gms | German Medical Science

Objectives: Candida albicans (CA) have frequently been associated with the formation of biofilms (BF) on biological and inert surfaces, such as intravascular catheters. Voriconazole (VRC) and anidulafungin (ANID) are the antifungal agents used to treat the resulting infections. BFs resistance to these antifungal agents may be associated with differential gene expression. Our aim was to investigate biofilm-associated gene expression in C. albicans exposed to VRC or ANID at various time points and identify new molecular targets for antifungal agents.

Methods: CA-M61, a well-documented BF-producing CA strain, was grown in RPMI at 106 blastoconidia/mL in 96-well plates at 37°C, 48h in order to produce BF. BF were then incubated for 12h, 18h, and 48h with VRC (128 or 512 mg/L) and ANID (0.25 or 1 mg/L). Total RNA was isolated from untreated (controls) and drug-treated CA BF. The amount of RNA recovered and extent of purity (A260/280) was measured spectrophotometrically. RNA integrity was checked by RNA gel electrophoresis. Differential expression of biofilmassociated genes at indicated time points was performed by RT-PCR. Changes in gene expression were calculated as a ratio of those expressed by untreated CA BF and to CA BF exposed to VRC and ANID. Actin gene transcript was used as internal control for gene normalization. Three genes were investigated: BGL2 (a cell wall metabolism gene), ECE1 (a cell elongation and hyphae formation gene) and SUN41 (a cell separation and hyphal elongation gene). A greater than 2-fold change was considered significant. Three independent experiments were performed.

Results: After incubation with VRC at 512mg/L for 12h and 18h, BGL2 was significantly down-regulated by 5.5-fold and 3.7-fold, respectively. Similarly, when CA BF were exposed to VRC at 128 mg/L for a 12h- and 18h-incubation period, BGL2 was down-regulated by 3.8-fold and 2.2-fold, respectively. In contrast, BGL2 and SUN41 were up-regulated by 2.1-fold and 2.3-fold, respectively, following exposure to VRC at 512 mg/L for 48h; whereas, ECE1 was significantly increased (3.1-fold) in response to ANID at 1mg/L at 48h incubation. BGL2 was also upregulated by 2.2-fold and 2.8-fold following exposure to ANID at 1 mg/L for 12h and 18h, respectively. Expression of SUN41 was significantly increased in response to ANID at 1mg/L (3.7-fold) and 0.25 mg/L (9.3-fold) after 12h of incubation with CA BF. ECE1 was downregulated by 11.2-fold in response to VRC at 512 mg/L after 18h of incubation, while SUN41 by 2.9-fold in response to VRC at 128 mg/L after 12h of incubation.

Conclusion: VRC caused down-regulation of three genes at 12-18hr of BF formation at both concentrations tested while AND caused up-regulation or indifference in gene expression at both concentrations tested.