El objetivo del estudio fue identificar posibles cambios en el perfil proteico de dos aislamientos clínicos de Candida guilliermondii, uno sensible y otro resistente al fluconazol, con el fin de discriminar las proteínas expresadas diferencialmente en relación con la resistencia a este antifúngico.
MétodosSe aislaron fracciones procedentes de extractos proteicos citoplásmicos y de membrana o pared de un aislamiento resistente (CIM >256) y de otro sensible (CIM=4) al fluconazol, analizándolos por electroforesis en gel de poliacrilamida (SDS-PAGE).
ResultadosSe identificaron cuatro bandas proteicas presentes en el aislamiento resistente al fluconazol y ausentes en el aislamiento sensible. En el extracto citoplásmico se encontraron tres proteínas, una de 16kDa, de igual peso molecular a YNK1p (nucleósido difosfato cinasa), otra de 37kDa de igual peso molecular a HEM13p (coproporfirinógeno III oxidasa) y a ADHp1 (alcohol deshidrogenasa) y una de 45kDa. En el extracto de membrana o pared se encontró una banda de 25kDa con peso molecular similar al de la HSP31p (cisteína proteasa).
DiscusiónEste es el primer estudio de análisis proteico de la resistencia al fluconazol realizado con C. guilliermondii. Se identificaron algunas proteínas posiblemente asociadas con la resistencia a este azol, y se detectaron cuatro bandas expresadas diferencialmente en el aislamiento resistente, tres de ellas correspondientes a proteínas identificadas previamente en otras especies de Candida como relacionadas a resistencia y, la cuarta, una proteína de 45kDa, no descrita previamente en otros estudios proteómicos realizados en este género; sin embargo, los estudios del análisis del perfil proteico de C. guilliermondii deben continuarse, desarrollando técnicas proteómicas más avanzadas y específicas, como el uso de 2D-DIGE y de espectrometría de masas.
The aim of this study was to identify possible changes in the protein profiles of two isolates of Candida guilliermondii, one sensitive and other resistant to fluconazole to recognize proteins differentially expressed in relation to the resistance to this antimycotic.
MethodsFor this purpose, fractions from cytoplasm and membrane bound protein extracts from one resistant (MIC>256) and another sensitive (MIC=4) isolates were obtained and analyzed by SDS-PAGE.
ResultsFour protein bands present in the resistant isolation and absent in the sensitive isolate were identified. In the cytoplasmic extract three proteins were identified, one of 16kDa with the same size to YNK1p (Nucleoside diphosphate kinase), other of 37kDa with similar size to HEM13p (Coproporphyrinogen III oxidase) and/or ADHp1 (Alcohol Dehydrogenase) and the last of 45kDa was not identified previously in other proteomic studies made with other Candida species. In the membrane extract, one band corresponding to 25kDa protein HSP31p (Cysteine protease) was found.
ConclusionThis is the first protein analysis study made in C. guilliermondii in which proteins potentially associated with resistance to fluconazole were found. In this research, four proteins bands differentially expressed and possibly associated to fluconazol resistance were identified. Three of these proteins, were previously described in other Candida species as related to azole resistance. The 45 kDa, hasn’t been previously reported in other proteomic studies and could be specific to this species. Despite these results, further studies should be conducted on the protein profile of C. guilliermondii using more advanced and specific proteomic techniques as 2D-DIGE and mass spectrometry.
