Fungal Melanin

85. Isolation of an Aspergillus fumigatus mutant strain with reduced virulence.

Axel A. Brakhage1, Andreas Koch¹, Axel Schmidt³ , Gerhard Wanner ⁴, Sucharit Bhakdi² and Bernhard Jahn².

¹Lehrstuhl fur Mikrobiologie, Universitat Munchen, 80638 Munchen, FRG ²Institut fur Medizinische Mikrobiologie, Universitat Mainz, 55101 Mainz, FRG ; ³ Institut fur Chemotherapie, Bayer AG, 42349 Wuppertal, FRG;

⁴Botanisches Institut, Universitat,Munchen, 80638 Munchen, FRG.

Aspergillus fumigatus is an important pathogen of the immunocompromised host causing pneumonia and invasive disseminated disease with high mortality. Since the survival of conidia in the host is prerequisite for establishing disease, we have been attempting to identify factors which are associated with conidia and, simultaneously, of importance for infection. Therefore, A. fumigatus mutant strains were isolated exhibiting altered conidia pigmentation. One mutant strain produced white conidia (white, W) and apparently lacked the pigment. Scanning electron microscopy revealed that conidia of the W mutant differed in their cell wall morphology from those of the wild type (WT). Luminol-dependent chemiluminescence was ten-fold higher when human neutrophils or monocytes were challenged with W conidia compared with WT conidia. Furthermore, W conidia were more susceptible to killing by oxidants in vitro. W conidia were also more efficiently damaged by human monocytes in vitro than WT conidia. ln a murine animal model, compared with the wild-type, conidia of the W mutant strain showed reduced virulence. These results suggest that pathways related to conidial pigmentation contribute to pathogenicity of A. fumigatus.

86. Fungal reactive armor: Redox buffering by melanin and Fe(II) Cryptococcus neoformans.

Eric S. Jacobson, McGuire Veterans Affairs Medical Center and Virginia Commonwealth University, Richmond, VA.

Melanin is an fungal extracellular redox buffer which, in principle, can neutralize antimicrobial oxidants generated by immunologic effector cells, but ist source Of reducing equivalents is not known. We wondered whether Fe(II) generated by the external ferric reductase of fungi might have the physiologic fimction of reducing fungal melanin and thereby promoting pathogenesis. Exposure to micromolar Fe(II) decreased the open circuit potential of a melanin film electrode from 0.00 V to -0.10 V, relative to a silver-silver chloride standard, and decreased the area of the cyclic voltammetric reduction wave by 50%, indicating reduction. Moreover, exposure to FE(II) increased electrochemical buffering by 44%, while exposure to millimolar dithionite reduced the film but did not increase buffering. The ratio of the amount of bound iron to the amount of the incremental increase in the following oxidation wave was approximately 1.0, suggesting that bound iron participates in buffefing. Light absorption by melanin suspensions or suspended, melanized C. neoformans was decreased 14% and 8%, respectively, by treatment with Fe(II), consistent with reduction of melanin. Cultures of C. neoformans grown in solubilized 1 mM Fe(III) generated 160 uM FE(II) in cultural supernatant. We infer that Fe(II) can reduce melanin under physiologic conditions; moreover, it binds to melanin and cooperatively increases redox buffering. The data support a model for physiologic redox cycling of fungal melanin, whereby electrons exported by the yeast to form extracellular FE(III) maintain the reducing capacity of the extracellular redox buffer.

87. The genetics of Gaeumannomyces graminis with particular reference to pigment production and pathogenicity.

Kelly, C.P^l., Osbourn, A.E². and Caten, C.E^l. ¹ School of Biological Sciences, University of Birmingham, Edgbaston,

Birmingham, B15 2TT, U.K. ² The Sainsbury Laboratory, Norwich Research Park, Colney, Norwich, NR7 4UH, U.K.

Pigmented hyphae of the take-all fungus Gaeumannomyces graminis (Gg) are observed both on infected host tissue and when the fungus is grown in vitro. The pigment has previously been partially purified and has properties characteristic of melanin. Growth of isolates in the presence of inhibitors of melanin biosynthesis such as tricyclazole and pyroquilon, give rise to characteristic changes in hyphal pigmentation, but did not affect growth rate. We are using two approaches to determine whether the pigment is necessary for pathogenicity and survival as a saprophyte. An albino and several pale mutants have been isolated following irradiation of protoplasts with ultra-violet light. Results suggest that melanin production is essential for pathogenicity in Gg. The second approach is to disrupt genes involved in melanin biosynthesis. A region of ~2.7kb has been subcloned and sequenced. Transformation mediated gene disruption of Gg will ultimately confirm the function and importance of this cloned DNA.

88. Construction of cDNA library and screening of genes that expressed specifically during appressorium formation of Colletotrichum lagenarium.

I. Kuroda, Y. Takano¹, I. Furusawa¹, O. Horino and Y. Kubo. Kyoto Prefectural University and ¹Kyoto University, Kyoto, Japan .

