more common nosocomial pathogen, Candida glabrata. Through the purification of each species and subsequent competition and other analyses, we determined that C. glabrata is capable of readily dominating mixed populations of C. auris and C. glabrata when exposed to hospital cleaners. This result suggests that exposure to antimicrobial compounds can preferentially select for low-level, stress-tolerant fungal pathogens. These findings indicate that clinical disinfection practices could contribute to the selection of tolerant, pathogenic microbes that persist within healthcare settings.

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.