Hans-Martin Dahse

Factor H related protein 1 (CFHR-1) inhibits complement C5 convertase activity and terminal complex formation

Homozygous deletion of a 84-kb genomic fragment in human chromosome 1 that encompasses the CFHR1 and CFHR3 genes represents a risk factor for hemolytic uremic syndrome (HUS) but has a protective effect in age-related macular degeneration (AMD). Here we identify CFHR1 as a novel inhibitor of the complement pathway that blocks C5 convertase activity and interferes with C5b surface deposition and MAC formation. This activity is distinct from complement factor H, and apparently factor H and CFHR1 control complement activation in a sequential manner. As both proteins bind to the same or similar sites at the cellular surfaces, the gain of CFHR1 activity presumably is at the expense of CFH-mediated function (inhibition of the C3 convertase). In HUS, the absence of CFHR1 may result in reduced inhibition of terminal complex formation and in reduced protection of endothelial cells upon complement attack. These findings provide new insights into complement regulation on the cell surface and biosurfaces and likely define the role of CFHR1 in human diseases.

Heterocyclic benzazole derivatives with antimycobacterial in vitro activity.

The series of 2-benzylsulfanyl derivatives of benzoxazole and benzothiazole were synthesized, evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis and non-tuberculous mycobacteria, and the activity expressed as the minimum inhibitory concentration (MIC) in micromol/L. The substances bearing two nitro groups (4e, 4f, 5e, 5f) or a thioamide group (4i, 4j, 5i, 5j) exhibited appreciable activity particularly against non-tuberculous strains. The most active compounds were subjected to the toxicity assay and were evaluated as moderately cytotoxic.

Conidial Dihydroxynaphthalene Melanin of the Human Pathogenic Fungus Aspergillus fumigatus Interferes with the Host Endocytosis Pathway.

Aspergillus fumigatus is the most important air-borne fungal pathogen of humans. The interaction of the pathogen with the hosts immune system represents a key process to understand pathogenicity. For elimination of invading microorganisms, they need to be efficiently phagocytosed and located in acidified phagolysosomes. However, as shown previously, A. fumigatus is able to manipulate the formation of functional phagolysosomes. Here, we demonstrate that in contrast to pigmentless pksP mutant conidia of A. fumigatus, the gray-green wild-type conidia inhibit the acidification of phagolysosomes of alveolar macrophages, monocyte-derived macrophages, and human neutrophil granulocytes. Therefore, this inhibition is independent of the cell type and applies to the major immune effector cells required for defense against A. fumigatus. Studies with melanin ghosts indicate that the inhibitory effect of wild-type conidia is due to their dihydroxynaphthalene (DHN)-melanin covering the conidia, whereas the hydrophobin RodA rodlet layer plays no role in this process. This is also supported by the observation that pksP conidia still exhibit the RodA hydrophobin layer, as shown by scanning electron microscopy. Mutants defective in different steps of the DHN-melanin biosynthesis showed stronger inhibition than pksP mutant conidia but lower inhibition than wild-type conidia. Moreover, A. fumigatus and A. flavus led to a stronger inhibition of phagolysosomal acidification than A. nidulans and A. terreus. These data indicate that a certain type of DHN-melanin that is different in the various Aspergillus species, is required for maximal inhibition of phagolysosomal acidification. Finally, we identified the vacuolar ATPase (vATPase) as potential target for A. fumigatus based on the finding that addition of bafilomycin which inhibits vATPase, led to complete inhibition of the acidification whereas the fusion of phagosomes containing wild-type conidia and lysosomes was not affected.

Botryorhodines A–D, antifungal and cytotoxic depsidones from Botryosphaeria rhodina, an endophyte of the medicinal plant Bidens pilosa

An endophytic fungus (Botryosphaeria rhodina) was isolated from the stems of the medicinal plant Bidens pilosa (Asteraceae) that is known for its anti-inflammatory, antiseptic and antifungal effects. The ethyl acetate extract of the fungal isolate exhibits significant antifungal activity as well as potent cytotoxic and antiproliferative effects against several cancer cell lines. Activity-guided fractionation resulted in the isolation of a complex of four depsidones, botryorhodines A-D and the auxin indole carboxylic acid. Botryorhodine A and B show moderate to weak cytotoxic activities against HeLa cell lines with a CC(50) of 96.97 microM and 36.41 microM, respectively. In addition, they also show antifungal activity against a range of pathogenic fungi such as Aspergillus terreus (MIC 26.03 microM for botryorhodine A and 49.70 microM for B) and the plant pathogen Fusarium oxysporum (MIC 191.60 microM for botryorhodine A and 238.80 microM for B). A potential role of the endophyte in modulating fungal populations living within or attacking the host plant is discussed.

Secretome analysis of Aspergillus fumigatus reveals Asp-hemolysin as a major secreted protein

Surface-associated and secreted proteins represent primarily exposed components of Aspergillus fumigatus during host infection. Several secreted proteins are known to be involved in defense mechanisms or immune evasion, thus, probably contributing to pathogenicity. Furthermore, several secreted antigens were identified as possible biomarkers for the verification of diseases caused by Aspergillus species. Nevertheless, there is only limited knowledge about the composition of the secretome and about molecular functions of particular proteins. To identify secreted proteins potentially essential for virulence, the core secretome of A. fumigatus grown in minimal medium was determined. Two-dimensional gel electrophoretic separation and subsequent MALDI-TOF-MS/MS analyses resulted in the identification of 64 different proteins. Additionally, secretome analyses of A. fumigatus utilizing elastin, collagen or keratin as main carbon and nitrogen source were performed. Thereby, the alkaline serine protease Alp1 was identified as the most abundant protein and hence presumably represents an important protease during host infection. Interestingly, the Asp-hemolysin (Asp-HS), which belongs to the protein family of aegerolysins and which was often suggested to be involved in fungal virulence, was present in the secretome under all growth conditions tested. In addition, a second, non-secreted protein with an aegerolysin domain annotated as Asp-hemolysin-like (HS-like) protein can be found to be encoded in the genome of A. fumigatus. Generation and analysis of Asp-HS and HS-like deletion strains revealed no differences in phenotype compared to the corresponding wild-type strain. Furthermore, hemolysis and cytotoxicity was not altered in both single-deletion and double-deletion mutants lacking both aegerolysin genes. All mutant strains showed no attenuation in virulence in a mouse infection model for invasive pulmonary aspergillosis. Overall, this study provides a comprehensive analysis of secreted proteins of A. fumigatus and a detailed characterization of hemolysin mutants.

