Dataset Information

Phagolysosomal proteomics identifies DHN-melanin-regulated processes in activated macrophage in response to Aspergillus fumigatus conidia

ABSTRACT: Infection by the human pathogenic fungus Aspergillus fumigatus is initialized by the outgrowth of asexual spores (conidia) into the lung tissue of the immunocompromised host following an inhalation of the airborne conidia. The resident phagocytes, the alveolar macrophages are the first immune cells to encounter invading conidia. However, A. fumigatus conidia employ versatile mechanisms to evade the host immune defense and establish a severe invasive infection. Previously, we showed that depending on the presence of conidial 1,8-dihydroxynaphthalene (DHN) melanin, A. fumigatus circumvents intracellular killing by manipulating the phagolysosomal maturation process. Here, by comparative dual proteomics we analyzed proteins of phagolysosomes containing melanized wild-type or non-melanized pksP mutant conidia. Bioinformatics compiled a regulatory module of differentially abundant proteins that mirrors processes targeted by the fungus for immune evasion. Those are i.e. vATPAse-driven phagolysosomal acidification, endocytic trafficking, signal transduction, energy metabolism and immune response. In detail, we found alterations of vATPase complex assembly and impaired abundances of mTOR and MAPK signaling molecules, Rab5 and Vamp8 mediators of endosomal trafficking as well as Lamp1 and cathepsin Z lysosomal markers.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Neosartorya Fumigata (aspergillus Fumigatus) Mus Musculus (mouse)

TISSUE(S): Conidium, Macrophage

SUBMITTER: Thomas Krüger

LAB HEAD: Axel A. Brakhage

PROVIDER: PXD006134 | Pride | 2018-03-08

REPOSITORIES: Pride

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Proteomics of Aspergillus fumigatus Conidia-containing Phagolysosomes Identifies Processes Governing Immune Evasion.

Schmidt Hella H Vlaic Sebastian S Krüger Thomas T Schmidt Franziska F Balkenhol Johannes J Dandekar Thomas T Guthke Reinhard R Kniemeyer Olaf O Heinekamp Thorsten T Brakhage Axel A AA

Molecular & cellular proteomics : MCP 20180305 6

Invasive infections by the human pathogenic fungus Aspergillus fumigatus start with the outgrowth of asexual, airborne spores (conidia) into the lung tissue of immunocompromised patients. The resident alveolar macrophages phagocytose conidia, which end up in phagolysosomes. However, A. fumigatus conidia resist phagocytic degradation to a certain degree. This is mainly attributable to the pigment 1,8-dihydroxynaphthalene (DHN) melanin located in the cell wall of conidia, which manip ...[more]

PMID: 29507050

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Project description:A. flavus keratitis is one of the predominant fungal infections affecting the tropical parts of the world. Melanin is a virulence factor in numerous pathogenic fungi and the melanin layer maintains the cell wall structure and provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile and virulence of melanized and non-melanized conidia of A. flavus. An environmental isolate and two clinical isolates were included in this study. L-DOPA melanin pathway-specific inhibitor kojic acid (KA) as used to prepare non-melanized conidia. Conidial surface proteins were extracted using 100% formic acid at 0 ºC and the extracted proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Formic acid extracts were analyzed in an Orbitrap Velos pro-mass spectrometer. Conidial surface morphology difference was examined using scanning electron microscopy (SEM). Furthermore, lactophenol and calcofluor staining were performed to check the morphology and staining efficiency of melanized conidia (MC) and non-melanized conidia (NMC). A Galleria mellonella insect model was used to examine the virulence efficiency of MC and NMC. Kojic acid treatment inhibited melanin synthesis in A. flavus and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathway were found only in the formic acid extracts of NMC. SEM analysis showed the conidial surface morphology difference between the NMC and MC indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining of MC and NMC. Evaluation of virulence of MC and NMC in G. mellonella model showed NMC was less virulent compared to MC . Our results showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer and hence melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. G. mellonella in vivo virulence assay also confirmed that the non-melanized conidia were susceptible to host defense as in other Aspergillus pathogens.

			Action	DRS
	LFQ-Afum_Mmus_UP.mgf	Mgf
	LFQ-Afum_Mmus_UP.msf	Msf
	LFQ-Afum_Mmus_UP.pdResult	Other
	LFQ-Afum_Mmus_UP.pep.xml	Pepxml
	LFQ-Afum_Mmus_UP.prot.xml	Xml

Dataset Information

Phagolysosomal proteomics identifies DHN-melanin-regulated processes in activated macrophage in response to Aspergillus fumigatus conidia

Dataset's files

Publications

Proteomics of <i>Aspergillus fumigatus</i> Conidia-containing Phagolysosomes Identifies Processes Governing Immune Evasion.

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