Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis.

Aspergillus species are globally ubiquitous saprophytes found in a variety of ecological niches. Almost 200 species of aspergilli have been identified, less than 20 of which are known to cause human disease. Among them, Aspergillus fumigatus is the most prevalent and is largely responsible for the increased incidence of invasive aspergillosis (IA) in the immunocompromised patient population. IA is a devastating illness, with mortality rates in some patient groups reaching as high as 90%. Studies identifying and assessing the roles of specific factors of A. fumigatus that contribute to the pathogenesis of IA have traditionally focused on single-gene deletion and mutant characterization. In combination with recent large-scale approaches analyzing global fungal responses to distinct environmental or host conditions, these studies have identified many factors that contribute to the overall pathogenic potential of A. fumigatus. Here, we provide an overview of the significant findings regarding A. fumigatus pathogenesis as it pertains to invasive disease.

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs.

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for future pandemics.

Aspergillus fumigatus LaeA-mediated phagocytosis is associated with a decreased hydrophobin layer.

Aspergillus fumigatus is the causal agent of the life-threatening disease invasive aspergillosis. A. fumigatus laeA deletants, aberrant in toxin biosynthesis and spore development, are decreased in virulence. Among other characteristics, the decreased virulence is associated with increased spore susceptibility to macrophage phagocytosis. Three characteristics, cell wall microbe-associated molecular patterns (MAMPs), secreted metabolites, and rodlet content, thought to be important in macrophage-Aspergillus spore interactions were examined. Flow cytometry analysis of wild-type and DeltalaeA spores did not reveal any differences in surface-accessible MAMPs, including beta-(1,3)-glucan, alpha-mannose, chitin, and other carbohydrate ligands. Blocking experiments with laminarin and mannan supported the conclusion that differences in cell wall carbohydrates were not responsible for enhanced DeltalaeA spore phagocytosis. Aspergillus spores have been reported to secrete metabolites affecting phagocytosis. Neither spent culture exchange, transwell, nor coincubation internalization experiments supported a role for secreted metabolites in the differential uptake of wild-type and DeltalaeA spores. However, sonication assays implicated a role for surface rodlet protein/hydrophobin (RodAp) in differential spore phagocytosis. A possible role of RodAp in enhanced DeltalaeA spore uptake was further assessed by RodAp extraction and quantification, where wild-type spores were found to contain 60% more RodAp than DeltalaeA spores. After removal of the surface rodlet layer, wild-type spores were phagocytosed at similar rates as DeltalaeA spores. We conclude that increased uptake of DeltalaeA resting spores is not associated with changes in secreted metabolite production of this mutant or surface carbohydrate availability but, rather, due to a decrease in the surface RodAp content of DeltalaeA spores. We theorize that RodAp acts as an antiphagocytic molecule, possibly via physicochemical means and/or by impeding MAMP recognition by macrophage receptors.

Elucidating the pathogenesis of spores from the human fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans was first described as a human fungal pathogen more than a century ago. One aspect of the C. neoformans infectious life cycle that has been the subject of earnest debate is whether the spores are pathogenic. Despite much speculation, no direct evidence has been presented to resolve this outstanding question. We present evidence that C. neoformans spores are pathogenic in a mouse intranasal inhalation model of infection. In addition, we provide mechanistic insights into spore-host interactions. We found that C. neoformans spores were phagocytosed by alveolar macrophages via interactions between fungal beta-(1,3)-glucan and the host receptors Dectin-1 and CD11b. Moreover, we discovered an important link between spore survival and macrophage activation state: intracellular spores were susceptible to reactive oxygen-nitrogen species. We anticipate these results will serve as the basis for a model to further investigate the pathogenic implications of infections caused by fungal spores.

HdaA, a class 2 histone deacetylase of Aspergillus fumigatus, affects germination and secondary metabolite production.

