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On-line version ISSN 1678-9946
Rev. Inst. Med. trop. S. Paulo vol.50 no.5 São Paulo Sept./Oct. 2008
Secreção de cinco enzimas extracelulares por amostras de agentes da cromoblastomicose
Thais Furtado de SouzaII; Maria Lúcia ScrofernekerI,II; Juliana Mônica da CostaI; Mariana CarissimiII; Valeriano Antonio CorbelliniIII
IGraduate Program in Medical Sciences, Universidade Federal do Rio Grande do Sul, Rio Grande do Sul State, Brazil
IIDepartment of Microbiology of the Institute of Basic Health Sciences of Universidade Federal do Rio Grande do Sul, Rio Grande do Sul State, Brazil
IIIDepartment of Physics and Chemistry of Universidade de Santa Cruz do Sul, Rio Grande do Sul State, Brazil
The gelatinase, urease, lipase, phospholipase and DNase activities of 11 chromoblastomycosis agents constituted by strains of Fonsecaea pedrosoi, F. compacta, Phialophora verrucosa, Cladosporium carrionii, Cladophialophora bantiana and Exophiala jeanselmei were analyzed and compared. All strains presented urease, gelatinase and lipase activity. Phospholipase activity was detected only on five of six strains of F. pedrosoi. DNase activity was not detected on the strains studied. Our results indicate that only phospholipase production, induced by egg yolk substrate, was useful for the differentiation of the taxonomically related species studied, based on their enzymatic profile.
Keywords: Chromoblastomycosis; Fonsecaea pedrosoi, Lipase; Phospholipase, DNAse; Urease; Gelatinase.
As atividades gelatinase, urease, lipase, fosfolipase e DNase de 11 agentes da cromoblastomicose constituídos por amostras de Fonsecaea pedrosoi, F. compacta, Phialophora verrucosa, Cladosporium carrionii, Cladophialophora bantiana e Exophiala jeanselmei foram analisadas e comparadas. Todas as amostras apresentaram atividade urease, gelatinase e lipase. A atividade fosfolipase foi detectada apenas em cinco das seis amostras de F. pedrosoi. A atividade DNase não foi detectada nas amostras estudadas. Os resultados indicam que para a diferenciação entre espécies taxonomicamente relacionadas estudadas, baseado no seu perfil enzimático, apenas a produção de fosfolipase, induzida pelo substrato com gema de ovo, foi útil.
Chromoblastomycosis is a subcutaneous fungal disease caused by dematiaceous fungi being characteristic of tropical and subtropical areas18,23. The disease occurs mainly among male agricultural laborers and primarily affects the lower limbs26,39. Infection begins with the traumatic implantation of conidia or hyphal fragments into subcutaneous tissues, producing initial lesions consisting of papules or nodules that become verrucous12. In Brazil, the most frequent etiological agents are Fonsecaea pedrosoi, Phialophora verrucosa and Cladosporium carrionii27,45. More rarely, Fonsecaea compacta, Exophiala jeanselmei and Rhinocladiella aquaspersa can also be isolated40.
The secretion of enzymes to the extracellular environment might be an important adaptive mechanism during the life cycle of fungi29. The first studies of fungal enzymatic activities aimed at establishing the role of enzymes in fungal pathogenicity, as well as their capacity to induce inflammatory reactions in the host40. It is logical to assume that these enzymes could act by making tissue invasion easier, but they could also participate in infection by eliminating some mechanisms of the immune defense and/or helping in the obtaining of nutrients, thus causing injuries to the human host2,3,10,38,43.
The enzymatic characterization of clinical isolates of chromoblastomycosis agents is important due to the fact that few studies have focused on this subject14,17,44. In the present study, we investigated the activity of five hydrolytic enzymes of 11 strains of chromoblastomycosis agents. The aim was to characterize the enzymatic profile of different species of chromoblastomycosis agents.
