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Subject: search.filters.subject.Dermatophytes

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    Multiple proteolytic enzyme production in keratinophilic fungi (preliminary investigations) : a thesis presented in fulfilment of the requirement for the degree of Master of Science in Microbiology, Massey University
    (Massey University, 1998) Burrows-Anderson, Damaris
    Superficial fungal infections can be acquired from a number of sources, e.g. animals, humans or from the soil. Many of the fungal species commonly associated with human disease arise from infection by species known commonly as dermatophytes, although infection from other non-dermatophytic keratinophilic fungi is becoming more common. Other species not commonly regarded as pathogenic have on occasion been found in human infection. Many of these opportunistic species are commonly found in soils. Isolation procedures employed in these studies were the hairbrush technique for small animals and the keratin-baiting technique for soil with samples being cultured on SDA containing antibiotics. Soil samples yielded 3 keratinophilic genera found in human infection (Microsporum spp.. Trichophyton spp. Aphanoascus sp.) while fungi isolated from animals yielded 3 fungal species. Microsporum canis. Microsporum cookei and Scopulariopsis brevicaulis. In these studies, various culture parameters e.g. pH, spore numbers and various hydrolysis techniques were examined in order to asses the production of proteolytic enzymes in vitro. Also in the course of these studies, the use of lactrimel medium as a suitable recovery agent for strains presenting atypical colony morphology and reduced proteolytic enzyme production was trialed with excellent results. The gelatin SDS-PAGE technique, mode of culture (shake and stationary) and the effect of substrate were analysed to compare the effects that these have on a range of keratinophilic fungi. Both pathogens and saprophytes were examined in an attempt to detect similarities in enzyme production which could be associated with the ability of various species to invade skin in vivo. A large body of data has been gathered demonstrating that the proteolytic enzymes produced by most keratinophilic fungi encompass a wide range of MW sizes and are not entirely predictable. This strongly suggests that when these fungi come into contact with a particular substrate, the ability of the strain to adapt may depend on the strains ability to produce a proteolytic enzyme capable of breaking down the substrates in the external environment providing nutrients for the growing fungi.
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    The multiple proteolytic enzymes of two microsporum species : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1995) Palmer, Jon Stuart
    Dermatophyte infections can be contracted from animals, humans or from the soil. In the genus Microsporum some species commonly are associated with cats & dogs but also often cause infections in humans. Others are regarded as non-pathogenic & are commonly isolated from the soil. The present studies investigated the production of proteolytic enzymes by the zoophilic species M.canis & the geophilic species M.cookei, in various cultural conditions which might affect expression of such enzymes, in an attempt to detect differences between the two that could be associated with the ability of M.canis to invade skin in vivo. Biochemical assays showed M.canis produced higher azocollytic & elastase activity in a keratin containing medium(BSW) than in Sabourauds Broth(SDB). In contrast, azocollytic & elastase activity of M.cookei in the two media was relatively similar. Azocollytic & elastase activity of both species peaked in the pH range 7-10 & azocollytic activity demonstrated highest activity around 45°C in both media. Both species produced some keratinolytic activity in BSW but not in SDB. Inhibition studies of azocollytic & elastase activity revealed the presence of an aspartic elastase with little or no azocollytic activity, which also was not detected using a substrate(gelatin) SDS-PAGE technique. Other proteinase types found were serine, cysteine & metalloproteinases. Using the gelatin-SDS-PAGE technique, the mode of culture(shake & stationary) & the effect of substrate, time & temperature were analysed to compare the effects these factors may have on proteolytic enzyme expression between the two species.Substrate proved to be the most important factor in the expression of gelatinases. Mode of culture in SDB demonstrated that some proteinases were expressed in shake culture sooner than in stationary cultures. M.canis in both SDB & BSW produced 6 bands between 85,000 Da & 13,000 Da. M.cookei in SDB produced 7 bands between 64,000 Da to 19,000 Da but in BSW only 5 bands between 61,500 Da to 19,000 Da. Inhibition studies revealed that both species expressed several metalloproteinases & serine proteinases in BSW which were not expressed in SDB cultures. It is suggested that these proteinases may be important factors in the ability of dermatophytes to colonise keratin & possibly, in the case of M.canis, to invade skin in vivo.
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    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
    (Massey University, 1994) Simpanya, Mukoma Francis
    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.