Inhibition of extracellular proteases and lipases from Candida albicans by hydroxytyrosol and oleuropein

Abstract

Gljivične Sap proteaze kao i Lip lipaze se ubrajaju u bitnije čimbenike virulencije medicinski značajnih gljivica. Stoga je inhibicija tvorbe i aktivnosti tih hidrolitičkih enzima jedan od važnijih faktora za eradikaciju mikoza. Predmet je ovog istraživanja bio je utvrditi mogućnosti modulacije aktivnosti izvanstaničnih Sap proteaza i Lip lipaza kvasca Candida albicans u in vitro uvjetima hidroksitirozolom i oleuropeinom. Prvi se korak sastojao od uzgoja kliničkih izolata vrste C. albicans na hranjivoj podlozi te nasađivanja i inkubacije uz indukciju proteaza odnosno lipaza u tekućem mediju a naposljetku i izolacije enzima u mediju filtracijom. Drugi korak bio je selekcija enzimatski najproduktivnijih sojeva na temelju kratke inkubacije alikvota filtrata sa odgovarajućim supstratom - BSA (goveđi serumski albumin) za proteaze, DMPTB (2,3-dimerkapto-1-propanol tributirat) za lipaze – te spektrofotometrijske analize uz pripadajuće kontrole. Treći korak sastojao se od ispitivanja utjecaja više koncentracija hidroksitirozola i oleuropeina na aktivnost ovih enzima, metodom sličnom onoj iz drugog koraka. Najproduktivnijim proizvođačem proteaza pokazao se klinički izolat sa oznakom 781, dok najproduktivnijeg proizvođača lipaza nije bilo moguće utvrditi. Obje tvari su pokazale inhibitorni učinak na Sap proteaze, s tim da je učinak hidroksitirozola bio bitno jači. Nije bilo moguće potvrditi njihov inhibitorni učinak na Lip lipaze budući da se nije uspjelo izdvojiti najproduktivniji soj, a moguće je da je i sama indukcija bila neuspješna. Pokušaj ustanovljivanja inhibitornog učinka na komercijalno nabavljenu lipazu srodne vrste, C. rugosa, nije polučio jasne rezultate.Fungal Sap proteases as well as Lip lipases are known as important virulence factors of medically important fungi. It is for this reason that the inhibition of synthesis and activity of these hydrolitic enzymes is an important target in mycosis eradication. The subject of this study was to determine the possibility of modulation of extracellular Sap proteases and Lip lipases of Candida albicans yeast in vitro by hydroxytyrosol and oleuropein. The first step consisted of the cultivation of clinical isolates of C. albicans on the culture medium, the inoculation and incubation followed by induction of proteases and lipases in liquid medium, and the isolation of enzymes from the medium by filtration. Subsequent step was composed of the selection of the enzymatically most productive strains based on short incubation of the filtrate aliquots with matching substrates – BSA (bovine serum albumin) for proteases, DMPTB (2,3-dimercapto-1-propanol tributyrate) for lipases – and the spectrophotometric analysis with corresponding controls. The final step was the examination of the impact that multiple concentrations of hydroxytyrosol and oleuropein have on the activity of these enzymes, by a method similar to the one from previous step. Clinical isolate designated as 781 has proven to be the most productive protease expressor, while it was not possible to determine the most productive lipase expressor. Both substances have shown inhibitory effect on Sap proteases, with the effect of hydroxytyrosol being significantly stronger. It was not possible to confirm their inhibitory effect on Lip lipases since the singling out of the most productive strain was unsuccessful, with the possibility that the induction itself was unsuccessful as well. The attempt to determine the inhibitory effect on the commercially purchased lipase of a related species, C. rugosa, did not yield clear results