Infection by Colletotrichum lagenarium requires appressorium differentiation. To identify genes that express preferentially during germination and appressorium formation, we constructed cDNA library and performed differential cDNA screening. Poly(A)+ RNA from appressorium-forming conidia incubated 6 hours was used to construct a directional cDNA library in LambdaGEM4. To identify specific cDNAs in appressorium-forming conidia, 12,000 cDNA clones from this cDNA library were applied to differential screening. In first screening, 125 cDNAs which did not hybridize to cDNA probes synthesized from poly(A)+ RNA of growing vegetative hyphae were selected. As second screening, 32 cDNAs which preferentially hybridized to cDNA probes synthesized from poly (A)+ RNA of appressorium-forming conidia at 6 hours were selected. Finally, some cDNAs were confirmed to be preferentially expressed during appressorium formation by RNA blot analysis. Two of these clones had strong signal were sequenced. One clone had high percentage of agreement with grg-1 glucose-repressive gene in Neurospora crassa, and the other had with ClpB the heat shock protein in E. coli.

89. Molecular phylogeny of graminicolous species of Heiminthosporium sensu lato, Bipolaris, Curvularia, Drechslera and Exserohilum.

Kiminori Shimizu, Chihiro Tanaka and Mtsuya Tsuda. Pesticide Research Institute, Faculty of Agriculture, Kyoto University, Kyoto 606-01, Japan.

Some graminicolous plant pathogenic fungi, Bipolaris, Curvularia, Drechslera and Exserohilum had been classified in Helminthosporium sensu lato. They are now treated as separate genera mentioned above. These fungi have melanin with 1,8-dihydroxynaphthalene as a precursor. We cloned and sequenced the Bipolaris maydis gene coding for the reductase gene involved in melanin biosynthesis (Brn1). The Brn1 gene contains one open reading frame, consisting of 3 exons separated by two introns, and the predicted Brnl polypeptide consists of 267 amino acids. This gene was contained only one copy per genome in B. maydis. By using this gene, we attempt to elucidate the taxonomical relationships in so-called graminicolous Helminthosporium species. The Brn1 gene of some selected members in four genera was amplified with partial sequences derived from that of B. maydis as primers in PCR and sequenced. Alignment of these sequences showed high similarities to each other through the entire sequence but intron regions. Maximum parsimony analysis using these sequences constructed four clusters. The cladogram well reflected the differentiation in this gene within these four genera. However, relationships within these four genera differ from those based on morphological characters.

90. The temporal transcriptional pattern of three melanin biosynthesis genes, PKS1, SCD1, and THR1 in appressorium-differentiating and non-differentiating conidia of Colletotrichum lagenarium .

Yoshitaka Takano1, Yasuyuki Kubo², Itaru Kuroda² and Iwao Furusawa¹. ¹ Laboratory of Plant Pathology, Faculty of

Agriculture, Kyoto University, Kyoto 606-01, Japan. ² Laboratory of Plant Pathology, Faculty of Agriculture,

Kyoto Prefectural University, Kyoto 606, Japan

A phytopathogenic fungus, Colletotrichum lagenarium produces melanized appressoria that display temperature-sensitive differentiation. Conidia incubated in water at 24 C germinated and germ tubes differentiated into melanized appressoria. On the other hand, conidia in water at 32 C germinated and elongated germ tubes without appressorium differentiation. Conidia in 0.1% yeast extract solution at 32 C germinated and developed into vegetative hyphae. Here, we investigated the temporal transcriptional pattern of cloned melanin biosynthesis genes, PKS1, SCD1, and THR1 in these differentiating and non differentiating conidia. During appressorium differentiation, de novo transcripts of the three melanin biosynthesis genes accumulated by 1-2 h after the start of conidial incubation at 24 C and began to decrease at 6 h. In conidia germinating in water at 32 C, the transcriptional pattern of that in appressorium-forming conidia, although no appressoria were formed. However, in conidia in 0.1% yeast extract solution at 32C, transcripts of the three melanin biosynthesis genes hardly accumulated.

91. Purification and analysis of expression pattern of melanin biosynthetic enzymes, scytalone dehydratase and 1,3,8-trihydroxynaphthalene reductase in Colletotrichum lagenarium.

Gento Tsuji, Toshiyuki Takeda¹, Yoshitaka Takano², Iwao Furusawa², Osamu Horino and Yasuyuki Kubo. Kyoto

Prefectural University, Kyoto 606, Japan. ¹Zen-Noh R&D, Kanagawa 254, Japan. ²Kyoto University, Kyoto 606, Japan.

Melanin biosynthesis of Colletotrichum lagenarium is essential for appressorial penetration of its host plants. In the melanin biosynthetic pathway, scytalone dehydratase and 1,3,8-trihydroxynaphthalene reductase catalyze the conversion of scytalone to 1,3,8-trihydroxynaphthalene and 1,3,8-trihydroxynaphthalene to vermelone, respectively. Previously, we cloned SCD1 gene coding for scytalone dehydratase and THR1 gene coding for 1,3,8-trihydroxynaphthalene reductase. In this study, the heterologuous expression vectors were constructed and the recombinant enzymes were purified. Polyclonal antibodies against purified recombinant scytalone dehydratase and 1,3,8-trihydroxynaphthalene reductase were prepared. By western blot analysis, scytalone dehydratase and 1,3,8-trihydroxynaphthalene were detected in melanized mycelia of C. lagenarium. In C. lagenarium, appressorial differenciation and melanization proceed synchronously. At present, we are investigating expression patterns of these enzymes during appressorium differentiation.