MATERIALS AND METODS
Storage and cultivation of strains: The strains of chromoblastomycosis agents Fonsecaea pedrosoi ATCC 46428, ATCC 46422, IMTSP 49, IMTSP 674, IQE 444.62 (19)*, MA; F. compacta IMTSP. 373; Phialophora verrucosa FMC.2214 (8); Cladosporium carrionii IMTSP. 680; Cladophialophora bantianum 2907-78 (13); Exophiala jeanselmei CROMO.HC8 (45) are patient isolates and were obtained from the Laboratory of Pathogenic Fungi of the Department of Microbiology of the Institute of Basic Health Sciences of Universidade Federal do Rio Grande do Sul, and stored on Sabouraud agar slants at 25 ºC. For all enzyme assays, the strains were previously cultured for 14 days on Sabouraud agar slants at 25 ºC.
Candida albicans ATCC 10231, Malassezia furfur IMTSP. 225, Nocardia brasiliensis IMTSP. 739 and Staphylococcus aureus ATCC 25923 were utilized as positive controls in the enzyme assays. C. albicans and S. aureus were previously cultured for 24h and M. furfur and N. brasi-liensis for 14 days. S. aureus was cultured on nutrient agar. C. albicans, M. furfur and N. brasiliensis were cultured on Sabouraud agar.
Enzymatic assays: Chromoblastomycosis agents strains previously cultured on Sabouraud agar were transferred to potato dextrose agar (PDA) slants at 35 ºC for 10 days. Conidial suspension was prepared by means of sterile saline solution (0.85%). Standard suspension was adjusted in sell UV-visible spectrophotometer (Spectrum Instruments Co. China, Shanghai) at 530 nm to 80-82%. After a 1:10 dilution, cell debris concentration yield corresponded to 10x0.4 - 5x104 UFC/mL. The suspension was added to Petri dishes containing specific medium to each enzyme tested for previous stimulation. After complete fungal growth on the surface of the agar plate (seven to 14 days), 5-mm culture discs were purchased and applied in triplicate onto other plates containing the specific medium. For the gelatinase assay, strains were grown in tubes containing 5 mL of test medium according to the method reported by KURTZMAN & FELL22. The strains were considered negative when the medium remained solid. They were considered positive when the gelatin liquefied. N. brasiliensis IMTSP 739 was utilized as positive control.
Urease activity was determined according to the method proposed by CHRISTENSEN6 and SEELIGER43. The strains were grown for seven days at 25 ºC and were considered positive when the medium became pinkish. N. brasiliensis IMTSP 739 was utilized as positive control.
The lipase assay was carried out according to the method described by MUHSIN et al.31. M. furfur IMTSP 225 was used as positive control. The lipase medium consisted of peptone 1%, sodium chloride 5%, calcium chloride 0.01% and agar 2%. Tween 80 was autoclaved separately and added to the medium at a final concentration of 1%. After inoculation, the plates were incubated at 25 °C for up to 30 days. Formation of a precipitate around the colonies demonstrated lipase production.
Phospholipase activity was determined according to the method described by PRICE et al.37. C. albicans ATCC 10231 was used as positive control. The medium consisted of peptone 1%, glucose 2%, sodium chloride 5.73%, calcium chloride 0.05% and agar 2%. The medium was autoclaved and then cooled to approximately 50 °C before the addition of sterile egg yolk at a final concentration of 4%. The plates were incubated at 25 °C for up to 30 days. Precipitation around the colonies attested phospholipase production.
DNase activity was determined according to the method proposed by LOPEZ-MARTINEZ et al.24. S. aureus ATCC 25923 was utilized as positive control. For the DNase assay, the isolates were inoculated on DNase plates, which were then incubated at 25 °C for up 30 days. The assay was considered positive when a degradation halo was visualized around the colonies after the addition of 5N HCl.
All strains of chromoblastomycosis agents presented lipase, urease and gelatinase and lack of DNase activity. Table 1 shows the presence/absence of enzymatic activities of the tested strains and controls.
Phospholipase was produced by most of F. pedrosoi strains; only strain 19 did not excrete the enzyme. The strains of other chromoblastomycosis agents were not able to degrade the substrate used.
The strains from chromoblastomycosis agents were phenotypically characterized by means of the secretory capacity of DNase, urease, lipase, phospholipase and gelatinase. The degradation pattern varied among different substrates.
