Septins coordinate cell wall integrity and lipid metabolism in a sphingolipid-dependent process.

Septins colocalize with membrane sterol-rich regions and facilitate recruitment of cell wall synthases during wall remodeling. We show that null mutants missing an Aspergillus nidulans core septin present in hexamers and octamers (ΔaspAcdc11, ΔaspBcdc3 or ΔaspCcdc12) are sensitive to multiple cell wall-disturbing agents that activate the cell wall integrity MAPK pathway. The null mutant missing the octamer-exclusive core septin (ΔaspDcdc10) showed similar sensitivity, but only to a single cell wall-disturbing agent and the null mutant missing the noncore septin (ΔaspE) showed only very mild sensitivity to a different single agent. Core septin mutants showed changes in wall polysaccharide composition and chitin synthase localization. Mutants missing any of the five septins resisted ergosterol-disrupting agents. Hexamer mutants showed increased sensitivity to sphingolipid-disrupting agents. Core septins mislocalized after treatment with sphingolipid-disrupting agents, but not after ergosterol-disrupting agents. Our data suggest that the core septins are involved in cell wall integrity signaling, that all five septins are involved in monitoring ergosterol metabolism, that the hexamer septins are required for sphingolipid metabolism and that septins require sphingolipids to coordinate the cell wall integrity response.

Azole resistance mechanisms and population structure of the human pathogen Aspergillus fumigatus on retail plant products.

Aspergillus fumigatus is a ubiquitous saprotroph and human-pathogenic fungus that is life-threatening to the immunocompromised. Triazole-resistant A. fumigatus was found in patients without prior treatment with azoles, leading researchers to conclude that resistance had developed in agricultural environments where azoles are used against plant pathogens. Previous studies have documented azole-resistant A. fumigatus across agricultural environments, but few have looked at retail plant products. Our objectives were to determine if azole-resistant A. fumigatus is prevalent in retail plant products produced in the United States (U.S.), as well as to identify the resistance mechanism(s) and population genetic structure of these isolates. Five hundred twenty-five isolates were collected from retail plant products and screened for azole resistance. Twenty-four isolates collected from compost, soil, flower bulbs, and raw peanuts were pan-azole resistant. These isolates had the TR34/L98H, TR46/Y121F/T289A, G448S, and H147Y cyp51A alleles, all known to underly pan-azole resistance, as well as WT alleles, suggesting that non-cyp51A mechanisms contribute to pan-azole resistance in these isolates. Minimum spanning networks showed two lineages containing isolates with TR alleles or the F46Y/M172V/E427K allele, and discriminant analysis of principle components identified three primary clusters. This is consistent with previous studies detecting three clades of A. fumigatus and identifying pan-azole-resistant isolates with TR alleles in a single clade. We found pan-azole resistance in U.S. retail plant products, particularly compost and flower bulbs, which indicates a risk of exposure to these products for susceptible populations and that highly resistant isolates are likely distributed worldwide on these products.IMPORTANCEAspergillus fumigatus has recently been designated as a critical fungal pathogen by the World Health Organization. It is most deadly to people with compromised immune systems, and with the emergence of antifungal resistance to multiple azole drugs, this disease carries a nearly 100% fatality rate without treatment or if isolates are resistant to the drugs used to treat the disease. It is important to determine the relatedness and origins of resistant A. fumigatus isolates in the environment, including plant-based retail products, so that factors promoting the development and propagation of resistant isolates can be identified.

Azole-resistant Aspergillus fumigatus in the environment: Identifying key reservoirs and hotspots of antifungal resistance.

