Aspergillus fumigatus Hypoxia Adaptation Is Critical for the Establishment of Fungal Keratitis.

Purpose: The poor visual outcomes associated with fungal keratitis (FK) underscore a need to identify fungal pathways that can serve as novel antifungal targets. In this report, we investigated whether hypoxia develops in the FK cornea and, by extension, if fungal hypoxia adaptation is essential for virulence in this setting. Methods: C57BL/6J mice were inoculated with Aspergillus fumigatus and Fusarium solani var. petroliphilum via topical overlay or intrastromal injection. At various time points post-inoculation (p.i.), animals were injected with pimonidazole for the detection of tissue hypoxia through immunofluorescence imaging. The A. fumigatus srbA gene was deleted through Cas9-mediated homologous recombination and its virulence was assessed in the topical infection model using slit-lamp microscopy and optical coherence tomography (OCT). Results: Topical inoculation with A. fumigatus resulted in diffuse pimonidazole staining across the epithelial and endothelial layers within 6 hours. Stromal hypoxia was evident by 48 hours p.i., which corresponded to leukocytic infiltration. Intrastromal inoculation with either A. fumigatus or F. solani similarly led to diffuse staining patterns across all corneal cell layers. The A. fumigatus srbA deletion mutant was unable to grow at oxygen levels below 3% in vitro, and corneas inoculated with the mutant failed to develop signs of corneal opacification, inflammation, or fungal burden. Conclusions: These results suggest that fungal antigen rapidly drives the development of corneal hypoxia, thus rendering fungal SrbA or related pathways essential for the establishment of infection. Such pathways may therefore serve as targets for novel antifungal intervention.

The Aspergillus fumigatus UPR is variably activated across nutrient and host environments and is critical for the establishment of corneal infection.

The Aspergillus fumigatus unfolded protein response (UPR) is a two-component relay consisting of the ER-bound IreA protein, which splices and activates the mRNA of the transcription factor HacA. Spliced hacA accumulates under conditions of acute ER stress in vitro, and UPR null mutants are hypovirulent in a murine model of invasive pulmonary infection. In this report, we demonstrate that a hacA deletion mutant (ΔhacA) is furthermore avirulent in a model of fungal keratitis, a corneal infection, and an important cause of ocular morbidity and unilateral blindness worldwide. Interestingly, we demonstrate that A. fumigatus hacA is spliced in infected lung samples, but not in the cornea, suggesting the amount of ER stress experienced by the fungus varies upon the host niche. To better understand how the UPR contributes to fungal cell biology across a spectrum of ER-stress levels, we employed transcriptomics on the wild-type and ΔhacA strains in glucose minimal media (low stress), glucose minimal media with dithiothreitol (high stress), and gelatin minimal media as a proxy for the nutrient stress encountered in the cornea (mid-level stress). These data altogether reveal a unique HacA-dependent transcriptome under each condition, suggesting that HacA activity is finely-tuned and required for proper fungal adaptation in each environment. Taken together, our results indicate that the fungal UPR could serve as an important antifungal target in the setting of both invasive pulmonary and corneal infections.

A host defense peptide mimetic, brilacidin, potentiates caspofungin antifungal activity against human pathogenic fungi.

Fungal infections cause more than 1.5 million deaths a year. Due to emerging antifungal drug resistance, novel strategies are urgently needed to combat life-threatening fungal diseases. Here, we identify the host defense peptide mimetic, brilacidin (BRI) as a synergizer with caspofungin (CAS) against CAS-sensitive and CAS-resistant isolates of Aspergillus fumigatus, Candida albicans, C. auris, and CAS-intrinsically resistant Cryptococcus neoformans. BRI also potentiates azoles against A. fumigatus and several Mucorales fungi. BRI acts in A. fumigatus by affecting cell wall integrity pathway and cell membrane potential. BRI combined with CAS significantly clears A. fumigatus lung infection in an immunosuppressed murine model of invasive pulmonary aspergillosis. BRI alone also decreases A. fumigatus fungal burden and ablates disease development in a murine model of fungal keratitis. Our results indicate that combinations of BRI and antifungal drugs in clinical use are likely to improve the treatment outcome of aspergillosis and other fungal infections.

Genomic and Phenotypic Trait Variation of the Opportunistic Human Pathogen Aspergillus flavus and Its Close Relatives.

