Michelle Momany

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

Polarisome meets spitzenkörper: microscopy, genetics, and genomics converge.

The impact of filamentous fungi on human welfare has never been greater. Fungi are acknowledged as the most economically devastating plant pathogens ([1][1]) and are attaining increasing notoriety for their ability to cause life-threatening infections in humans ([57][2], [71][3]), and fungal

Analysis of septins across kingdoms reveals orthology and new motifs.

BackgroundSeptins are cytoskeletal GTPase proteins first discovered in the fungus Saccharomyces cerevisiae where they organize the septum and link nuclear division with cell division. More recently septins have been found in animals where they are important in processes ranging from actin and microtubule organization to embryonic patterning and where defects in septins have been implicated in human disease. Previous studies suggested that many animal septins fell into independent evolutionary groups, confounding cross-kingdom comparison.ResultsIn the current work, we identified 162 septins from fungi, microsporidia and animals and analyzed their phylogenetic relationships. There was support for five groups of septins with orthology between kingdoms. Group 1 (which includes S. cerevisiae Cdc10p and human Sept9) and Group 2 (which includes S. cerevisiae Cdc3p and human Sept7) contain sequences from fungi and animals. Group 3 (which includes S. cerevisiae Cdc11p) and Group 4 (which includes S. cerevisiae Cdc12p) contain sequences from fungi and microsporidia. Group 5 (which includes Aspergillus nidulans AspE) contains sequences from filamentous fungi. We suggest a modified nomenclature based on these phylogenetic relationships. Comparative sequence alignments revealed septin derivatives of already known G1, G3 and G4 GTPase motifs, four new motifs from two to twelve amino acids long and six conserved single amino acid positions. One of these new motifs is septin-specific and several are group specific.ConclusionOur studies provide an evolutionary history for this important family of proteins and a framework and consistent nomenclature for comparison of septin orthologs across kingdoms.

Polarity in filamentous fungi: establishment, maintenance and new axes

Germ tube emergence in filamentous fungi appears to be similar to bud emergence in yeast. Several key proteins (e.g. Cdc42, septins, Bni1 formin, Rho1 and Rho3) play common roles in polarity establishment and early polarity maintenance in both processes. Although germ tube extension, which can be thought of as extreme polarity maintenance, uses some of the same genes, they are likely to be regulated differently. Mutations in polarity maintenance genes often lead to a split tip in filamentous fungi, a phenotype without an analogue in yeast. Cell cycle regulation differs between tip splitting and subapical branching, but in both processes filamentous fungi maintain several axes of polar growth simultaneously.

Distinct roles for dectin-1 and TLR4 in the pathogenesis of Aspergillus fumigatus keratitis.

Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1β and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that β-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1−/− corneas have impaired IL-1β and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high β-glucan. In contrast to Dectin 1−/− mice, cellular infiltration into infected TLR2−/−, TLR4−/−, and MD-2−/− mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4−/− mice, but not TLR2−/− or MD-2−/− mice. We also found that TRIF−/− and TIRAP−/− mice exhibited no fungal-killing defects, but that MyD88−/− and IL-1R1−/− mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which β-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1β, and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing.

Landmarks in the early duplication cycles of Aspergillus fumigatus and Aspergillus nidulans: polarity, germ tube emergence and septation.

When the spores of filamentous fungi break dormancy, nuclear division is accompanied by a series of ordered morphological events including the switch from isotropic to polar growth, the emergence of a second germ tube from the conidium and septation. Correlation of these morphological events with nuclear number allows them to serve as duplication cycle landmarks. Early duplication cycle landmarks have been characterized in Aspergillus nidulans, but not in other filamentous fungi. To learn more about duplication cycle control in filamentous fungi, a study was undertaken to compare the timing of landmarks in Aspergillus fumigatus and A. nidulans. Nuclear duplication took approximately 45 min in A. fumigatus, with mitosis occupying roughly 5% of this period. Under the same conditions, nuclear duplication in A. nidulans took approximately 60 min, with mitosis occupying roughly 4% of this period. In A. fumigatus the isotropic to polar switch preceded the first mitosis in 22% of cells, while in A. nidulans the isotropic to polar switch did not occur until after the first mitosis. In both A. fumigatus and A. nidulans the earliest emergence of a second germ tube from the conidium occurred after the third mitotic division. However, by the fifth mitosis only 19% of A. fumigatus conidia had a second germ tube, compared to 98% of A. nidulans conidia. In both A. fumigatus and A. nidulans, formation of the first septum occurred after the fourth mitotic division. In all experiments a few cells lagged behind the others in nuclear number. In this delayed group, it was common to see landmark events at an earlier mitotic division. Differences in nuclear number when identical landmarks occur in A. fumigatus versus A. nidulans, and uncoupling of mitotic division and landmarks in delayed cells suggest that nuclear division and morphogenesis lie in parallel pathways, perhaps coordinated by checkpoints.

Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth

The opportunistic mycopathogen Aspergillus fumigatus expresses both glucosylceramide and galactosylceramide (GlcCer and GalCer), but their functional significance in Aspergillus species is unknown. We here identified and characterized a GlcCer from Aspergillus nidulans, a non‐pathogenic model fungus. Involvement of GlcCer in fungal development was tested on both species using a family of compounds known to inhibit GlcCer synthase in mammals. Two analogs, D‐threo‐1‐phenyl‐2‐palmitoyl‐3‐pyrrolidinopropanol (P4) and D‐threo‐3′,4′‐ethylenedioxy‐P4, strongly inhibited germination and hyphal growth. Neutral lipids from A. fumigatus cultured in the presence of these inhibitors displayed a significantly reduced GlcCer/GalCer ratio. These results suggest that synthesis of GlcCer is essential for normal development of A. fumigatus and A. nidulans.

Harnessing glycosylation to improve cellulase activity

Cellulases and hemicellulases are responsible for the turnover of plant cell wall polysaccharides in the biosphere, and thus form the foundation of enzyme engineering efforts in biofuels research. Many of these carbohydrate-active enzymes from filamentous fungi contain both N-linked and O-linked glycosylation, the extent and heterogeneity of which depends on growth conditions, expression host, and the presence of glycan trimming enzymes in the secretome, all of which in turn impact enzyme activity. As the roles of glycosylation in enzyme function have not been fully elucidated, here we discuss the potential roles of glycosylation on glycoside hydrolase enzyme structure and function after secretion. We posit that glycosylation, instead of hindering cellulase engineering, can be used as an additional tool to enhance enzyme activity, given deeper understanding of its molecular-level role in biomass deconstruction.

Mapping Woronin body position in Aspergillus nidulans

The positions of all Woronin bodies in five germlings of Aspergillus nidulans prepared by plunge freezing and freeze substitution were determined by transmission electron microscopy. As expected, Woronin bodies were found near septa. High numbers of morphologically identical organelles were also found in apical regions. To verify that these organelles were authentic Woronin bodies, we used antibodies raised against the Neurospora crassa Woronin body matrix protein Hex1. Anti-Hex1 antibodies labeled Woronin bodies at septa and in apical regions of A. nidulans. In germlings that had not yet formed septa, at least fifty percent of Woronin bodies were found within 2.5 μm of the tip. In germ tubes that had formed septa, the total number of Woronin bodies remained the same, but only twenty percent were near the tip. Our results clearly establish that Woronin bodies are found in apical regions of Aspergillus germ tubes and suggest that Woronin bodies are transported from the apex to the more basal regions of the cell immediately before or during septation.

Fungal antioxidant pathways promote survival against neutrophils during infection

Filamentous fungi are a common cause of blindness and visual impairment worldwide. Using both murine model systems and in vitro human neutrophils, we found that NADPH oxidase produced by neutrophils was essential to control the growth of Aspergillus and Fusarium fungi in the cornea. We demonstrated that neutrophil oxidant production and antifungal activity are dependent on CD18, but not on the β-glucan receptor dectin-1. We used mutant A. fumigatus strains to show that the reactive oxygen species-sensing transcription factor Yap1, superoxide dismutases, and the Yap1-regulated thioredoxin antioxidant pathway are each required for protection against neutrophil-mediated oxidation of hyphae as well as optimal survival of fungal hyphae in vivo. We also demonstrated that thioredoxin inhibition using the anticancer drug PX-12 increased the sensitivity of fungal hyphae to both H2O2- and neutrophil-mediated killing in vitro. Additionally, topical application of PX-12 significantly enhanced neutrophil-mediated fungal killing in infected mouse corneas. Cumulatively, our data reveal critical host oxidative and fungal anti-oxidative mediators that regulate hyphal survival during infection. Further, these findings also indicate that targeting fungal anti-oxidative defenses via PX-12 may represent an efficacious strategy for treating fungal infections.