Conidial Melanin of the Human-Pathogenic Fungus Aspergillus fumigatus Disrupts Cell Autonomous Defenses in Amoebae

Abstract

The human-pathogenic fungus Aspergillus fumigatus is a ubiquitous saprophyte that causes fatal lung infections in immunocompromised individuals. Following inhalation, conidia are ingested by innate immune cells and can arrest phagolysosome maturation. How this virulence trait could have been selected for in natural environments is unknown. Here, we found that surface exposure of the green pigment 1,8-dihydroxynaphthalene-(DHN)-melanin can protect conidia from phagocytic uptake and intracellular killing by the fungivorous amoeba Protostelium aurantium and delays its exocytosis from the nonfungivorous species Dictyostelium discoideum. To elucidate the antiphagocytic properties of the surface pigment, we followed the antagonistic interactions of A. fumigatus conidia with the amoebae in real time. For both amoebae, conidia covered with DHN-melanin were internalized at far lower rates than were seen with conidia lacking the pigment, despite high rates of initial attachment to nonkilling D. discoideum. When ingested by D. discoideum, the formation of nascent phagosomes was followed by transient acidification of phagolysosomes, their subsequent neutralization, and, finally, exocytosis of the conidia. While the cycle was completed in less than 1 h for unpigmented conidia, the process was significantly prolonged for conidia covered with DHN-melanin, leading to an extended intracellular residence time. At later stages of this cellular infection, pigmented conidia induced enhanced damage to phagolysosomes and infected amoebae failed to recruit the ESCRT (endosomal sorting complex required for transport) membrane repair machinery or the canonical autophagy pathway to defend against the pathogen, thus promoting prolonged intracellular persistence in the host cell and the establishment of a germination niche in this environmental phagocyte. IMPORTANCE Infections with Aspergillus fumigatus are usually acquired by an inhalation of spores from environmental sources. How spores of a saprophytic fungus have acquired abilities to withstand and escape the phagocytic attacks of innate immune cells is not understood. The fungal surface pigment dihydroxynaphtalene-melanin has been shown to be a crucial factor for the delay in phagosome maturation. Here, we show that this pigment also has a protective function against environmental phagocytes. Pigmented conidia escaped uptake and killing by the fungus-eating amoeba Protostelium aurantium. When ingested by the nonfungivorous phagocyte Dictyostelium discoideum, the pigment attenuated the launch of cell autonomous defenses against the fungal invader, such as membrane repair and autophagy, leading to prolonged intracellular retention. Membrane damage and cytoplasmic leakage may result in an influx of nutrients and thus may further promote intracellular germination of the fungus, indicating that A. fumigatus has acquired some of the basic properties of intracellular pathogens.

Characterisation of Aspergillus fumigatus Endocytic Trafficking within Airway Epithelial Cells Using High-Resolution Automated Quantitative Confocal Microscopy

“…In accordance with previous studies, phagosomelysosome fusion was found to increase in A. fumigatus pksP and rodA null mutants in both A549 and 16HBE airway epithelial cells, which is linked with a significant decrease in fungal survival [5,39,44,[63][64][65][66]. In A549 cells, no difference in spore uptake or phagosomelysosome fusion was observed in the rodB null mutant compared to wild type, even though both processes were enhanced in bronchial epithelial cells [55]. This high-resolution livecell imaging approach has also enabled us to observe digested A. fumigatus spores within airway epithelial cell phagolysosomes for the first time, as reported for spores contained within macrophages [14].…”

“…In-vitro confrontation assays are typically visualized by microscopy, and manually observed and quantified, which is time consuming, where user variability and error occur across biological replicates. Additionally, these methodologies typically use macrophages as the model cell type to study fungal-host interaction and are performed using fixed samples which can alter the outcome of the interaction of Aspergillus with the host [51][52][53][54][55]. Here, we have combined a live-cell labelling strategy with automated 4D live-cell imaging to visualize and measure critical events required for Aspergillus clearance by the airway epithelium.…”

“…Here, we were able to additionally follow that most of the spores contained within fully acidified phagosomes from airway epithelial cells were killed. It is likely that spore killing within acidified phagosomes is due to the permeation of acids and enzymes through the spore wall, with the formation of pores on the spore surface as reported for other model systems [55]. To determine the suitability of our image rendering approach to quantify phagosome acidification, the capability of A. fumigatus cell surface mutants to acidify phagosomes was determined and compared by manual counts.…”

Regulatory Effects of Nitric Oxide on Reproduction and Melanin Biosynthesis in Onion Pathogenic Fungus Stemphylium Eturmiunum

“…While in the ubiquitous humanpathogenic fungus Aspergillus fumigatus, the pathogen that causes fatal lung infection in immunocompromised individuals, produces both DOPA and DHN melanins (Langfelder et al 2003). The presence of DHN melanin in conidia protects from phagocytic uptake and ROS-induced intracellular killing by frugivorous amoeba Protostelium aurantium and disrupts its autonomous defense (Ferling et al 2020). In Sporothrix schenckii, production of DOPA melanin in the presence of tyrosine in fungal cell confers more resistance to nitrogen-derived oxidants and UV irradiation (Almeida-Paes et al 2012).…”

The Pathogenesis of Aspergillus fumigatus, Host Defense Mechanisms, and the Development of AFMP4 Antigen as a Vaccine

“…The mechanism includes phagocytosis, reactive oxygen species (ROS) generations mediated by nicotinamide adenine dinucleotide phosphate-oxidase (NADPH), lactoferrin production, and neutrophil extracellular traps (NETs) formation ( Schoen et al 2019 ; Souza et al 2019 ; Shopova et al 2020 ). ROS production responds to swollen conidia, but not resting conidia, through NADPH oxidase activation ( Ferling et al 2020 ; Khani et al 2020 ). It has been demonstrated that the ROS-producing complex plays a crucial fungicidal role during A. fumigatus infection ( Shen et al 2016 ).…”