Aspergillus fumigatus is an opportunistic human pathogen that causes aspergillosis, a spectrum of environmentally acquired respiratory illnesses. It has a cosmopolitan distribution and exists in the environment as a saprotroph on decaying plant matter. Azoles, which target Cyp51A in the ergosterol synthesis pathway, are the primary class of drugs used to treat aspergillosis. Azoles are also used to combat plant pathogenic fungi. Recently, an increasing number of azole-naive patients have presented with pan-azole-resistant strains of A. fumigatus. The TR34/L98H and TR46/Y121F/T289A alleles in the cyp51A gene are the most common ones conferring pan-azole resistance. There is evidence that these mutations arose in agricultural settings; therefore, numerous studies have been conducted to identify azole resistance in environmental A. fumigatus and to determine where resistance is developing in the environment. Here, we summarize the global occurrence of azole-resistant A. fumigatus in the environment based on available literature. Additionally, we have created an interactive world map showing where resistant isolates have been detected and include information on the specific alleles identified, environmental settings, and azole fungicide use. Azole-resistant A. fumigatus has been found on every continent, except for Antarctica, with the highest number of reports from Europe. Developed environments, specifically hospitals and gardens, were the most common settings where azole-resistant A. fumigatus was detected, followed by soils sampled from agricultural settings. The TR34/L98H resistance allele was the most common in all regions except South America where the TR46/Y121F/T289A allele was the most common. A major consideration in interpreting this survey of the literature is sampling bias; regions and environments that have been extensively sampled are more likely to show greater azole resistance even though resistance could be more prevalent in areas that are under-sampled or not sampled at all. Increased surveillance to pinpoint reservoirs, as well as antifungal stewardship, is needed to preserve this class of antifungals for crop protection and human health.

Diversity of opisthokont septin proteins reveals structural constraints and conserved motifs.

BACKGROUND: Septins are cytoskeletal proteins important in cell division and in establishing and maintaining cell polarity. Although septins are found in various eukaryotes, septin genes had the richest history of duplication and diversification in the animals, fungi and protists that comprise opisthokonts. Opisthokont septin paralogs encode modular proteins that assemble into heteropolymeric higher order structures. The heteropolymers can create physical barriers to diffusion or serve as scaffolds organizing other morphogenetic proteins. How the paralogous septin modules interact to form heteropolymers is still unclear. Through comparative analyses, we hoped to clarify the evolutionary origin of septin diversity and to suggest which amino acid residues were responsible for subunit binding specificity. RESULTS: Here we take advantage of newly sequenced genomes to reconcile septin gene trees with a species phylogeny from 22 animals, fungi and protists. Our phylogenetic analysis divided 120 septins representing the 22 taxa into seven clades (Groups) of paralogs. Suggesting that septin genes duplicated early in opisthokont evolution, animal and fungal lineages share septin Groups 1A, 4 and possibly also 1B and 2. Group 5 septins were present in fungi but not in animals and whether they were present in the opisthokont ancestor was unclear. Protein homology folding showed that previously identified conserved septin motifs were all located near interface regions between the adjacent septin monomers. We found specific interface residues associated with each septin Group that are candidates for providing subunit binding specificity. CONCLUSIONS: This work reveals that duplication of septin genes began in an ancestral opisthokont more than a billion years ago and continued through the diversification of animals and fungi. Evidence for evolutionary conservation of ~ 49 interface residues will inform mutagenesis experiments and lead to improved understanding of the rules guiding septin heteropolymer formation and from there, to improved understanding of development of form in animals and fungi.

Transcript levels of the Aspergillus fumigatus Cdc42 module, polarisome, and septin genes show little change from dormancy to polarity establishment.

Aspergillus fumigatus is the most common airborne pathogen causing fatal mycoses in immunocompromised patients. During the first 8 hours of development A. fumigatus conidia break dormancy, expand isotopically, establish an axis of polarity, and begin to extend germ tubes in a polar manner. The transition from isotropic to polar growth is critical for tissue invasion and pathogenesis. In the current work, we used two-color microarrays to examine the A. fumigatus transcriptome during early development, focusing on the isotropic to polar switch. The most highly regulated transcripts in the isotropic to polar switch did not include known polarity genes. Transcripts encoding the Cdc42 module, polarisome components, and septins, known to be critical players in polarity, showed relatively steady levels during the isotropic to polar switch. Indeed, these transcripts were present in dormant conidia, and their levels changed little from dormancy through germ tube emergence. Not only did the isotropic to polar switch show little change in the expression of key polarity genes of the Cdc42 module, polarisome, and septins, it also showed the lowest overall levels of both up- and downregulation in early development.