Human complement factor H-related protein 4 binds and recruits native pentameric C-reactive protein to necrotic cells

Human complement factor H-related protein 4 (CFHR4) is a plasma glycoprotein which appears in two isoforms. CFHR4 is a member of the factor H protein family, and shares structural similarity and sequence homology with the other CFHR proteins and with the complement regulator factor H. Given the structural and sequence similarity, we hypothesized that similar to factor H, CFHR4 binds to C-reactive protein (CRP). We have recombinantly expressed the two CFHR4 isoforms and analyzed their binding to both native and denatured, monomeric CRP. Here, we show that both CFHR4 isoforms bind in the presence of calcium to native pentameric CRP, but not to modified CRP. This is in contrast to factor H, which binds to modified CRP independent of calcium. Comparison of the two CFHR4 isoforms and a recombinant CFHR4 fragment for CRP binding indicates that the first domain of CFHR4 is relevant for this interaction. Interaction of the native proteins was demonstrated by co-precipitation of CFHR4 and CRP from serum of sepsis patients with elevated CRP levels. CFHR4 bound to necrotic cells and was localized in necrotic tumor tissue as demonstrated by immunohistological analyses. In addition, CFHR4 facilitated binding of native CRP to the surface of necrotic cells. Altogether these data identify CFHR4 as a novel ligand for native CRP, and suggest a role for CFHR4 in opsonization of necrotic cells.

Cytotoxic Pheofungins from an Engineered Fungus Impaired in Posttranslational Protein Modification

What makes a fungus blush? The deletion of a gene that is required for global protein N-acetylation triggers the production of unprecedented metabolites in Aspergillus nidulans. The pronounced red pigmentation of the engineered mutant is caused by pheofungins (benzothiazinone chromophores), the biogenesis of which is strikingly similar to those of pheomelanins found in red bird feathers and hair of Celtic origin.

Secreted pH-Regulated Antigen 1 of Candida albicans Blocks Activation and Conversion of Complement C3

The complement system forms the first defense line of innate immunity and is activated within seconds upon infection by human pathogenic yeast Candida albicans. In this study, we identified a new complement evasion strategy used by C. albicans. The fungus secretes a potent complement inhibitor, pH-regulated Ag 1 (Pra1), which in the direct surrounding of the pathogen binds to fluid-phase C3 and blocks cleavage of C3 to C3a and C3b, as shown by ELISA, native gel electrophoresis, and Western blotting. Consequently, complement activation via the alternative and classical pathways is inhibited. In addition, the release of the anaphylatoxins C3a and C5a, as well as C3b/iC3b surface deposition, is reduced, as demonstrated by Western blotting, ELISA, confocal microscopy, and flow cytometry. By reducing C3b/iC3b levels at the yeast surface, Pra1 decreases complement-mediated adhesion, as well as uptake of C. albicans by human macrophages, as shown by flow cytometry. Thus, Pra1 is, to our knowledge, the first potent fungal complement inhibitor that favors C. albicans immune escape by inactivating and controlling host complement attack at the level of C3.

Furanocembranoids from the Soft Corals Sinularia asterolobata and Litophyton arboreum

The new cembranoid diterpene danielid (1) along with 3α‐ethoxyfuranocembranoid 2, pukalide (3), 13α‐acetoxypukalide (4), furanocembranoid 5, and furanosesquiterpene 6 have been isolated from the soft coral Sinularia asterolobata. The furanocembranoid diterpene 11β,12β‐epoxypukalide (7) and the sesquiterpene (−)‐bicyclogermacrene (8) have been obtained from the soft coral Litophyton arboreum. The structures were elucidated primarily by NMR spectroscopy. The furanocembranoids 2, 4, and 5 show good antiproliferative activities against the cell lines L‐929 and K‐562, and weak cytotoxic effects on HeLa cells.

Exploiting Enzymatic Promiscuity to Engineer a Focused Library of Highly Selective Antifungal and Antiproliferative Aureothin Analogues

Aureothin is a shikimate-polyketide hybrid metabolite from Streptomyces thioluteus with a rare nitroaryl moiety, a chiral tetrahydrofuran ring, and an O-methylated pyrone ring. The antimicrobial and antitumor activities of aureothin have caught our interest in modulating its structure as well as its bioactivity profile. In an integrated approach using mutasynthesis, biotransformation, and combinatorial biosynthesis, a defined library of aureothin analogues was generated. The promiscuity of the polyketide synthase assembly line toward different starter units and the plasticity of the pyrone and tetrahydrofuran ring formation were exploited. A selection of 15 new aureothin analogues with modifications at the aryl residue, the pyrone ring, and the oxygenated backbone was produced on a preparative scale and fully characterized. Remarkably, various new aureothin derivatives are less cytotoxic than aureothin but have improved antiproliferative activities. Furthermore, we found that the THF ring is crucial for the remarkably selective activity of aureothin analogues against certain pathogenic fungi.