Melanin in the human pathogenic fungus Aspergillus fumigatur : Discovery of a novel melanin in the fungus and the use of conidial melanin as a target for camelid heavy-chain antibodies

摘要

Aspergillus fumigatus is the causative agent of the invasive aspergillosis which is often fatal for immunocompromised patients. This work aimed at: (i) studying the role of melanisation as pathogenicity mechanism and (ii) detecting the fungus with the help of a melanin specific antibody. A camelid antibody from a recombinant VHH-domain library was successfully selected against melanised conidia applying phage-display and a newly developed filtration approach. Its recombinant production in Escherichia coli by high cell density fermentation provided a pure antibody which bound with high affinity to melanised conidia of different Aspergillus species. A less stringent binding to synthetic DOPA-melanin and Sepia-melanin was observed in ELISA studies. Furthermore, the biotinylated antibody demonstrated melanisation of conidia and hyphae by an immunofluorescent staining approach. Furthermore, melanisation of A. fumigatus was studied by means of deletion mutants of genes coding for key enzymes of the DHN-melanin and tyrosine degradation pathway. The loss of a functional Arp2 enzyme, a reductase involved in the DHN-melanin biosynthesis, did not result in a distinct phenotype from wild type despite the colour change of the conidia from grey-green to pinkish. Hence, the modified DHN-melanin is as protective as the grey-green wild-type pigment.By the deletion of the genes encoding homogentisate dioxygenase (HmgA) and 4-hydroxyphenylpyruvate dioxygenase (HppD) it was shown that homogentisic acid is the major intermediate in the L-tyrosine degradation pathway which yields in the formation of pyomelanin. Despite activation of the genes by tyrosine and during infiltrational growth in the mouse lung and despite the increased sensitivity of the germlings of the hppD strain to ROI, the mutant was not attenuated in its virulence in the mouse infection model. Hence, this work provides the proof for the presence of pyomelanin in A. fumigatus.