Rite of passage: a bZIP transcription factor must transit the cell apex to become competent.

In the filamentous fungus Aspergillus nidulans BrlA triggers the central developmental pathway that controls the transition from vegetative growth to asexual reproduction. Upstream regulators including the bZIP transcription factor FlbB activate the expression of brlA. Previous work has established that FlbB localizes to both the apex of the hypha, where it interacts with and is anchored by FlbE, and to nuclei, with highest levels in the nucleus closest to the apex and successively lower levels in nuclei further away from the apex. In this issue, Herrero-Garcia et al. dissect the roles of these two FlbB pools and the mechanisms underlying their localization and activity. Using a photoactivatable tag, they demonstrate that FlbB moves from the tip into the apical nucleus. Through a series of deletion constructs, they show that import of FlbB into the nucleus requires a bipartite NLS, that FlbB localization at the tip requires actin and that the FlbB tip-high gradient appears to be mass action dependent as the gradient is lost with FlbB constitutive upregulation. They show that while the pool of FlbB at the apex is required for triggering asexual development, the tip high nuclear gradient is not required.

The Evolutionary Origins and Ancestral Features of Septins.

Septins are a family of membrane-associated cytoskeletal GTPases that play crucial roles in various cellular processes, such as cell division, phagocytosis, and organelle fission. Despite their importance, the evolutionary origins and ancestral function of septins remain unclear. In opisthokonts, septins form five distinct groups of orthologs, with subunits from multiple groups assembling into heteropolymers, thus supporting their diverse molecular functions. Recent studies have revealed that septins are also conserved in algae and protists, indicating an ancient origin from the last eukaryotic common ancestor. However, the phylogenetic relationships among septins across eukaryotes remained unclear. Here, we expanded the list of non-opisthokont septins, including previously unrecognized septins from rhodophyte red algae and glaucophyte algae. Constructing a rooted phylogenetic tree of 254 total septins, we observed a bifurcation between the major non-opisthokont and opisthokont septin clades. Within the non-opisthokont septins, we identified three major subclades: Group 6 representing chlorophyte green algae (6A mostly for species with single septins, 6B for species with multiple septins), Group 7 representing algae in chlorophytes, heterokonts, haptophytes, chrysophytes, and rhodophytes, and Group 8 representing ciliates. Glaucophyte and some ciliate septins formed orphan lineages in-between all other septins and the outgroup. Combining ancestral-sequence reconstruction and AlphaFold predictions, we tracked the structural evolution of septins across eukaryotes. In the GTPase domain, we identified a conserved GAP-like arginine finger within the G-interface of at least one septin in most algal and ciliate species. This residue is required for homodimerization of the single Chlamydomonas septin, and its loss coincided with septin duplication events in various lineages. The loss of the arginine finger is often accompanied by the emergence of the α0 helix, a known NC-interface interaction motif, potentially signifying the diversification of septin-septin interaction mechanisms from homo-dimerization to hetero-oligomerization. Lastly, we found amphipathic helices in all septin groups, suggesting that curvature-sensing is an ancestral trait of septin proteins. Coiled-coil domains were also broadly distributed, while transmembrane domains were found in some septins in Group 6A and 7. In summary, this study advances our understanding of septin distribution and phylogenetic groupings, shedding light on their ancestral features, potential function, and early evolution.

The septin AspB in Aspergillus nidulans forms bars and filaments and plays roles in growth emergence and conidiation.

