J Med Microbiol 52 (2003), 479-481; DOI: 10.1099/jmm.0.04933-0
© 2003 Society for General Microbiology
ISSN 0022-2615
Biosynthesis of chondroitinase and hyaluronidase by different strains of Paracoccidioides brasiliensis
C. M. de Assisl,
R. F. Gandra2,
W. Gambale2,
M. T. Shimizu3 and
C. R. Paula3
1Instituto Adolfo Lutz, São Paulo, SP, Brazil 2Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, SP, Brazil 3Faculdade de Odontologia, Universidade Estadual de São Paulo, São José dos Campos, SP, Brazil#dReceived 3 April 2002 Accepted 5 February 2003
Correspondence: C. R. Paula (crpmieol{at}uom.com.br)
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Abstract
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The biosynthesis of chondroitinase and hyaluronidase by different isolates of Paracoccidioides brasiliensis was investigated in 20 strains isolated from patients (17 strains), a penguin (Pygocelis adeliae, one strain), an armadillo (Dasypus novemcinctus, one strain) and the environment (dog food, one strain). All the P. brasiliensis isolates studied had the ability to produce chondroitinase and hyaluronidase, although differences in colony morphology and enzyme production were detected among them. These results suggest that further investigations should be carried out in the clinical field in order to clarify the potential role of P. brasiliensis enzyme production in the pathogenesis of paracoccidioidomycosis.
Photographs showing halo formation by Paracoccidioides brasiliensis isolates on chondroitin and hyaluronic acid agar plates are available as supplementary material in JMM Online.
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Introduction
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Paracoccidioides brasiliensis is one of the most prevalent invasive fungal pathogens in Latin America. The chronic form of paracoccidioidomycosis afflicts the lungs and can disseminate to other organs and tissues, forming secondary lesions in the mucous membranes, skin, lymph nodes and adrenal glands. The acute form is characterized by a significant rate of mortality, due primarily to reticuloendothelial system organ hypertrophy (Borges-Walmsley et al., 2002).
Molecular aspects of P. brasiliensis dimorphism, diagnosis, epidemiology, taxonomy, genetics and immunology have been the focus of intensive research (San-Blas & Niño-Vega, 2001; San-Blas et al., 2002). It produces various enzymes, such as proteases (Mendes-Giannini et al., 1990; Puccia & Travassos, 1991; Vaz et al., 1994; Carmona et al., 1995; de Assis et al., 1999), elastase and collagenase (Bedoya-Escobar et al., 1993), phospholipases (Heise & Cardoso de Almeida, 1994; San-Blas & Niño-Vega, 2001) and urease (Nogueira, 1959). Several micro-organisms produce hydrolytic enzymes and structures that help in their pathogenicity (Schaechter et al., 1993). Chondroitinase and hyaluronidase are believed to play a role in the pathogenicity of fungi, particularly species of the genus Candida (Shimizu et al., 1995). These enzymes are also considered as important pathogenic factors of bacteria that cause oral infections (Hershon, 1971; Takeuchi et al., 1982; Tam et al., 1982).
In this study, we investigated whether P. brasiliensis was able to secrete chondroitinase and hyaluronidase, using chondroitin sulphate type A and sodium hyaluronate as substrates.
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Methods
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Twenty different isolates of P. brasiliensis were studied, of which 17 were recovered from patients, one from the environment (dog food probably contaminated with soil in Uberlândia, Brazil) (Ferreira et al., 1990), one from an Adelie penguin (Pygocelis adeliae) (Garcia et al., 1993) and one from a nine-banded armadillo (Dasypus novemcinctus) (Vidal et al., 1995). Sixteen of these isolates were provided by the Fungal Culture Collection of the Institute of Tropical Medicine of São Paulo, São Paulo, Brazil. Cultures were grown on agar slants of bacto-peptone/glucose, pH 5.5, at 27 °C for 30 days and then transferred onto Petri dishes with basic medium to evaluate enzyme production. A strain of Candida albicans from our stock (ICB-12A) was used as a positive control for chondroitinase and hyaluronidase production.