Fungal diseases affect millions of humans annually, yet fungal pathogens remain understudied. The mold Aspergillus flavus can cause both aspergillosis and fungal keratitis infections, but closely related species are not considered clinically relevant. To study the evolution of A. flavus pathogenicity, we examined genomic and phenotypic traits of two strains of A. flavus and three closely related species, Aspergillus arachidicola (two strains), Aspergillus parasiticus (two strains), and Aspergillus nomiae (one strain). We identified >3,000 orthologous proteins unique to A. flavus, including seven biosynthetic gene clusters present in A. flavus strains and absent in the three nonpathogens. We characterized secondary metabolite production for all seven strains under two clinically relevant conditions, temperature and salt concentration. Temperature impacted metabolite production in all species, whereas salinity did not affect production of any species. Strains of the same species produced different metabolites. Growth under stress conditions revealed additional heterogeneity within species. Using the invertebrate fungal disease model Galleria mellonella, we found virulence of strains of the same species varied widely; A. flavus strains were not more virulent than strains of the nonpathogens. In a murine model of fungal keratitis, we observed significantly lower disease severity and corneal thickness for A. arachidicola compared to other species at 48 h postinfection, but not at 72 h. Our work identifies variations in key phenotypic, chemical, and genomic attributes between A. flavus and its nonpathogenic relatives and reveals extensive strain heterogeneity in virulence that does not correspond to the currently established clinical relevance of these species. IMPORTANCE Aspergillus flavus is a filamentous fungus that causes opportunistic human infections, such as aspergillosis and fungal keratitis, but its close relatives are considered nonpathogenic. To begin understanding how this difference in pathogenicity evolved, we characterized variation in infection-relevant genomic, chemical, and phenotypic traits between strains of A. flavus and its relatives. We found extensive variation (or strain heterogeneity) within the pathogenic A. flavus as well as within its close relatives, suggesting that strain-level differences may play a major role in the ability of these fungi to cause disease. Surprisingly, we also found that the virulence of strains from species not considered to be pathogens was similar to that of A. flavus in both invertebrate and murine models of disease. These results contrast with previous studies on Aspergillus fumigatus, another major pathogen in the genus, for which significant differences in infection-relevant chemical and phenotypic traits are observed between closely related pathogenic and nonpathogenic species.

High-resolution crystal structure and chemical screening reveal pantothenate kinase as a new target for antifungal development.

Fungal infections are the leading cause of mortality by eukaryotic pathogens, with an estimated 150 million severe life-threatening cases and 1.7 million deaths reported annually. The rapid emergence of multidrug-resistant fungal isolates highlights the urgent need for new drugs with new mechanisms of action. In fungi, pantothenate phosphorylation, catalyzed by PanK enzyme, is the first step in the utilization of pantothenic acid and coenzyme A biosynthesis. In all fungi sequenced so far, this enzyme is encoded by a single PanK gene. Here, we report the crystal structure of a fungal PanK alone as well as with high-affinity inhibitors from a single chemotype identified through a high-throughput chemical screen. Structural, biochemical, and functional analyses revealed mechanisms governing substrate and ligand binding, dimerization, and catalysis and helped identify new compounds that inhibit the growth of several Candida species. The data validate PanK as a promising target for antifungal drug development.

Redesigning the Aspergillus nidulans xylanase regulatory pathway to enhance cellulase production with xylose as the carbon and inducer source.

BACKGROUND: Biomass contains cellulose (C6-sugars), hemicellulose (C5-sugars) and lignin. Biomass ranks amongst the most abundant hydrocarbon resources on earth. However, biomass is recalcitrant to enzymatic digestion by cellulases. Physicochemical pretreatment methods make cellulose accessible but partially destroy hemicellulose, producing a C5-sugar-rich liquor. Typically, digestion of pretreated LCB is performed with commercial cellulase preparations, but C5-sugars could in principle be used for "on site" production of cellulases by genetically engineered microorganism, thereby reducing costs. RESULTS: Here we report a succession of genetic interventions in Aspergillus nidulans that redesign the natural regulatory circuitry of cellulase genes in such a way that recombinant strains use C5-sugar liquors (xylose) to grow a vegetative tissue and simultaneously accumulate large amounts of cellulases. Overexpression of XlnR showed that under xylose-induction conditions only xylanase C was produced. XlnR overexpression strains were constructed that use the xynCp promoter to drive the production of cellobiohydrolases, endoglucanases and ß-glucosidase. All five cellulases accumulated at high levels when grown on xylose. Production of cellulases in the presence of pretreated-biomass C5-sugar liquors was investigated, and cellulases accumulated to much higher enzyme titers than those obtained for traditional fungal cell factories with cellulase-inducing substrates. CONCLUSIONS: By replacing expensive substrates with a cheap by-product carbon source, the use of C5-sugar liquors directly derived from LCB pretreatment processes not only reduces enzyme production costs, but also lowers operational costs by eliminating the need for off-site enzyme production, purification, concentration, transport and dilution.