In yeast, septins form rings at the mother-bud neck and function as diffusion barriers. In animals, septins form filaments that can colocalize with other cytoskeletal elements. In the filamentous fungus Aspergillus nidulans there are five septin genes, aspA (an ortholog of Saccharomyces cerevisiae CDC11), aspB (an ortholog of S. cerevisiae CDC3), aspC (an ortholog of S. cerevisiae CDC12), aspD (an ortholog of S. cerevisiae CDC10), and aspE (found only in filamentous fungi). The aspB gene was previously reported to be the most highly expressed Aspergillus nidulans septin and to be essential. Using improved gene targeting techniques, we found that deletion of aspB is not lethal but results in delayed septation, increased emergence of germ tubes and branches, and greatly reduced conidiation. We also found that AspB-green fluorescent protein (GFP) localizes as rings and collars at septa, branches, and emerging layers of the conidiophore and as bars and filaments in conidia and hyphae. Bars are found in dormant and isotropically expanding conidia and in subapical nongrowing regions of hyphae and display fast movements. Filaments form as the germ tube emerges, localize to hyphal and branch tips, and display slower movements. All visible AspB-GFP structures are retained in ΔaspD and lost in ΔaspA and ΔaspC strains. Interestingly, in the ΔaspE mutant, AspB-GFP rings, bars, and filaments are visible in early growth, but AspB-GFP rods and filaments disappear after septum formation. AspE orthologs are only found in filamentous fungi, suggesting that this class of septins might be required for stability of septin bars and filaments in highly polar cells.

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-1beta and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that beta-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-1beta and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high beta-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 beta-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1beta, and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing.

Development stage-specific proteomic profiling uncovers small, lineage specific proteins most abundant in the Aspergillus Fumigatus conidial proteome.

BACKGROUND: The pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores) in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process. RESULTS: To gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development. Four different stages were examined: dormant conidia, isotropically expanding conidia, hyphae in which germ tube emergence has just begun, and pre-septation hyphae. To enrich for glycan-linked cell wall proteins we used an alkaline cell extraction method. Shotgun proteomic resulted in the identification of 375 unique gene products with high confidence, with no evidence for enrichment of cell wall-immobilized and secreted proteins. The most interesting discovery was the identification of 52 proteins enriched in dormant conidia including 28 proteins that have never been detected in the A. fumigatus conidial proteome such as signaling protein Pil1, chaperones BipA and calnexin, and transcription factor HapB. Additionally we found many small, Aspergillus specific proteins of unknown function including 17 hypothetical proteins. Thus, the most abundant protein, Grg1 (AFUA_5G14210), was also one of the smallest proteins detected in this study (M.W. 7,367). Among previously characterized proteins were melanin pigment and pseurotin A biosynthesis enzymes, histones H3 and H4.1, and other proteins involved in conidiation and response to oxidative or hypoxic stress. In contrast, expanding conidia, hyphae with early germ tubes, and pre-septation hyphae samples were enriched for proteins responsible for housekeeping functions, particularly translation, respiratory metabolism, amino acid and carbohydrate biosynthesis, and the tricarboxylic acid cycle. CONCLUSIONS: The observed temporal expression patterns suggest that the A. fumigatus conidia are dominated by small, lineage-specific proteins. Some of them may play key roles in host-pathogen interactions, signal transduction during conidial germination, or survival in hostile environments.

Analysis of Cyp51 protein sequences shows 4 major Cyp51 gene family groups across fungi.

Azole drugs target fungal sterol biosynthesis and are used to treat millions of human fungal infections each year. Resistance to azole drugs has emerged in multiple fungal pathogens including Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, and Aspergillus fumigatus. The most well-studied resistance mechanism in A. fumigatus arises from missense mutations in the coding sequence combined with a tandem repeat in the promoter of cyp51A, which encodes a cytochrome P450 enzyme in the fungal sterol biosynthesis pathway. Filamentous members of Ascomycota such as A. fumigatus have either 1 or 2 of 3 Cyp51 paralogs (Cyp51A, Cyp51B, and Cyp51C). Most previous research in A. fumigatus has focused on Cyp51A due to its role in azole resistance. We used the A. fumigatus Cyp51A protein sequence as the query in database searches to identify Cyp51 proteins across fungi. We found 435 Cyp51 proteins in 295 species spanning from early-diverging fungi (Blastocladiomycota, Chytridiomycota, Zoopagomycota, and Mucormycota) to late-diverging fungi (Ascomycota and Basidiomycota). We found these sequences formed 4 major Cyp51 groups: Cyp51, Cyp51A, Cyp51B, and Cyp51C. Surprisingly, we found all filamentous Ascomycota had a Cyp51B paralog, while only 50% had a Cyp51A paralog. We created maximum likelihood trees to investigate the evolution of Cyp51 in fungi. Our results suggest Cyp51 is present in all fungi with 3 paralogs emerging in Pezizomycotina, including Cyp51C which appears to have diverged from the progenitor of the Cyp51A and Cyp51B groups.

