Efeito paradoxal da caspofungina em Candida spp
Arquivos
Data
2010
Autores
Bizerra, Fernando César 
Orientadores
Colombo, Arnaldo Lopes
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O efeito paradoxal (EP) é caracterizado pelo crescimento celular em
concentrações de equinocandinas acima da concentração inibitória mínima (CIM).
Apesar da resistência a esta classe de drogas ser um fenômeno pouco comum em
isolados de Candida spp., a ocorrência do EP é freqüentemente observada entre
isolados desta levedura durante os testes de susceptibilidade às equinocandinas. O
objetivo deste estudo foi analisar a freqüência do EP da caspofungina (CAS) entre
isolados de Candida spp. e avaliar os aspectos morfológicos e os mecanismos
moleculares relacionados a este fenômeno. Para tanto, foram utilizados 77 isolados de
Candida spp., incluindo 21 isolados de C. albicans, 22 de C. tropicalis, 23 de C.
parapsilosis e 11 de C. orthopsilosis. Todos isolados foram submetidos ao teste de
susceptibilidade da CAS, segundo recomendações do Clinical Laboratory Standards
Institute (CLSI, documento M27-A3), para determinação da CIM e avaliar a presença
do EP. Entre os isolados que apresentaram EP da CAS, foram selecionados dois
isolados de cada espécie para aplicação dos demais testes propostos neste estudo.
Os ensaios de concentração fungicida mínima (CFM), curva de morte e ensaio de
viabilidade celular foram utilizados para avaliar o comportamento das células fúngicas
durante o EP. Analisamos ainda as alterações ultraestruturais e bioquímicas da parede
celular durante o EP e a expressão de genes relacionados à síntese da parede celular
durante este fenômeno. Entre os 77 isolados de Candida spp. analisados, 42 (54,5%)
apresentaram o EP da CAS em concentrações que variaram de 4,0 a 32,0 µg/mL de
CAS. Os ensaios de viabilidade celular mostraram que as células do EP
apresentaram-se viáveis, mesmo quando expostas a altas concentrações de CAS (16
µg/mL). Os ensaios de curva de morte provaram ser mais discriminatórios que MFC
para caracterizar o EP, principalmente para isolados de C. parapsilosis e C.
orthopsilosis. As quatro espécies de Candida analisadas neste estudo apresentaram
um padrão semelhante de alterações ultraestruturais e bioquímicas da parede celular
durante o EP. De forma geral, durante o EP houve uma diminuição de 2,7 a 7,8 vezes
no conteúdo de β-1,3-glucana e um aumento de 4,0 a 6,6 vezes no conteúdo de
quitina da parede celular. Sobre a análise ultraestrutural por microscopia eletrônica, as
células do EP apresentaram aumento do tamanho celular, formação de aglomerados
celulares, ausência de filamentação e anormalidades no septo de brotamento. Além
disso, a parede celular das células do EP apresentou um predomínio da camada mais
eletrodensa, indicando uma diminuição da camada de β-1,3-glucana. Por fim, os
resultados da análise da expressão gênica sugerem que o aumento da síntese de
quitina em células do EP de C. albicans é regulado pelas vias PKC, HOG e
Calcineurina-Ca2+. Nossos resultados apresentaram detalhes sobre as alterações
morfológicas e ultraestruturais durante o EP, mostrando que as diferentes espécies
analisadas apresentaram resposta celular semelhante durante o EP da CAS. Podemos
concluir ainda que as células do EP possuem mecanismos adaptativos responsáveis
pelo aumento do conteúdo de quitina na parede celular, em resposta à inibição de β-
1,3-glucana. Esses mecanismos permitem que as células do EP sobrevivam a altas
concentrações de CAS, sugerindo que a estimulação da síntese de quitina pode
representar um mecanismo de escape à atividade desta droga.
