Comparative enzyme studies of Microsporum canis and Microsporum cookei in relation to their pathogenicity and diversity : a thesis presented in partial fulfillment of the requirement for the degree of Doctor of Philosophy in Microbiology at Massey University

Infections by dermatophytes can be contracted from animals, humans, soil or contaminated fomites. In the genus Microsporum, some species e.g. M. canis are commonly associated with cats and dogs which act as an important reservoir for human infections. Others, e.g. M. cookei are nonpathogenic and found in the soil. The present studies have investigated the incidence of these ecologically contrasting species on cats, dogs and in the soil, their enzyme expression, and enzyme types as identified by proteinase inhibitors, gelatin/SDS-PAGE and multilocus enzyme electrophoresis, and have led to an investigation of their phenotypic variation. The primary aim was to attempt to detect differences in enzyme production which might be related to mechanisms of pathogenicity of M. canis. Isolation procedures employed were the hairbrush technique for small animals and the keratin-baiting technique for soil with samples being cultured on SDA containing antibiotics. Soil samples revealed 19 fungal genera, three being of keratinolytic fungi, representing 50% of total isolations. Trichophyton species were the most common (39% samples) but M. cookei was isolated from 6.8%. Fungi isolated from cats and dogs represented 20 genera, with the predominant isolates being keratinolytic fungi (51.9% of total samples). Cats were the major carriers of keratinolytic fungi (Chrysosporium, Microsporum and Trichophyton). M. canis was frequently isolated (18.5% of cats) and its distribution had a seasonal variation, with a peak appearing in May-June. All isolates of M. canis were of the "-" mating type. M. cookei isolates were of both the "+" and "-" mating types, but "+" types were predominant. Biochemical assays showed that M. canis produced higher proteinase and keratinase activities in shake cultures than in stationary cultures. Elastase activity was greater in stationary cultures. M. cookei's proteinase and keratinase activities were lower but again greater in shake cultures. There was no detectable keratinase activity in stationary cultures of M. cookei, and no significant difference in elastinolytic activity between shake and stationary cultures. Growth in shake culture produces the "pseudo-parasitic" morphology which mimics that found in infection, therefore, the differing enzyme expression of the two Microsporum species may be a reflection of their differing ecological roles. Characterisation of the enzymes with chemical inhibitors revealed that M. canis and M. cookei produced serine proteinases, but only M. canis produced cysteine and probably aspartic and metallo-proteinases. The serine and cysteine proteinases are considered likely to be of particular significance in the pathogenesis of M. canis infections. Using substrate copolymerised gel electrophoresis (gelatin\SDS-PAGE), shake and stationary cultures were again compared for enzyme expression. Among the six different Mr proteinases (122 KDa, 64 KDa, 62 KDa, 44 KDa, 36 KDa, and 28 KDa) expressed by M. canis, three (122 KDa, 62 KDa and 28 KDa) were found to be more highly expressed in shake cultures. In contrast, M. cookei isolates expressed seven different proteinases (67 KDa, 64 KDa, 63 KDa, 62 KDa. 54 KDa, 52 KDa, and 42 KDa), of which two (67 KDa, 64 KDa) were expressed only in stationary cultures and one (52 KDa) although expressed in shake cultures was more highly expressed in stationary cultures. Possibly the high and low Mr proteinases expressed by M. canis are more important in its pathogenicity than the middle range proteinases also detected in M. cookei. Multilocus enzyme electrophoresis using starch gels and examining eight enzymes, showed M. canis to be phenotypically more diverse than M. cookei as measured by the normalised Shannon-Wiener diversity statistic. M. canis showed a substantial within population variability (84.9%) by geographical region, with a moderate level (21.7%) of interpopulation differentiation. Cluster analysis confirmed this diversity and also revealed a possible grouping of isolates from clinical infections, and based on the accumulated data of these studies, EST pnenotype 9 although present in a few carrier isolates was commonly associated with isolates from clinical cases and perhaps deserves further investigation