Project description:We have developed a monoclonal antibody (mAb) C7 that reacts with Als3p and enolase present in Candida albicans cell wall and exerts three anti-Candida activities: candidacidal activity and inhibition of both adhesion and filamentation. To investigate the mode of action of mAb C7 on fungal viability, we examined changes in the genome-wide gene expression profile of C. albicans grown in presence of a subinhibitory concentration of mAb C7 (12.5 µg/ml) by using microarrays. A total of 49 genes were found to be differentially expressed upon treatment with mAb C7. Of these, 28 were found to be up-regulated and 21 down-regulated. The categories of up-regulated genes with the largest number of variations were those involved in iron uptake or related to iron homeostasis (42.86%), while the energy-related group accounted for 38.10% of the down-regulated genes (8/21). Results were validated by real Time PCR. Since these effects resembled those found under iron-limited conditions, the activity of mAb C7 on C. albicans mutants with deletions in key genes implicated in the three iron acquisition systems described in this yeast was also assessed. Only mutants lacking TPK1 and TPK2 genes were less sensitive to the candidacidal effect of mAb C7. FeCl3 or hemin at concentrations ≥ 7.8µM reversed the candidacidal effect of mAb C7 on C. albicans, on a concentration dependent manner. The results presented in this study provide evidence that the candidacidal effect of mAb C7 is related to the blockage of the reductive iron uptake pathway of C. albicans. A saturated culture of C. albicans grown overnight was diluted to an optical density at 600 nm of approximately 0.1 and divided in two aliquots. One of them was used untreated as control and the second one was treated with a subinhibitory concentration (12.5 µg/ml) of monoclonal antibody C7 . Both cultures were incubated for 18 h at 37ºC before harvesting cells. Antibody added and control samples were obtained each time. The experiment was repeated once. Dye-swap technique was used for hybridization and four arrays were analyzed to compare the expresion of over six thousands genes in response to antibody C7.
Project description:N2 young adult animals were analyzed four hours after exposure to wild-type Candida albicans DAY185, heat-killed C. albicans DAY185 and heat-killed Escherichia coli OP50, all on Brain Heart Infusion (BHI) agar. It was necessary to use heat-killed E. coli OP50 as a control for these experiments because live E. coli OP50 (the normal nematode food source) is pathogenic to nematodes on BHI agar. These data identify the C. elegans genes that are differentially regulated during nematode infection with a human fungal pathogen.
Project description:We have developed a monoclonal antibody (mAb) C7 that reacts with Als3p and enolase present in Candida albicans cell wall and exerts three anti-Candida activities: candidacidal activity and inhibition of both adhesion and filamentation. To investigate the mode of action of mAb C7 on fungal viability, we examined changes in the genome-wide gene expression profile of C. albicans grown in presence of a subinhibitory concentration of mAb C7 (12.5 µg/ml) by using microarrays. A total of 49 genes were found to be differentially expressed upon treatment with mAb C7. Of these, 28 were found to be up-regulated and 21 down-regulated. The categories of up-regulated genes with the largest number of variations were those involved in iron uptake or related to iron homeostasis (42.86%), while the energy-related group accounted for 38.10% of the down-regulated genes (8/21). Results were validated by real time PCR. Since these effects resembled those found under iron-limited conditions, the activity of mAb C7 on C. albicans mutants with deletions in key genes implicated in the three iron acquisition systems described in this yeast was also assessed. Only mutants lacking TPK1 gene, and TPK2 to a lesser extent were less sensitive to the candidacidal effect of mAb C7. FeCl3 or hemin at concentrations ≥ 7.8µM reversed the candidacidal effect of mAb C7 on C. albicans, on a concentration dependent manner. The results presented in this study provide evidence that the candidacidal effect of mAb C7 is related to the blockage of the reductive iron uptake pathway of C. albicans.
Project description:In C. albicans the sirtuin Hst3p is responsible for removing the acetyl group of the Lys 56 of the H3 histone which is particularly abundant in yeasts and contributes to fungal genome integrity and virulence. Here we identified the H3K56ac patterns across Candida albicans genome in cells maintained for 28 h in YPD at 25°C in the presence or absence of 10 mM nicotinamide (used in this study as Hst3p inhibitor). Chromatin immunoprecipitation was carried out using an anti-H3K56ac antibody, whereas input samples were taken before addition the antibody. Two biological replicates were performed for each condition.