sodium-dodecyl-sulfate and Aspergillosis

Mitogen-activated protein kinase (MAPK) signaling pathways are involved in the regulation of various cellular responses in eukaryotes. In fungal pathogens they are of special interest because of their possible contribution to pathogenicity. Bioinformatic analysis of the genome of the most prevalent airborne human pathogenic fungus Aspergillus fumigatus, revealed the presence of four distinct MAPK-encoding genes. Here, we present the detailed functional analysis of one of these MAPKs, MpkA. Comparative analysis revealed similarities of MpkA with MAPKs involved in cell wall integrity signaling of other fungi. Accordingly, the analysis of mpkA deletion mutants revealed severe sensitivity of the mutants against cell wall active compounds, drastical alterations of the fungal morphology and increased resistance against oxidative stress. The expression of mpkA was induced by cell wall damaging conditions. Despite its involvement in cell wall signaling no influence on virulence of the deletion of mpkA was observed in a murine infection model.

Topics: Animals; Antifungal Agents; Artificial Gene Fusion; Aspergillosis; Aspergillus fumigatus; Benzenesulfonates; beta-Galactosidase; Caffeine; Cell Wall; Congo Red; Diamide; Gene Deletion; Gene Expression Regulation, Fungal; Genes, Reporter; Genetic Complementation Test; Hydrogen Peroxide; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Oxidants; Oxidative Stress; Signal Transduction; Sodium Dodecyl Sulfate; Survival Analysis; Virulence; Vitamin K 3

A serine proteinase (Alp) from the culture supernate of a clinical isolate of Aspergillus fumigatus was purified to virtual homogeneity at a yield of 41%. The procedure involved affinity chromatography on agarose-epsilon-amino-caproyl-D-tryptophan methyl ester. Alp had an estimated mol. wt of 32 Kda and the pI was determined at pH 7.9. The enzyme was fully inhibited by phenylmethyl sulphonyl fluoride, chymostatin and alpha-1-proteinase inhibitor, and it was largely inhibited by alpha-1-anti-chymotrypsin. Partial inhibition was observed with tosyl-phenylalanine chloromethyl ketone, but tosyl-lysine chloromethyl ketone was ineffective. Thus, Alp may be identical with the major chymotryptic activity of A. fumigatus, which has already been described. The N-terminal sequence of 25 amino acids revealed an 88% homology of Alp with the subtilisin-related proteinase of A. oryzae. Alp acted on casein over a broad range from pH 5.5 to 11.5 and also acts to a lesser extent on haemoglobin and serum albumin. The enzyme degraded elastin and a synthetic elastase substrate; hence, it may be identical with the previously described elastinolytic activity of the fungus. At pH 7.3 and a concentration of 1 microgram/ml, Alp was not toxic for Vero cells, but it efficiently detached such cells from a plastic surface. Specific antibodies against Alp were detected by enzyme immunoassay in the sera of patients and Alp-antigen was demonstrated by immunofluorescence in mycotic human lung. In addition, a second proteinase (Exalp) with extremely alkaline activity, and an aspartic proteinase of A. fumigatus are described.

Topics: Amino Acid Sequence; Animals; Antibodies, Fungal; Antigens, Fungal; Aspergillosis; Aspergillus fumigatus; Chromatography, Affinity; Chymotrypsin; Guinea Pigs; Humans; Hydrogen-Ion Concentration; Immunoenzyme Techniques; Kinetics; Male; Middle Aged; Sequence Homology, Nucleic Acid; Serine Endopeptidases; Serine Proteinase Inhibitors; Sodium Dodecyl Sulfate; Substrate Specificity; Vero Cells