Liliane Alves ScheidI; Débora Alves Nunes MarioI; Paulo Guilherme Markus LopesI; Érico de LoretoII; Carlos Eduardo Blanco LinaresIII; Janio Morais SanturioIV; Sydney Hartz AlvesI, IV
IPharmaceutical Sciences Post-Graduate Program, Universidade Federal de Santa Maria. Santa Maria, RS IIDepartment of Chemistry, Universidade Federal de Santa Maria. Santa Maria, RS IIIDepartment of Health Sciences, Universidade Regional do Alto Uruguai Campus de Frederico Westphalen. Santa Maria, RS IVDepartment of Microbiology and Parasitology. Universidade Federal de Santa Maria. Santa Maria, RS
ABSTRACT
INTRODUCTION: The phospholipase activity in Candida albicans and Candida dubliniensis isolated from oral candidiasis cases were studied.
METHODS: The phospholipase activity was evaluated in egg yolk agar.
RESULTS: All the C. albicans isolates (n = 48) showed phospholipase activity (mean Pz = 0.66). However, none of the C. dubliniensis isolates (n = 24) showed this activity.
CONCLUSIONS: The authors discuss whether these findings are a true characteristic of C. dubliniensis or a consequence of the methodology employed, which includes the possibility that NaCl may have inhibited the enzymatic activity of C. dubliniensis.
Key-words: Candida albicans. Candida dubliniensis. Phospholipase activity.
RESUMO
INTRODUÇÃO: Avaliou-se a atividade fosfolipásica em Candida albicans e Candida dubliniensisisoladas de casos de candidíase oral.
MÉTODOS: A atividade de fosfolipase foi avaliada em ágar gema de ovo.
RESULTADOS: Todos os isolados de C. albicans (nº = 48) evidenciaram atividade fosfolipásica (média Pz = 0.66). Todavia, nenhum isolado de C. dubliniensis (nº= 24) demonstrou esta atividade.
CONCLUSÕES: Os autores discutem se estes achados são uma característica verdadeira de C. dubliniensis ou uma conseqüência da metodologia empregada, a qual inclui a possibilidade de que o NaCl seja inibidor da atividade enzimática de C. dubliniensis.
Palavras-chaves: Candida albicans. Candida dubliniensis. Atividade de fosfolipase.
The virulence of yeast species is a multifactorial property dependent on many different virulence factors, including adhesion to the host cells, formation of hyphae, phenotypic switching and production of hydrolytic enzymes such as proteinases and phospholipases1-3. Candida albicansshows these putative virulence factors, but there has been less investigation of these phenotypic characteristics in Candida dubliniensis, particularly its phospholipase activity. So far, only two studies have on reported the production of phospholipases, and only in a limited quantity of C. dubliniensis2,4. This study aimed to compare the phospholipase activity of C. albicans and C. dubliniensis that were isolated from clinical specimens.
We studied twenty-four clinical strains of C. dubliniensis and forty-eight strains of C. albicans, both recovered from oral candidiasis lesions in AIDS patients. The C. dubliniensis isolates were obtained from different medical centers in Brazil between 1998 and 2007. The C. albicans isolates were obtained from the University Hospital of Santa Maria (Santa Maria, Rio Grande do Sul, Brazil) between 1995 and 2005. All the isolates were maintained at -70 °C. The results from phenotypic identification tests on C. dubliniensis were confirmed by genotypic methods using randomly amplified polymorphic DNA (RAPD) and using the primers CDU (5′ GCGATCCCC3′) and B-14 (5′ GATCAAGTC3′) (Bauer et al5). C. albicans isolates were identified using classical methods. The production of phospholipases by C. albicans and C. dubliniensis was investigated by means of culture media6 and techniques described by Price et al7 and Saramanayake et al8. C. albicans CBS 2730, C. albicans 2630 and C. dubliniensis CBS 7987 were included as controls.
Phospholipase activity of C. albicans was detected in all 48 of our assayed strains, which resulted in Pz ranges from 0.41 to 0.80 (mean = 0.66). However, phospholipase activity for C. dubliniensiswas absent. The colonies of C. albicans developed dense growth, which resulted in a cream-like opaque zone around the colonies in the medium. In contrast, C. dubliniensis colonies scarcely developed, and no opaque halo surrounding the colonies was observed. None of the C. dubliniensisstrains showed phospholipase activity.
Phospholipase exoenzymes are considered to play an important role in the pathogenesis of opportunistic fungi, as well as an active role in the invasion of host tissue during candidiasis9. By cleaving phospholipids, phospholipase destabilizes the membrane and promotes cell lysis3. Since Price et al7 described a plate method for detection of phospholipase activity in C. albicans, it has become the traditional screening method for phospholipase activity among Candida species, as well as for other yeast-like fungi such as Cryptococcus neoformans3 and Malassezia pachydermatis6. In the present study, we demonstrated that C. dubliniensis did not have any extracellular phospholipase activity. In our opinion, this result requires new studies, because the medium indicated by Price et al7 is composed of 5.73 gram % NaCl, which seems to be an inhibitory factor for C. dubliniensis growth. We previously demonstrated that Sabouraud broth with 6.5% NaCl was inhibitory for C. dubliniensis, and then proposed a simple test for C. dubliniensisscreening1.
We therefore assume that the phospholipase activity of C. dubliniensis cannot be assayed using Price’s method7, which could explain the scarcity of studies focusing on this virulence factor in relation to the biology of C. dubliniensis. Results similar to ours were reported by Hannula et al4, applying the same methodology that we used; however, those authors did not emphasize this finding. More recently, Fotedar & Al-Hedaithy2 studied 87 C. dubliniensis strains and observed that none showed phospholipase activity, even after prolonging the incubation for seven days. Under those conditions, they reported that colonies with dense growth formed but did not have any enzymatic activity in Price’s medium7. The relationship between NaCl and scarce growth was also observed for C. albicans in a hypertonic medium containing 6.5% NaCl, which explains why incubation took four days in that test1. C. dubliniensis, which is more sensitive, showed no growth under these conditions. Studies focusing on virulence factors have shown that C. dubliniensis is less virulent than C. albicans2,9. Would the phospholipase be the main cause of this characteristic? Based on our observations, new and more rigorous studies, such as biochemical analysis using radiometric or colorimetric assays, are needed in order to answer this question. In addition, gene cloning will elucidate the presence, activity and role of extracellular phospholipase as a virulence factor relating to C. dubliniensis3.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
REFERENCES
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Address to:
Prof. Sydney Hartz Alves
Rua dos Andradas 1985/201. 97010-033
Santa Maria, RS, Brazil
Telefax: 55 55 3220-8906
Received in 09/12/2009
Accepted in 18/01/2010
