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Assembly and disassembly of Aspergillus fumigatus conidial rodlets.

ABSTRACT: The rodlet structure present on the Aspergillus fumigatus conidial surface hides conidia from immune recognition. In spite of the essential biological role of the rodlets, the molecular basis for their self-assembly and disaggregation is not known. Analysis of the soluble forms of conidia-extracted and recombinant RodA by NMR spectroscopy has indicated the importance of disulfide bonds and identified two dynamic regions as likely candidates for conformational change and intermolecular interactions during conversion of RodA into the amyloid rodlet structure. Point mutations introduced into the RODA sequence confirmed that (1) mutation of a single cysteine was sufficient to block rodlet formation on the conidial surface and (2) both presumed amyloidogenic regions were needed for proper rodlet assembly. Mutations in the two putative amyloidogenic regions retarded and disturbed, but did not completely inhibit, the formation of the rodlets in vitro and on the conidial surface. Even in a disturbed form, the presence of rodlets on the surface of the conidia was sufficient to immunosilence the conidium. However, in contrast to the parental conidia, long exposure of mutant conidia lacking disulfide bridges within RodA or expressing RodA carrying the double (I115S/I146G) mutation activated dendritic cells with the subsequent secretion of proinflammatory cytokines. The immune reactivity of the RodA mutant conidia was not due to a modification in the RodA structure, but to the exposure of different pathogen-associated molecular patterns on the surface as a result of the modification of the rodlet surface layer. The full degradation of the rodlet layer, which occurs during early germination, is due to a complex array of cell wall bound proteases. As reported earlier, this loss of the rodlet layer lead to a strong anti-fumigatus host immune response in mouse lungs.

SUBMITTER: Valsecchi I

PROVIDER: S-EPMC7389560 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:Aspergillus fumigatus, a filamentous fungus producing bluish-green conidia, is an important opportunistic pathogen that primarily affects immunocompromised patients. Conidial pigmentation of A. fumigatus significantly influences its virulence in a murine model. In the present study, six genes, forming a gene cluster spanning 19 kb, were identified as involved in conidial pigment biosynthesis in A. fumigatus. Northern blot analyses showed the six genes to be developmentally regulated and expressed during conidiation. The gene products of alb1 (for "albino 1"), arp1 (for "aspergillus reddish-pink 1"), and arp2 have high similarity to polyketide synthases, scytalone dehydratases, and hydroxynaphthalene reductases, respectively, found in the dihydroxynaphthalene (DHN)-melanin pathway of brown and black fungi. The abr1 gene (for "aspergillus brown 1") encodes a putative protein possessing two signatures of multicopper oxidases. The abr2 gene product has homology to the laccase encoded by the yA gene of Aspergillus nidulans. The function of ayg1 (for "aspergillus yellowish-green 1") remains unknown. Involvement of the six genes in conidial pigmentation was confirmed by the altered conidial color phenotypes that resulted from disruption of each gene in A. fumigatus. The presence of a DHN-melanin pathway in A. fumigatus was supported by the accumulation of scytalone and flaviolin in the arp1 deletant, whereas only flaviolin was accumulated in the arp2 deletants. Scytalone and flaviolin are well-known signature metabolites of the DHN-melanin pathway. Based on DNA sequence similarity, gene disruption results, and biochemical analyses, we conclude that the 19-kb DNA fragment contains a six-gene cluster which is required for conidial pigment biosynthesis in A. fumigatus. However, the presence of abr1, abr2, and ayg1 in addition to alb1, arp1, and arp2 suggests that conidial pigment biosynthesis in A. fumigatus is more complex than the known DHN-melanin pathway.

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Assembly and disassembly of Aspergillus fumigatus conidial rodlets.

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