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.

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.

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 ß-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.