Evidence for the agricultural origin of resistance to multiple antimicrobials in Aspergillus fumigatus, a fungal pathogen of humans.

Pathogen resistance to clinical antimicrobial agents is an urgent problem. The fungus Aspergillus fumigatus causes 300,000 life-threatening infections in susceptible humans annually. Azoles, which are widely used in both clinical and agricultural settings, are currently the most effective treatment, but resistance to clinical azoles is emerging worldwide. Here, we report the isolation and analysis of azole-sensitive and azole-resistant A. fumigatus from agricultural environments in the southeastern United States (USA) and show that the USA pan-azole-resistant isolates form a clade with pan-azole-resistant isolates from the United Kingdom, the Netherlands, and India. We show that several pan-azole-resistant isolates from agricultural settings in the USA and India also carry alleles with mutations conferring resistance to agricultural fungicides from the benzimidazole (MBC) and quinone outside inhibitor (QoI) classes. We further show that pan-azole-resistant A. fumigatus isolates from patients in clinical settings in the USA, India, and the Netherlands also carry alleles conferring resistance to MBC and QoI agricultural fungicides. The presence of markers for resistance to agricultural-use fungicides in clinical A. fumigatus isolates is strong evidence for an agricultural origin of pan-azole resistance in patients. The presence of multiple fungicide-resistance alleles in agricultural and clinical isolates further suggests that the unique genetics of the pan-azole-resistant clade enables the evolution and/or persistence of antimicrobial resistance mutations leading to the establishment of multifungicide-resistant isolates.

Immunogenicity and protective efficacy of a pan-fungal vaccine in preclinical models of aspergillosis, candidiasis, and pneumocystosis.

Invasive fungal infections cause over 1.5 million deaths worldwide. Despite increases in fungal infections as well as the numbers of individuals at risk, there are no clinically approved fungal vaccines. We produced a "pan-fungal" peptide, NXT-2, based on a previously identified vaccine candidate and homologous sequences from Pneumocystis, Aspergillus,Candida, and Cryptococcus. We evaluated the immunogenicity and protective capacity of NXT-2 in murine and nonhuman primate models of invasive aspergillosis, systemic candidiasis, and pneumocystosis. NXT-2 was highly immunogenic and immunized animals had decreased mortality and morbidity compared to nonvaccinated animals following induction of immunosuppression and challenge with Aspergillus, Candida, or Pneumocystis. Data in multiple animal models support the concept that immunization with a pan-fungal vaccine prior to immunosuppression induces broad, cross-protective antifungal immunity in at-risk individuals.

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.

Septins AspA and AspC are important for normal development and limit the emergence of new growth foci in the multicellular fungus Aspergillus nidulans.

Septins are cytoskeletal proteins found in fungi, animals, and microsporidia, where they form multiseptin complexes that act as scaffolds recruiting and organizing other proteins to ensure normal cell division and development. Here we characterize the septins AspA and AspC in the multicellular, filamentous fungus Aspergillus nidulans. Mutants with deletions of aspA, aspC, or both aspA and aspC show early and increased germ tube and branch emergence, abnormal septation, and disorganized conidiophores. Strains in which the native aspA has been replaced with a single copy of aspA-GFP driven by the native septin promoter or in which aspC has been replaced with a single copy of aspC-GFP driven by the native promoter show wild-type phenotypes. AspA-GFP and AspC-GFP show identical localization patterns as discrete spots or bars in dormant and expanding conidia, as rings at forming septa and at the bases of emerging germ tubes and branches, and as punctate spots and filaments in the cytoplasm and at the cell cortex. In conidiophores, AspA-GFP and AspC-GFP localize as diffuse bands or rings at the bases of emerging layers and conidial chains and as discrete spots or bars in newly formed conidia. AspA-GFP forms abnormal structures in DeltaaspC strains while AspC-GFP does not localize in DeltaaspA strains. Our results suggest that AspA and AspC interact with each other and are important for normal development, especially for preventing the inappropriate emergence of germ tubes and branches. This is the first report of a septin limiting the emergence of new growth foci in any organism.

