Lead (pb) toxicity in saccharomyces cerevisiae: the role of cell wall

O chumbo é utilizado em muitos produtos, tais como baterias, gasolina, tintas e corantes, resultando na sua libertação no meio ambiente. Neste trabalho, foi examinado o papel da parede celular da levedura Saccharomyces cerevisiae como uma barreira ou como alvo da toxicidade do chumbo. A biodisponibilidade do Pb é muito reduzida pelos componentes do meio de cultura YEPD, o que dificulta a avaliação da toxicidade deste elemento em concentrações ambientalmente realistas. Para avaliar a toxicidade de Pb em S. cerevisiae, em condições de crescimento, foram efetuadas diferentes diluições (10-100 vezes) do meio YEPD, as quais foram misturadas com várias concentrações de Pb (0,1-1,0 mmol/l). Observou-se que o YEPD diluído 25 vezes constituía a melhor condição de compromisso entre o crescimento celular e a precipitação de Pb. Os genes CWP1 e CWP2 codificam para duas grandes manoproteínas da parede celular da levedura S. cerevisiae; a deleção destes genes CWP aumenta a permeabilidade da parede celular. A suscetibilidade de células de levedura interrompidas no gene CWP1 (estirpe cwp1Δ) ou CWP2 (estirpe cwp2Δ) foi comparada com a da estirpe, isogénica, selvagem (WT). Verificou-se que o crescimento das estirpes cwp1Δ e cwp2Δ, no meio de cultura YEPD 25 vezes diluído, na presença de Pb, não diferiu do crescimento da estirpe WT. Este resultado sugere que a alteração da permeabilidade da parede celular não altera a sensibilidade de células de levedura ao Pb. Foi investigada o impacto do Pb na parede celular de levedura. Para este efeito, comparou-se a suscetibilidade ao dodecil sulfato de sódio (SDS), ao calcofluor (CFW) e a uma enzima que degrada a parede da célula (liticase), em células da estirpe WT não expostas ou expostas a Pb durante 4, 8 ou 24 h. Além disso, o conteúdo de quitina da parede celular de levedura foi investigada por coloração das células com CFW. Os resultados não mostraram uma alteração da suscetibilidade ao SDS e ao CFW, nas células tratadas com Pb; contudo, nas células tratadas durante 24 h com Pb, observou-se um aumento da sensibilidade à liticase e um aumento da coloração com CFW. Estes resultados sugerem que o chumbo interage com a parede celular da levedura e influencia a sua composição. Deve ser levado a cabo trabalho adicional a fim de confirmar estes resultados.

Lead is used in many products like batteries, gasoline, paints and dyes, resulting in leakage in the environment. In this work, the role of the cell wall of Saccharomyces cerevisiae as a barrier or as a target of Pb toxicity was examined. Pb bioavailability is greatly reduced by YEPD medium components, which difficult the evaluation of Pb toxicity under environmentally realistic concentrations. To assess Pb toxicity to S. cerevisiae, in growing conditions, different dilutions (10-100 times) of the YEPD medium were made and mixed with several Pb concentrations (0.1-1.0 mmol/l). It was observed that YEPD 25 times diluted was the best compromising condition between cell growth and Pb precipitation. CWP1 and CWP2 genes encode two major mannoproteins of yeast cell wall of S. cerevisiae; deletion of these CWP genes enhances cell wall permeability. The susceptibility of yeast cells deleted on CWP1 (cwp1Δ strain) or CWP2 genes (cwp2Δ strain) was compared with the isogenic wild-type (WT) strain. It was found that the growth of cwp1Δ and cwp2Δ strains, in 25 times diluted YEPD medium, in presence of Pb did not differ from the growth of the WT strain of S. cerevisiae. This result suggests that the modification of cell wall permeability does not affect the sensitivity of yeast cells to Pb. The impact of Pb on yeast cell wall was investigated. For this purpose the susceptibility to sodium dodecyl sulphate (SDS), calcofluor white (CFW) and cell wall-degrading enzyme (lyticase) were compared in cells of the WT strain not exposed or exposed to Pb for 4, 8 and 24 h. In addition, the chitin content of yeast cell wall was investigated by staining the cells with CFW. The results showed no modification of the susceptibility to SDS and CFW, an increased sensitivity to lyticase and an increase of cell staining with CFW, in Pb treated cells for 24 h. These results suggest that Pb interacts with yeast cell wall and influences its composition. A further work should be carried out in order to confirm these results.

Lood wordt gebruikt in vele producten zoals batterijen, brandstof, verf en kleurstoffen, waardoor het ook in de natuur terecht komt. In dit werk werd de rol van de celwand van Saccharomyces cerevisiae als barrière en doelwit voor Pb toxiciteit onderzocht. De bio-beschikbaarheid van Pb verlaagd door componenten van YEPD medium, waardoor onderzoek van de toxiciteit in biologisch realistische concentraties bemoeilijkt wordt. Om de Pb toxiciteit bij S. cerevisiae in groeiomstandigheden te onderzoeken, werden verschillende verdunningen (10-100 keer) van het YEPD medium gemaakt, waaraan Pb in verschillende concentraties (0,1-1,0 mmol/l) werd toegevoegd. Uit waarnemingen bleek dat 25 keer verdund YEPD medium de beste compromis is tussen celgroei en Pb precipitatie/ complexvorming. De genen CWP1 en CWP2 coderen voor twee belangrijke mannoproteinen van de celwand van S. cerevisiae; verwijderen van deze CWP genen verhoogt de permeabiliteit van de celwand. De gevoeligheid van gistcellen zonder het CWP1 gen (cwp1Δ stam) of zonder het CWP2 gen (cwp2Δ stam) werd vergeleken met de gevoeligheid van het isogene wilde type (WT). De groei van de cwp1Δ en de cwp2Δ stam, in 25 keer verdund YEPD medium, in aanwezigheid van Pb, verschilde niet van de groei van WT S. cerevisiae. Dit verondersteld dat modificatie van de permeabiliteit van de celwand de sensitiviteit van gistcellen voor Pb niet beïnvloedt. De impact van Pb op de celwand van gistcellen werd onderzocht. Hiervoor werd de gevoeligheid voor natrium dodecyl sulfaat (SDS), calcofluor white (CFW) en het celwand afbrekend enzym lyticase bij gistcellen van het wilde type onderzocht. De gevoeligheid werd vergeleken tussen cellen die niet blootgesteld werden aan Pb en blootgesteld werden aan Pb op de tijdstippen 4, 8 en 24 uur. Ook werd de hoeveelheid chitine in de gistcelwand onderzocht met behulp van kleuring met CFW. De resultaten tonen aan dat de gevoeligheid voor SDS en CFW, bij cellen 24 u blootgesteld aan Pb, niet verandert en de gevoeligheid voor lyticase toeneemt na 24 uur. Dit veronderstelt dat lood interactie aangaat met de celwand van gist en een invloed heeft op de samenstelling van de celwand. Verder onderzoek moet uitgevoerd worden om deze resultaten te bevestigen.