Secretion of chondroitinase and hyaluronidase (Smith & Willett, 1968; Shimizu et al., 1995) by P. brasiliensis isolates was tested by cultivation on medium consisting of 15 g Noble agar (Difco), 10 g neopeptone (Difco) and 40 g glucose (Merck) dissolved in water to 1000 ml final volume. The medium was autoclaved at 121 °C for 15 min and cooled to 46 °C. Aqueous solutions of 5 % bovine albumin fraction V (Sigma) and either 4 mg bovine trachea chondroitin sulphate type A ml-1 (Sigma) for the chondroitinase assay or 2 mg human umbilical cord sodium hyaluronate ml-1 (Sigma) for the hyaluronidase assay were added to the medium. All the solutions were previously sterilized by Millipore filtration (0.20 µm pore size). The substrates were added to cooled media to give a final concentration of 400 µg ml-1; bovine albumin fraction V was added to a final concentration of 1 %. Final culture pH was 6.8 ± 0.1. Chondroitin agar was poured onto Petri dishes and left at room temperature. Plates were inverted at 4 °C in a moist atmosphere for up to 1 month before use. The test medium was initially translucent.
Plates were inoculated with P. brasiliensis and C. albicans and subsequently incubated at 27 °C for 1 month. All the isolates were placed on test media in duplicate. Petri dishes were observed daily and checked for the presence of a transparent ring around the colonies. The formation of a halo (zone of precipitation) around the colony was considered to indicate enzyme production. Enzyme activities for hyaluronidase (Hz) and chondroitin sulphatase (Cz) were determined as the ratio of the colony diameter to the total diameter of the colony and zone of precipitation, as described by Price et al. (1982) for the production of phospholipase by C. albicans. Therefore, Hz or Cz = 1.00 would be an enzyme activity-negative strain (index = 1); Hz or Cz < 1.00 and 
0.64 would be a positive strain (index = 2) and Hz or Cz < 0.64 would be a strongly positive strain (index = 3) for the production of the enzyme. Total degraded chondroitin or hyaluronidase was visualized by flooding the plates with 2 M acetic acid for 10 min. Photographs showing halo formation are available as supplementary material in JMM Online.
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Results and Discussion
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Hyaluronidase and chondroitin sulphatase are considered virulence factors for either dissemination or tissue penetration, favouring the dispersion of infecting micro-organisms. These virulence factors catalyse depolymerization of hyaluronic acid and chondroitin sulphate, basic constituents of tissue (Schaechter et al., 1993).
Twenty isolates of P. brasiliensis were grown on chondroitin agar and hyaluronic acid agar at 27 °C for 30 days. Colonies of P. brasiliensis isolates 1–8 and 10–20 showed an ivory-coloured, smooth morphology and exhibited wrinkled surfaces, with no aerial mycelia. Some isolates of P. brasiliensis 1, 3–8, 10 and 20, however, grew as cotton colonies with unwrinkled, flat, woolly, white surfaces and aerial mycelia.
All the P. brasiliensis isolates revealed chondroitinase and hyaluronidase activity when grown on chondroitin agar or hyaluronic acid agar as appropriate. The chondroitinase activity index of P. brasiliensis isolates 1–6 and 9–19 was 2 and that of isolates 7, 8 and 20 was 3 (Table 1). The hyaluronidase activity index of isolates 1–6, 8 and 11–20 was 2, while that of isolates 7, 9 and 10 was 3 (Table 1).
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Table 1. Morphology and enzyme activities of P. brasiliensis strains Cz and Hz values of enzyme activity were determined according to Price et al. (1982). Cz or Hz = 1 indicates absence of enzyme activity (index = 1); Cz or Hz 0.64 and < 1.0 indicates positive enzyme activity (index = 2); Cz or Hz < 0.64 indicates strongly positive enzyme activity (index = 3).
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Production of these enzymes is a major virulence factor for some bacteria, for instance Streptococcus species. Some Streptococcus strains are highly invasive, mainly due to hyaluronidase production (Schaechter et al., 1993). It was verified that, amongst oral bacteria, production of hyaluronidase and chondroitin sulphatase is associated with periodontal disease (Tam et al., 1982). Shimizu et al. (1995) suggested that the production of such enzymes is related to the pathogenicity of some Candida species.
In conclusion, the 20 isolates studied presented chondroitinase and hyaluronidase production, although differences among them were observed with respect to colony morphology and enzyme activity. We believe that the production of these enzymes might be associated with the pathogenicity of P. brasiliensis strains, mainly in seriously compromised patients. Our results suggest that further investigations should be carried out in the clinical field in order to clarify the potential role of P. brasiliensis enzyme production in the pathogenesis of paracoccidioidomycosis.
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Acknowledgments
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We are grateful to Professor Dr Carlos da Silva Lacaz for providing us with P. brasiliensis strains and Satiko Uehara for her technical assistance. This research was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).
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References
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Abstract
Introduction