The paradoxical growth effect (PG) has been described for echinocandins and is characterized by cellular growth at drug concentrations above the minimal inhibitory concentrations (MIC). Despite resistance to this class of drugs is an unusual phenomenon among Candida isolates, occurrence of PG is frequently observed during susceptibility tests for echinocandins. The aim of this study was to analyze the frequency of PG for caspofungin (CAS) among Candida strains and also to evaluate the morphological aspect, as well as the molecular mechanisms related to this phenomenon. Seventy-seven strains of Candida spp., including 21 isolates of C. albicans, 22 isolates of C. tropicalis, 23 isolates of C. parapsilosis and 11 isolates of C. orthopsilosis were studied. All strains were subjected to susceptibility tests of CAS, as described in the Clinical Laboratory Standards Institute (CLSI, document M27-A3), to determine the MIC and evaluate the presence of PG. Afterwards, two isolates each of Candida albicans, C. tropicalis, C. orthopsilosis and C. parapsilosis, all of which displayed PG in response to caspofungin, were subjected to MIC, minimal fungicidal concentration (MFC) and time-kill curve assays to evaluate the PG. We also analyzed the ultrastructural and biochemical changes in the cell wall of PG cells and the gene expression related to the synthesis of cell wall during this phenomenon. Of the 77 Candida isolates initially screened, 42 isolates (54.5%) displayed the phenomenon of PG in concentrations ranging from 4.0 to 32.0 µg/mL CAS. C. parapsilosis demonstrated the higher frequency of PG, followed by C. albicans, C. tropicalis and C. orthopsilosis. Cell viability assays showed that the PG cells were viable, even when exposed to high CAS concentrations (16 µg/mL). The time-kill curve assays were more discriminatory than MFC in detecting the PG effect, especially for C. parapsilosis and C. orthopsilosis isolates. The four different Candida species studied demonstrated similar alterations in the cell wall components and ultrastructure associated with PG. In PG cells, β-1,3-glucan content decreased 2.7- to 7.8-fold, whereas chitin content increased 4.0- to 6.6-fold. Electron microscopy study of the PG cells revealed morphological alterations, clumping and enlarged cells, absence of filamentation, abnormal septa and accumulation of chitin in the cell wall. Also, PG cells basically exhibited a single dark high density layer in the cell wall, indicating loss of the β-1,3-glucan layer. Finally, the analysis of gene expression suggests that the increase of chitin synthesis in PG cells of C. albicans is regulated by PKC, HOG and Calcineurin-Ca2+ pathways. Our results showed details about the morphological and ultrastructural alterations during PG, demonstrating that different Candida spp. analyzed presented similar cellular response during PG of CAS. Our findings also suggest that the PG cells have a compensatory mechanism responsible for the increase of chitin content in the cell wall, in response to β-1,3-glucan inhibition. These mechanisms allow the survival of PG cells to high concentrations of CAS, suggesting that stimulation of chitin synthesis may represent a rescue mechanism against caspofungin activity.
The paradoxical growth effect (PG) has been described for echinocandins and is characterized by cellular growth at drug concentrations above the minimal inhibitory concentrations (MIC). Despite resistance to this class of drugs is an unusual phenomenon among Candida isolates, occurrence of PG is frequently observed during susceptibility tests for echinocandins. The aim of this study was to analyze the frequency of PG for caspofungin (CAS) among Candida strains and also to evaluate the morphological aspect, as well as the molecular mechanisms related to this phenomenon. Seventy-seven strains of Candida spp., including 21 isolates of C. albicans, 22 isolates of C. tropicalis, 23 isolates of C. parapsilosis and 11 isolates of C. orthopsilosis were studied. All strains were subjected to susceptibility tests of CAS, as described in the Clinical Laboratory Standards Institute (CLSI, document M27-A3), to determine the MIC and evaluate the presence of PG. Afterwards, two isolates each of Candida albicans, C. tropicalis, C. orthopsilosis and C. parapsilosis, all of which displayed PG in response to caspofungin, were subjected to MIC, minimal fungicidal concentration (MFC) and time-kill curve assays to evaluate the PG. We also analyzed the ultrastructural and biochemical changes in the cell wall of PG cells and the gene expression related to the synthesis of cell wall during this phenomenon. Of the 77 Candida isolates initially screened, 42 isolates (54.5%) displayed the phenomenon of PG in concentrations ranging from 4.0 to 32.0 µg/mL CAS. C. parapsilosis demonstrated the higher frequency of PG, followed by C. albicans, C. tropicalis and C. orthopsilosis. Cell viability assays showed that the PG cells were viable, even when exposed to high CAS concentrations (16 µg/mL). The time-kill curve assays were more discriminatory than MFC in detecting the PG effect, especially for C. parapsilosis and C. orthopsilosis isolates. The four different Candida species studied demonstrated similar alterations in the cell wall components and ultrastructure associated with PG. In PG cells, β-1,3-glucan content decreased 2.7- to 7.8-fold, whereas chitin content increased 4.0- to 6.6-fold. Electron microscopy study of the PG cells revealed morphological alterations, clumping and enlarged cells, absence of filamentation, abnormal septa and accumulation of chitin in the cell wall. Also, PG cells basically exhibited a single dark high density layer in the cell wall, indicating loss of the β-1,3-glucan layer. Finally, the analysis of gene expression suggests that the increase of chitin synthesis in PG cells of C. albicans is regulated by PKC, HOG and Calcineurin-Ca2+ pathways. Our results showed details about the morphological and ultrastructural alterations during PG, demonstrating that different Candida spp. analyzed presented similar cellular response during PG of CAS. Our findings also suggest that the PG cells have a compensatory mechanism responsible for the increase of chitin content in the cell wall, in response to β-1,3-glucan inhibition. These mechanisms allow the survival of PG cells to high concentrations of CAS, suggesting that stimulation of chitin synthesis may represent a rescue mechanism against caspofungin activity.
Descrição
Citação
BIZERRA, Fernando César. Efeito paradoxal da caspofungina em Candida spp.: mecanismos moleculares e aspectos morfológicos. 2010. 142 f. Tese (Doutorado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2010.