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This chapter establishes a catalog of the proteases secreted by Aspergillus fumigatus, briefly describes their various properties, and examines their biological functions, ranging from protein digestion into short peptides and assimilable amino acids to specific proteolysis during infection. The proteases include all enzymes that catalyze the cleavage of peptide bonds (CO-NH) of proteins, digesting them into peptides or free amino acids. The proteases can be further divided into endoproteases and exoproteases. From a physiological point of view, Aspergillus secreted proteases can be classified as either acidic, neutral, or basic. Efficient protein degradation of hard keratin by hydrolytic enzymes has to be accompanied by simultaneous reduction of cysteine disulfide bridges. Although it can be stated that individual proteases such as Alp1, Mep, and Pep1 are not essential for tissue invasion, it cannot be ruled out that secreted proteases do not contribute to the establishment of invasive aspergillosis. A. fumigatus also does not possess specific large gene families encoding secreted proteases. Aspartic proteases of the A1 family and carboxypeptidases of the S10 family are multiple in many fungi. The emergence of multigenic families is most frequently due to ancient gene duplication processes allowing organisms to better adapt to different environmental conditions, and marked differences occur from one pathogenic species to another.
Hydrophobicity plots of two pepsin-like proteases, Pep1 (MER001437) and AP1 (MER082513) (A1 family) ( Table 1 ). Pep1 is a secreted enzyme with a hydrophobic signal sequence. In contrast, AP1 has no hydrophobic signal sequence at its beginning. For the plot, the program TopPred, available at http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html, was used. The Pep1 signal peptidase cleavage site is indicated by a vertical arrow.
Phylogenetic relationships of aspartic proteases (A1 family) from A. fumigatus, A. nidulans, A. terreus, and S. cer-evisiae. Sequences of the well-characterized A. niger PepA (MER00919) and A. oryzae PepO (MER00920) were also included. Phylogenetic analyses were performed as previously described by Zaugg et al. (2008) . Three well-supported monophyletic groups correspond to MEROPS identifiers A01.026, A01.018, and A01.077. The other proteases are not assigned to specific identifiers. Nonprotease homologs in A. nidulans and A. terreus are indicated by an asterisk.
Immunofluorescence reaction of aspartic protease antigen at the surface of hyphae and conidiophores of A. fumigatus. Indirect immunofluorescence was achieved with anti-Pep1 rabbit antiserum.
Phylogenetic relationships of A. fumigatus carboxypeptidases with other biochemically well-characterized carboxy-peptidases of the S10 family. The tree is rooted with plant carboxypeptidases of the S10.005 subfamily. Phylogenetic analyses were performed as previously described ( Zaugg et al., 2008 ). Abbreviations: Tru, Tricho phyton rubrum; Afu, A. fumigatus; Aory, A. oryzae; Anid, A. nidulans; Anig, A. niger; Pja, Penicillium janthinellum; Scer, S. cerevisiae; Psat, Pisum sativum; Taes, Triticum aestivum. MEROPS identifiers are as follows: AfuCp1, MER079359; AfuCp2, MER079360; AfuCp3, MER079361; AfuCp4, MER079362; AfuCp5, MER079363; AfuCp6, MER082516; AfuCp7, MER082530; AfuCp8, MER032646; AfuCp9, MER032643; AoryS1, MER016549; AnidCpyA, MER090176, AnigCpD1, MER027994; AnigCpD2, MER000415; PjaS1, MER000412; ScerCpY, MER002010; TruScpB, MER079400; TruScpC, MER079401. TruScpA ( Zaugg et al., 2008 ) is not registered in MEROPS.
Direct mycological examination of an infected nail, showing A. oryzae hyphae and conidiophores. Nail scrapings were examined in a dissolving solution containing a fluorochrome ( Monod et al., 1989 ). In situ identification of the fungus was performed using a PCR and sequencing method ( Monod et al., 2006 ).
Reactivity of mycelia in the lung of an immunocompromised mouse infected with A. fumigatus. Indirect immunofluorescence was achieved with anti-Alp1 rabbit antiserum.
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