The DNase assay was not useful in this study for the detection of extracellular activity of this enzyme, differently from the observation made by LOPEZ-MARTINEZ et al.24, when secretion levels could be evaluated by dermatophytes isolated from acute and chronic cases.
Phospholipases hydrolyze phospholipids, molecules common to all cell membranes1,36,37. Phospholipase has been used as a virulence marker for some pathogenic fungi such as Candida albicans16,19,20,21,37,46, Cryptococcus neoformans5,8,45, Aspergillus fumigatus2, Trichophyton rubrum13 and Microsporum canis25. According to their phospholipase activity, the strains analyzed can be placed in two groups. The first one, which is characterized by positive phospholipase activity, includes F. pedrosoi MA, IMTSP. 674, ATCC (46422, 46428) and IMTSP 49. The negative results of the other strains (F. pedrosoi 19, F. compacta 373, P. verrucosa 8, C. carrionii 680, C. bantianum 13 and E. jeanselmei 45) could be related to the enzyme-substrate specificity rather than to the absence of this enzyme on the profile of these strains. The genotypic expression of the enzyme was not investigated, but negative results of phospholipase production, described for other pathogenic fungi, were associated with the restriction of enzyme activity to the intracellular environment11.
Extracellular proteinases of saprophytic microorganisms are primarily secreted to break down or decompose complex materials into nutrients readily available to the cells or to compete with other environmental bacteria, parasites or fungi9. Direct virulence factors for pathogenic microorganisms include hydrolyzing proteins of host cell membranes to facilitate adhesion and tissue invasion, or damaging cells and molecules of the host defense system to avoid or resist antimicrobial attack30,32,41. Peptidases are believed to contribute to microbial virulence by destroying host tissues and digesting immunologically important proteins, such as antibodies and complement factors4,42. Gelatinase activity was only qualified in this study, and its potential for differentiating chromoblastomycosis agents is controversial, since its use for this purpose has already been described, particularly for the characterization of Cladosporium carrionii and Exophiala jeanselmei15, showing no significant differences between known pathogenic and nonpathogenic isolates14. PALMEIRA et al.34 found that medium composition could induce the synthesis and secretion of distinct proteolytic enzymes by F. pedrosoi conidia.
All analyzed strains presented precipitation haloes for lipolytic activity. Tween 80, utilized as carbon source, accumulated efficiently as biomass and stimulated lipase excretion by the fungus. The use of lipase activity for differentiating fungal genera closely related to mycotic infection is well reported in the literature, especially concerning dermatophytes33. According to other studies, an elevated rate of seborrheic dermatitis was related to lipase production on different substrates and under pH effect, for instance on Mallassezia furfur35, or to the virulence of Candida albicans strains43. Greater lipase activity was also verified frequently on isolates from chronic mycotic infections24. Nevertheless, further investigation is necessary to confirm these findings, with inclusion of more strains of the same species as well as of other genera (Wangiella, Rhinocladiella).
All strains presented urease activity in this work, showing that urea is a suitable nitrogen source to these strains. These results are in agreement with those observed in dematiaceous fungi17. Urease is a metalloenzyme that catalyzes the hydrolysis of urea to ammonia and carbamate. This reaction can result in an increase in pH under physiological conditions28. In Cryptococcus neoformans, a significant human pathogenic fungus that produces large amounts of urease, this activity is involved in the pathogenesis of cryptococcosis, but its importance may be specific to the species and/or infection site7.
Our results indicate that for differentiation among the taxonomically related species studied based on their enzymatic profile, only phospholipase production, induced by egg yolk substrate, was useful. Nevertheless, the production of enzymes that degrade substrates found in animal tissues is an indicative of this activity during an infectious process.
The authors are grateful to Propesq/UFRGS for their financial support.
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Maria Lúcia Scroferneker
Rua Vasco da Gama 176/604
90420-111 Porto Alegre, RS, Brasil
Fax: +55 51 3308 3155
Received: 1 November 2007
Accepted: 1 September 2008