Game and player: C. albicans biofilm lifestyle and extracellular DNA

Abstract

DNA is as a structural component of bacterial biofilms extracellular matrix (ECM). Although evidences have shown that DNA may play a role in C. albicans biofilms, further studies are required to understand the contribution of extracellular DNA (eDNA) in C. albicans biofilm lifestyle. Herein we aimed to determine the eDNA content of C. albicans SC5314 biofilm ECM and the effect of DNase I and exogenous DNA treatments on biofilm formation and biofilm cells susceptibility to antifungals. First, for eDNA estimation in C. albicans biofilm ECM, biofilms were formed under flow conditions for 48 h. ECM was isolated and its DNA and protein contents were determined. Second, DNase (0.02 - 2 mg/ml) and exogenous DNA (10 - 2560 ng/ml) were added at different stages of biofilm development (microtiter plate model under static conditions). The effect of 24 h treatments was evaluated in terms of biofilm biomass by crystal violet assay (A550). Third, for antifungal testing, biofilms (in 96-well plates) were challenged with amphotericin B (0.06 - 16 mg/l), caspofungin (0.008 to 2 mg/l), and fluconazole (4 - 1024 mg/l) alone or in combination with DNase (0.125 mg/ml) or exogenous DNA (320 ng/ml). Sessile minimum inhibitory concentrations (SMIC) were determined at 80 % inhibition compared to drug-free controls using the XTT reduction assay. RPMI medium was used in all the assays. On one hand, C. albicans biofilms ECM contained 3045.4 ± 227.3 ng eDNA/mg of protein. On the other hand, DNase or exogenous DNA treatments did not affect further biofilm development by C. albicans adherent cells. In contrast, DNase (> 0.03 mg/ml) promoted a general biomass reduction on C. albicans preformed biofilms, as indicated by the reduction of A550 compared with the control. Furthermore addition of exogenous DNA (> 160 ng/ml) to preformed biofilms led to an increase in biofilm biomass, similarly assessed by the higher A550 readings compared with control biofilms. Finally, DNase I (0.125 mg/ml) did not change C. albicans biofilm cells susceptibility to fluconazole, but increased their susceptibility to amphotericin B and caspofungin, as indicated by the lower SMIC compared to biofilms grown without DNase. In contrast, exogenous DNA (320 ng/ml) did not affect C. albicans biofilm cells susceptibility against these antifungals.