Histone deacetylases (HDACs) play an important role in regulation of gene expression through histone modifications. Here we show that the Aspergillus fumigatus HDAC HdaA is involved in regulation of secondary metabolite production and is required for normal germination and vegetative growth. Deletion of the hdaA gene increased the production of several secondary metabolites but decreased production of gliotoxin whereas over-expression hdaA increased production of gliotoxin. RT-PCR analysis of 14 nonribosomal peptide synthases indicated HdaA regulation of up to nine of them. A mammalian cell toxicity assay indicated increased activity in the over-expression strain. Neither mutant affected virulence of the fungus as measured by macrophage engulfment of conidia or virulence in a neutropenic mouse model.

H3K9 methylation regulates growth and development in Aspergillus fumigatus.

In most species, chromatin remodeling mediates critical biological processes ranging from development to disease states. In fungi within the genus Aspergillus, chromatin remodeling may regulate expression of metabolic gene clusters, but other processes regulated by chromatin structure remain to be elucidated. In many eukaryotic species, methylation of lysine 9 of histone 3 (H3K9) is a hallmark of heterochromatin formation and subsequent gene silencing. The sole H3K9 methyltransferase in Schizosaccharomyces pombe is Clr4. We report that disruption of the Clr4 homolog in the pathogenic mold Aspergillus fumigatus (ClrD), which is involved in both mono- and trimethylation of H3K9, results in several growth abnormalities. Developmental defects in DeltaAfclrD include reduction in radial growth, reduction in conidial production, and delayed conidiation after developmental competence mediated by delayed expression of brlA, the master regulator of conidiophore development. Sensitivity of DeltaAfclrD to 6-azauracil suggests that ClrD influences transcriptional processing in A. fumigatus. Despite growth abnormalities, macrophage assays suggest ClrD may be dispensable for host interactions.

Defects in conidiophore development and conidium-macrophage interactions in a dioxygenase mutant of Aspergillus fumigatus.

Oxygenated fatty acids, or oxylipins, play an essential role in physiological signaling and developmental processes in animals, plants, and fungi. Previous characterization of three Aspergillus fumigatus dioxygenases (PpoA, PpoB, and PpoC), similar in sequence to mammalian cyclooxygenases, showed that PpoA is responsible for the production of the oxylipins 8R-hydroperoxyoctadecadienoic acid and 5S,8R-dihydroxy-9Z,12Z-octadecadienoic acid and that PpoC is responsible for 10R-hydroxy-8E,12Z-hydroperoxyoctadecadienoic acid. Here, Delta ppo mutants were characterized to elucidate the role of fungal dioxygenases in A. fumigatus development and host interactions. The Delta ppoC strain displayed distinct phenotypes compared to those of other Delta ppo mutants and the wild type, including altered conidium size, germination, and tolerance to oxidative stress as well as increased uptake and killing by primary alveolar macrophages. These experiments implicate oxylipins in pathogen development and suggest that Delta ppoC represents a useful model for studying the A. fumigatus-host interaction.

Loss of CclA, required for histone 3 lysine 4 methylation, decreases growth but increases secondary metabolite production in Aspergillus fumigatus.

Secondary metabolite (SM) production in filamentous fungi is mechanistically associated with chromatin remodeling of specific SM clusters. One locus recently shown to be involved in SM suppression in Aspergillus nidulans was CclA, a member of the histone 3 lysine 4 methylating COMPASS complex. Here we examine loss of CclA and a putative H3K4 demethylase, HdmA, in the human pathogen Aspergillus fumigatus. Although deletion of hdmA showed no phenotype under the conditions tested, the cclA deletant was deficient in tri- and di-methylation of H3K4 and yielded a slowly growing strain that was rich in the production of several SMs, including gliotoxin. Similar to deletion of other chromatin modifying enzymes, ΔcclA was sensitive to 6-azauracil indicating a defect in transcriptional elongation. Despite the poor growth, the ΔcclA mutant had wild-type pathogenicity in a murine model and the Toll-deficient Drosophila model of invasive aspergillosis. These data indicate that tri- and di-methylation of H3K4 is involved in the regulation of several secondary metabolites in A. fumigatus, however does not contribute to pathogenicity under the conditions tested.