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The role of RodAp-conserved cysteine residues in the Aspergillus fumigatus conidial surface organization

ABSTRACT: Immune inertness of Aspergillus fumigatus conidia, an airborne fungal pathogen, is attributed to its surface rodlet-layer made up of RodAp, a protein belonging to the hydrophobin family, characterized by eight conserved cysteine residues forming four disulfide bonding. Earlier, we showed that the conserved cysteine residue point (ccrp) mutations result in conidia devoid of the rodlet-layer. Here we extended our study in comparing ccrp-mutation with RODA deletion on their mutant conidial surface organization, permeability and immunoreactivity. Western blot using anti-RodAp antibodies indicated the absence of RodAp in the cytoplasm of ccrp-mutant conidia. Upon immunolabelling, ccrp-mutant conidia showed strong positivity for -(1,3)-glucan and weak positivity for -(1,3)-glucan, which was reverse in ∆rodA conidia, and the parental strain conidia were negative; all of their conidial cell wall permeability was similar. Proteomic analyses of the conidial surface exposed proteins of the ccrp-mutants, although lower in number, showed more similarities with the parental strain, but a significant difference with the RODA deletion mutant strain. Further, ccrp-mutant conidia are less immunostimulatory compared to ∆rodA conidia. Together, our data suggest that (i) the conserved cysteine residues are essential for the trafficking of RodAp and the organization of rodlet-layer on the conidial surface, and (ii) point-mutation could be an alternative strategy to study the proteins involved in the organization of fungal cell wall.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Neosartorya Fumigata (aspergillus Fumigatus)

SUBMITTER: Thomas Krüger

LAB HEAD: Axel A. Brakhage

PROVIDER: PXD020136 | Pride | 2021-02-18

REPOSITORIES: Pride

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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.

Project description:Aerial conidia of Ascomycetes and Basidiomycetes are coated with an amphipathic hydrophobin layer commonly called rodlet layer, that renders their surface hydrophobic and resist wetting, thus facilitating effective dispersal in the air. Fungal hydrophobins are small secreted proteins with specific hydropathy patterns and eight cysteine residues ordered in a particular manner which define the two well characterized classes I and II of hydrophobins and a third intermediate class (Kershaw and Talbot, 1998. Littlejohn et al, 2012; Jensen et al, 2010). Wessels 1994). Class I assemblies are generally water insoluble, have an amyloid structure and form the patterned rodlet layer. Class II are smaller proteins than class I and are soluble in aqueous ethanol mixtures of SDS. The intermediate class is a rather heterogenous tote class which would gather non-class I and non-class II hydrophobins without a defined structure. In addition to the role of the rodlet in the fungal life during conidiogenesis and conidial dispersal in the air, it was shown that rodlets can have a role in fungal – host interactions and favour the pathogenic behavior of the fungal pathogens in humans, plant and insect hosts (Talbot et al., 1996, Aimanianda et al, 2009; Zhang et al, 2011). In Aspergillusfumigatus rodlets immunosilence the conidium and their loss from the conidial surface initiates the host immune response (Aimanianda et al., 2009). Our study is focused on the Rod A protein which forms the rodlet structure on the surface of the conidium of A. fumigatus. In spite of their prominent morphological and biological role, the molecular basis for rodlet self-assembly and desagregation in this fungal species has not been understood yet. Point mutations in the rodA gene have helped understanding the mechanism which drives hydrophobin proteins to assemble into highly ordered structures anchored to the conidium surface. Moreover, the biochemical events responsible for the lysis of the rodlets have been investigated.

			Action	DRS
	AF1-1-a.raw	Raw
	AF1-1-b.raw	Raw
	AF1-1-c.raw	Raw
	AF1-1.fasta	Fasta
	AF1-1.mgf	Mgf

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The role of RodAp-conserved cysteine residues in the Aspergillus fumigatus conidial surface organization

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