Production of Extracellular Traps against Aspergillus fumigatus In Vitro and in Infected Lung Tissue Is Dependent on Invading Neutrophils and Influenced by Hydrophobin RodA

Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs) consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A. fumigatus, but suggest that it does not play a major role in killing this fungus. Instead, NETs may have a fungistatic effect and may prevent further spreading

Hydrophobins—Unique Fungal Proteins

International audienc

Innate Recognition of Fungal Cell Walls

The emergence of fungal infections as major causes of morbidity and mortality in immunosuppressed individuals has prompted studies into how the host recognizes fungal pathogens. Fungi are eukaryotes and as such share many similarities with mammalian cells. The most striking difference, though, is the presence of a cell wall that serves to protect the fungus from environmental stresses, particularly osmotic changes [1]. This task is made challenging because the fungus must remodel itself to allow for cell growth and division, including the conversion to different morphotypes, such as occurs during germination of spherical spores into filamentous hyphae. The cell wall also connects the fungus with its environment by triggering intracellular signaling pathways and mediating adhesion to other cells and extracellular matrices. Here, important facts and concepts critical for understanding innate sensing of the fungal cell wall by mammalian pathogens are reviewed

Novel insights into host-fungal pathogen interactions derived from live-cell imaging

Acknowledgments The authors acknowledge funding from the Wellcome Trust (080088, 086827, 075470 and 099215) including a Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377 and FP7-2007–2013 grant agreement HEALTH-F2-2010-260338–ALLFUN to NARG.Peer reviewedPublisher PD

SUN proteins belong to a novel family of β-(1,3)-glucan-modifying enzymes involved in fungal morphogenesis

BACKGROUND: SUN proteins are involved in yeast morphogenesis, but their function is unknown. RESULTS: SUN protein plays a role in the Aspergillus fumigatus morphogenesis. Biochemical properties of recombinant SUN proteins were elucidated. CONCLUSION: Both Candida albicans and Aspergillus fumigatus sun proteins show a β-(1,3)-glucanase activity. SIGNIFICANCE: The mode of action of SUN proteins on β-(1,3)-glucan is unique, new, and original. In yeasts, the family of SUN proteins has been involved in cell wall biogenesis. Here, we report the characterization of SUN proteins in a filamentous fungus, Aspergillus fumigatus. The function of the two A. fumigatus SUN genes was investigated by combining reverse genetics and biochemistry. During conidial swelling and mycelial growth, the expression of AfSUN1 was strongly induced, whereas the expression of AfSUN2 was not detectable. Deletion of AfSUN1 negatively affected hyphal growth and conidiation. A closer examination of the morphological defects revealed swollen hyphae, leaky tips, intrahyphal growth, and double cell wall, suggesting that, like in yeast, AfSun1p is associated with cell wall biogenesis. In contrast to AfSUN1, deletion of AfSUN2 either in the parental strain or in the AfSUN1 single mutant strain did not affect colony and hyphal morphology. Biochemical characterization of the recombinant AfSun1p and Candida albicans Sun41p showed that both proteins had a unique hydrolysis pattern: acting on β-(1,3)-oligomers from dimer up to insoluble β-(1,3)-glucan. Referring to the CAZy database, it is clear that fungal SUN proteins represent a new family of glucan hydrolases (GH132) and play an important morphogenetic role in fungal cell wall biogenesis and septation

Role of Germination in Murine Airway CD8+ T-Cell Responses to Aspergillus Conidia

