F. Brouta

Purification and characterization of a 43.5 kDa keratinolytic metalloprotease from Microsporum canis.

A keratinolytic protease secreted by a feline clinical isolate of Microsporum canis cultivated in a broth containing feline keratin as the sole nitrogen source was purified from the culture filtrate by affinity chromatography on bacitracin-agarose and by hydrophobic chromatography on octyl-agarose. The enzyme had an apparent molecular mass of 43.5 kDa and the pI was 7.7. It had a significant activity against keratin azure, elastin-Congo red and denatured type I collagen (azocoll). Using the latter substrate, the optimum pH was around 8 and the apparent optimum temperature around 50 degrees C. The protease was strongly inhibited by 1,10-phenanthroline, phosphoramidon and EDTA. The first 13 N-terminal amino acid sequence showed a 61% homology with that of the extracellular metalloprotease of Aspergillus fumigatus and with the neutral protease I of A. oryzae, confirming that this 43.5 kDa keratinase is a metalloprotease. This keratinolytic metalloprotease could be a virulence-related factor involved in pathophysiological mechanisms of M. canis dermatophytosis.

Secreted Metalloprotease Gene Family of Microsporum canis

ABSTRACT Keratinolytic proteases secreted by dermatophytes are likely to be virulence-related factors. Microsporum canis, the main agent of dermatophytosis in dogs and cats, causes a zoonosis that is frequently reported. Using Aspergillus fumigatus metalloprotease genomic sequence (MEP) as a probe, three genes (MEP1, MEP2, and MEP3) were isolated from an M. canis genomic library. They presented a quite-high percentage of identity with both A. fumigatus MEP and Aspergillus oryzae neutral protease I genes. At the amino acid level, they all contained an HEXXH consensus sequence, confirming that these M. canis genes (MEP genes) encode a zinc-containing metalloprotease gene family. Furthermore, MEP3 was found to be the gene encoding a previously isolated M. canis 43.5-kDa keratinolytic metalloprotease, and was successfully expressed as an active recombinant enzyme in Pichia pastoris. Reverse transcriptase nested PCR performed on total RNA extracted from the hair of M. canis-infected guinea pigs showed that at least MEP2 and MEP3 are produced during the infection process. This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes.

Humoral and cellular immune response to a Microsporum canis recombinant keratinolytic metalloprotease (r-MEP3) in experimentally infected guinea pigs.

In order to better understand the host-fungus relationship in Microsporum canis dermatophytosis and to identify major fungal antigens, the immune response to a crude exoantigen preparation and to a purified recombinant keratinolytic metalloprotease (r-MEP3) was evaluated in guinea pigs experimentally infected with M. canis. Humoral and cellular immune responses were assessed from day 0 to day 57 post-infection (PI), the former by enzyme-linked immunosorbent assay (ELISA) and the latter via a lymphocyte proliferation assay. Infected guinea pigs developed humoral and cellular responses to both M. canis exoantigen and r-MEP3, while no specific immune response to these antigens was observed in control animals. This is the first report on the development of both humoral and cell-mediated immune responses to a purified keratinase in M. canis dermatophytosis.

A RETROSPECTIVE EPIDEMIOLOGICAL SURVEY OF CANINE AND FELINE DERMATOPHYTOSIS IN BELGIUM OVER THE PERIOD 1996–2001

Cyptococcus neoformans has been subdivided into 3 varieties: Cr neofonnans var. grubii (serotype A), var. neoformans, (serotypes D), hybrid (serotype A D ) and var. gattii (serotypes B, C). 340 clinical, environmental and veterinary isolates from Argentina, Brazil, Chile, Colonibia, Mexico, Peru, Venezuela, Guatemala and Spain were divided by PCR-fingerprinting with a minisatellite specific primer (hf13) and RFLP analysis of the orotidine monophosphate pyrophosphorylase ( C R 4 . 5 ) and phospholipase (PLBZ) genes into 8 niolecular types. The majority of the isolates 68.2% (n = 232) were VNI (var. grubii, serotype A). This is in accordance with the fact that this variety causes the majority of all human cryptococcal infections worldwide. 5.6% (n= 19) were VNII (var. grubii, serotype A); 4.0% (n = 14) VNIII (AD hybrid); 2.0% (n = 7) VNIV (var. neofonnans, serotype D); 3.5% (n = 12) and VGI; 6.2% (n = 21) VGII; 9.2% (11 = 3 1) VGIII, 1.5% (n = 5) VGIV (all var. gatti i , serotypes B and C). VNIII, AD hybrid isolates, mainly from Chile and Spain, revealed two RFLP patterns one corresponding to VNI, VNII and VNIV and the other to VNII and VNIV suggesting different recombination events between var. grubii and var. neoformans leading to diploid or triploid strains. These findings suggest an epidemiological link between the old and the new world and support an ongoing speciation within the Cx neofmnans complex. A RETROSPECTIVE EPIDEMIOLOGICAL SURVEY OF CANINE AND FELINE DERMATOPHYTOSIS IN BELGIUM OVER THE PERIOD 1996-2001

IN VIVO EXPRESSION OF A MICROSPORUM CANIS 43.5 KDA METALLOPROTEASE IN INFECTED GUINEA PIGS

listed by 500-5000 CFU of fungi per cbm. Most freqLle& the flats were colonized by molds of the genera finiciIliun1, Cladosporium and Aspergillus (A. n i p , A. versifo/or,A. ustus). In several apartments the potentially mycotosic mold Stachybolris chartarum was found. Some flats \ \ u x ~ inhabited with molds of the genera Mucor, Rhizopus, Afternaria, Steniphyliurn, Amenzonium, Phoma, Botrytis and other. A wide spectrum of fungal species with pot~n t i a l allergenic, pathogenic and toxigenic activity was revealed in wet dwellings in St. Petersburg.