Septins From Protists to People.

Septin GTPases form nonpolar heteropolymers that play important roles in cytokinesis and other cellular processes. The ability to form heteropolymers appears to be critical to many septin functions and to have been a major driver of the high conservation of many septin domains. Septins fall into five orthologous groups. Members of Groups 1-4 interact with each other to form heterooligomers and are known as the "core septins." Representative core septins are present in all fungi and animals so far examined and show positional orthology with monomer location in the heteropolymer conserved within groups. In contrast, members of Group 5 are not part of canonical heteropolymers and appear to interact only transiently, if at all, with core septins. Group 5 septins have a spotty distribution, having been identified in specific fungi, ciliates, chlorophyte algae, and brown algae. In this review we compare the septins from nine well-studied model organisms that span the tree of life (Homo sapiens, Drosophila melanogaster, Schistosoma mansoni, Caenorhabditis elegans, Saccharomyces cerevisiae, Aspergillus nidulans, Magnaporthe oryzae, Tetrahymena thermophila, and Chlamydomonas reinhardtii). We focus on classification, evolutionary relationships, conserved motifs, interfaces between monomers, and positional orthology within heteropolymers. Understanding the relationships of septins across kingdoms can give new insight into their functions.

Sporulation environment drives phenotypic variation in the pathogen Aspergillus fumigatus.

Aspergillus fumigatus causes more than 300,000 life-threatening infections annually and is widespread across varied environments with a single colony producing thousands of conidia, genetically identical dormant spores. Conidia are easily wind-dispersed to new environments where they can germinate and, if inhaled by susceptible hosts, cause disease. Using high-throughput single-cell analysis via flow cytometry we analyzed conidia produced and germinated in nine environmentally and medically relevant conditions (complete medium, minimal medium, high temperature, excess copper, excess iron, limited iron, excess salt, excess reactive oxygen species, and limited zinc). We found that germination phenotypes vary among genetically identical individuals, that the environment of spore production determines the size of spores and the degree of germination heterogeneity, and that the environment of spore production impacts virulence in a Galleria mellonella host.

Vaccine-Induced Protection in Two Murine Models of Invasive Pulmonary Aspergillosis.

Life-threatening, invasive fungal infections (IFIs) cause over 1.5 million deaths worldwide and are a major public health concern with high mortality rates even with medical treatment. Infections with the opportunistic fungal pathogen, Aspergillus fumigatus are among the most common. Despite the growing clinical need, there are no licensed vaccines for IFIs. Here we evaluated the immunogenicity and protective efficacy of an A. fumigatus recombinant protein vaccine candidate, AF.KEX1, in experimental murine models of drug-induced immunosuppression. Immunization of healthy mice with AF.KEX1 and adjuvant induced a robust immune response. Following AF.KEX1 or sham immunization, mice were immunosuppressed by treatment with either cortisone acetate or hydrocortisone and the calcineurin inhibitor, tacrolimus. To test vaccine efficacy, immunosuppressed mice were intranasally challenged with A. fumigatus conidia (Af293) and weight and body temperature were monitored for 10 days. At study termination, organism burden in the lungs was evaluated by quantitative PCR and Gomori's methanamine silver staining. In both models of immunosuppression, AF.KEX1 vaccinated mice experienced decreased rates of mortality and significantly lower lung organism burden compared to non-vaccinated controls. The lung fungal burden was inversely correlated with the peak anti-AF.KEX1 IgG titer achieved following vaccination. These studies provide the basis for further evaluation of a novel vaccine strategy to protect individuals at risk of invasive aspergillosis due to immunosuppressive treatments.