Pulmonary exposure to Aspergillus fumigatus has been associated with morbidity and mortality, particularly in immunocompromised individuals. A. fumigatus conidia produce β-glucan, proteases, and other immunostimulatory factors upon germination. Murine models have shown that the ability of A. fumigatus to germinate at physiological temperature may be an important factor that facilitates invasive disease. We observed a significant increase in IFN-γ-producing CD8+ T cells in bronchoalveolar lavage fluid (BALF) of immunocompetent mice that repeatedly aspirated A. fumigatus conidia in contrast to mice challenged with A. versicolor, a species that is not typically associated with invasive, disseminated disease. Analysis of tissue sections indicated the presence of germinating spores in the lungs of mice challenged with A. fumigatus, but not A. versicolor. Airway IFN-γ+CD8+ T-cells were decreased and lung germination was eliminated in mice that aspirated A. fumigatus conidia that were formaldehyde-fixed or heat-inactivated. Furthermore, A. fumigatus particles exhibited greater persistence in the lungs of recipient mice when compared to non-viable A. fumigatus or A. versicolor, and this correlated with increased maintenance of airway memory-phenotype CD8+ T cells. Therefore, murine airway CD8+ T cell-responses to aspiration of Aspergillus conidia may be mediated in part by the ability of conidia to germinate in the host lung tissue. These results provide further evidence of induction of immune responses to fungi based on their ability to invade host tissue

Diffusion of hydrophobin proteins in solution and interactions with a graphite surface

<p>Abstract</p> <p>Background</p> <p>Hydrophobins are small proteins produced by filamentous fungi that have a variety of biological functions including coating of spores and surface adhesion. To accomplish these functions, they rely on unique interface-binding properties. Using atomic-detail implicit solvent rigid-body Brownian dynamics simulations, we studied the diffusion of HFBI, a class II hydrophobin from <it>Trichoderma reesei</it>, in aqueous solution in the presence and absence of a graphite surface.</p> <p>Results</p> <p>In the simulations, HFBI exists in solution as a mixture of monomers in equilibrium with different types of oligomers. The oligomerization state depends on the conformation of HFBI. When a Highly Ordered Pyrolytic Graphite (HOPG) layer is present in the simulated system, HFBI tends to interact with the HOPG layer through a hydrophobic patch on the protein.</p> <p>Conclusions</p> <p>From the simulations of HFBI solutions, we identify a tetrameric encounter complex stabilized by non-polar interactions between the aliphatic residues in the hydrophobic patch on HFBI. After the formation of the encounter complex, a local structural rearrangement at the protein interfaces is required to obtain the tetrameric arrangement seen in HFBI crystals. Simulations performed with the graphite surface show that, due to a combination of a geometric hindrance and the interaction of the aliphatic sidechains with the graphite layer, HFBI proteins tend to accumulate close to the hydrophobic surface.</p

Mechanisms of redundancy and specificity of the Aspergillus fumigatus Crh transglycosylases

Transglycosylases strengthen the fungal cell wall by forming a rigid network of crosslinks. Here, Fang et al. show that the five Crh transglycosylases of Aspergillus fumigatus are dispensable for cell wall integrity in vitro, and solve the crystal structure of Crh5 in complex with chitooligosaccharides

Aspergillus fumigatus Acetate Utilization Impacts Virulence Traits and Pathogenicity

This is the final version. Available on open access from the American Society for Microbiology via the DOI in this recordData availability: The RNA-seq data set can be accessed at NCBI’s Short Read Archive under the Bioproject identifier (ID) PRJNA668271.Aspergillus fumigatus is a major opportunistic fungal pathogen of immunocompromised and immunocompetent hosts. To successfully establish an infection, A. fumigatus needs to use host carbon sources, such as acetate, present in the body fluids and peripheral tissues. However, utilization of acetate as a carbon source by fungi in the context of infection has not been investigated. This work shows that acetate is metabolized via different pathways in A. fumigatus and that acetate utilization is under the regulatory control of a transcription factor (TF), FacB. A. fumigatus acetate utilization is subject to carbon catabolite repression (CCR), although this is only partially dependent on the TF and main regulator of CCR CreA. The available extracellular carbon source, in this case glucose and acetate, significantly affected A. fumigatus virulence traits such as secondary metabolite secretion and cell wall composition, with the latter having consequences for resistance to oxidative stress, antifungal drugs, and human neutrophil-mediated killing. Furthermore, deletion of facB significantly impaired the in vivo virulence of A. fumigatus in both insect and mammalian models of invasive aspergillosis. This is the first report on acetate utilization in A. fumigatus, and this work further highlights the importance of available host-specific carbon sources in shaping fungal virulence traits and subsequent disease outcome, and a potential target for the development of antifungal strategies