CRISPR/Cas9-Mediated Gene Replacement in the Fungal Keratitis Pathogen Fusarium solani var. petroliphilum.

Fungal keratitis (FK) is a site-threatening infection of the cornea associated with ocular trauma and contact lens wear. Members of the Fusarium solani species complex (FSSC) are predominant agents of FK worldwide, but genes that support their corneal virulence are poorly understood. As a means to bolster genetic analysis in FSSC pathogens, we sought to employ a CRISPR/Cas9 system in an FK isolate identified as Fusarium petroliphilum. Briefly, this approach involves the introduction of two components into fungal protoplasts: (1) A purified Cas9 protein complexed with guide RNAs that will direct the ribonuclease to cut on either side of the gene of interest, and (2) a "repair template" comprised of a hygromycin resistance cassette flanked by 40 bp of homology outside of the Cas9 cuts. In this way, Cas9-induced double strand breaks should potentiate double homologous replacement of the repair template at the desired locus. We targeted a putative ura3 ortholog since its deletion would result in an easily discernable uracil auxotrophy. Indeed, 10% of hygromycin-resistant transformants displayed the auxotrophic phenotype, all of which harbored the expected ura3 gene deletion. By contrast, none of the transformants from the repair template control (i.e., no Cas9) displayed the auxotrophic phenotype, indicating that Cas9 cutting was indeed required to promote homologous integration. Taken together, these data demonstrate that the in vitro Cas9 system is an easy and efficient approach for reverse genetics in FSSC organisms, including clinical isolates, which should enhance virulence research in these important but understudied ocular pathogens.

Genomic Characterization of Candidate Division LCP-89 Reveals an Atypical Cell Wall Structure, Microcompartment Production, and Dual Respiratory and Fermentative Capacities.

Recent experimental and bioinformatic advances enable the recovery of genomes belonging to yet-uncultured microbial lineages directly from environmental samples. Here, we report on the recovery and characterization of single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) representing candidate phylum LCP-89, previously defined based on 16S rRNA gene sequences. Analysis of LCP-89 genomes recovered from Zodletone Spring, an anoxic spring in Oklahoma, predicts slow-growing, rod-shaped organisms. LCP-89 genomes contain genes for cell wall lipopolysaccharide (LPS) production but lack the entire machinery for peptidoglycan biosynthesis, suggesting an atypical cell wall structure. The genomes, however, encode S-layer homology domain-containing proteins, as well as machinery for the biosynthesis of CMP-legionaminate, inferring the possession of an S-layer glycoprotein. A nearly complete chemotaxis machinery coupled to the absence of flagellar synthesis and assembly genes argues for the utilization of alternative types of motility. A strict anaerobic lifestyle is predicted, with dual respiratory (nitrite ammonification) and fermentative capacities. Predicted substrates include a wide range of sugars and sugar alcohols and a few amino acids. The capability of rhamnose metabolism is confirmed by the identification of bacterial microcompartment genes to sequester the toxic intermediates generated. Comparative genomic analysis identified differences in oxygen sensitivities, respiratory capabilities, substrate utilization preferences, and fermentation end products between LCP-89 genomes and those belonging to its four sister phyla (Calditrichota, SM32-31, AABM5-125-24, and KSB1) within the broader FCB (Fibrobacteres-Chlorobi-Bacteroidetes) superphylum. Our results provide a detailed characterization of members of the candidate division LCP-89 and highlight the importance of reconciling 16S rRNA-based and genome-based phylogenies.IMPORTANCE Our understanding of the metabolic capacities, physiological preferences, and ecological roles of yet-uncultured microbial phyla is expanding rapidly. Two distinct approaches are currently being utilized for characterizing microbial communities in nature: amplicon-based 16S rRNA gene surveys for community characterization and metagenomics/single-cell genomics for detailed metabolic reconstruction. The occurrence of multiple yet-uncultured bacterial phyla has been documented using 16S rRNA surveys, and obtaining genome representatives of these yet-uncultured lineages is critical to our understanding of the role of yet-uncultured organisms in nature. This study provides a genomics-based analysis highlighting the structural features and metabolic capacities of a yet-uncultured bacterial phylum (LCP-89) previously identified in 16S rRNA surveys for which no prior genomes have been described. Our analysis identifies several interesting structural features for members of this phylum, e.g., lack of peptidoglycan biosynthetic machinery and the ability to form bacterial microcompartments. Predicted metabolic capabilities include degradation of a wide range of sugars, anaerobic respiratory capacity, and fermentative capacities. In addition to the detailed structural and metabolic analysis provided for candidate division LCP-89, this effort represents an additional step toward a unified scheme for microbial taxonomy by reconciling 16S rRNA gene-based and genomics-based taxonomic outlines.