Underlying mechanisms of the effect of minocycline against Candida albicans biofilms

Abstract

Minocycline (MH) is a broad-spectrum antimicrobial agent and semisynthetic tetracycline derivative, which has been widely used in the clinic due to its efficacy. Having the strongest anti-microbial effect, MH exceeded the traditional scope of antibiotics and its previously unknown antifungal activity is also gradually being discovered. To preliminarily investigate the inhibitory effect of MH on Candida albicans (C. albicans), changes of cell growth, hyphal formation and transition, biofilm production and signaling pathway gene expression of C. albicans in the presence of MH were assessed in the present study. An XTT reduction assay was performed to quantitatively detect the metabolic activity of biofilms and evaluate the inhibition of MH on this. The results suggested that biofilm formation was clearly inhibited by 67% (P<0.0001) in the presence of 250 µg/ml MH, while mature biofilms were not significantly affected. In addition, MH inhibited the transition from yeast to hypha in a dose-dependent manner. Furthermore, reverse transcription-quantitative PCR revealed that several hyphae- and adhesion-specific genes associated with the Ras/cyclic (c)AMP/protein kinase A (PKA) pathway were differentially expressed following MH treatment, including downregulation of ras family GTPase (RAS1), adenylyl cyclase-associated protein 1 (CAP1), thiamin pyrophosphokinase 1 (TPK1), adenylate cyclase (CDC35), transcription factor (TEC1), agglutinin-like protein 3 (ALS3) and hyphal wall protein 1 (HWP1) and upregulation of EFG1 (enhanced filamentous growth protein 1 gene) and PDE2 (high-affinity phosphodiesterase gene). The most obviously changed genes were TPK1, HWP1 and RAS1, downregulated by 0.33-, 0.48- and 0.55-fold, respectively. It was suggested that MH is associated with alterations in the morphology of C. albicans, such as the repression of hypha and biofilm formation of cells, and MH affected the Ras/cAMP pathway to regulate the expression of cAMP-associated genes.

Keywords: Candida albicans; anti-biofilm; antifungal activity; minocycline; morphology.

Figure 1

Antifungal effects of 4 TE derivatives. MH had a marked antifungal activity against C. albicans and DO had a slight antifungal activity against C. albicans, while TE and TGC had no antifungal activity. TE, tetracycline; MH, minocycline; DO, doxycycline; C. albicans, Candida albicans; TGC, tigecycline.

Figure 2

Effects of different concentrations of MH on fungal cell growth of the Candida albicans strain ATCC 90028. ^*P<0.05; ^**P<0.01. OD, optical density; MH, minocycline.

Figure 3

(A) Effects of MH on hyphal formation in C. albicans. Cells (1x10⁶ cells/ml) were grown in Spider or RPMI 1640 media supplemented with 20% FBS. Photomicrographs of C. albicans ATCC 90028 grown at 37˚C for 4 h. MH had an obvious trend to inhibit filamentous growth as compared with cells incubated in drug-free medium (magnification, x400). (B) The hyphae rate was counted in a minimum of 200 cells from each sample following 4 h of cultivation. The results indicated that MH was able to inhibit hyphae formation at all concentrations, with the effects of 125 and 250 µg/ml MH being most significant. ^**P<0.01; ^***P<0.001 vs. control. MH, minocycline; C. albicans, Candida albicans; FBS, fetal bovine serum.

Figure 4

(A) Light microscopy images of biofilm formation and mature biofilm. With the increase in the MH concentration, the inhibition of biofilm formation was more obvious. At 250 µg/ml MH, there was a marked decrease in the density of biofilms. By contrast, in the presence of MH, mature biofilms exhibited no changes obvious changes observed by standard light microscopy (magnification, x1,000) (B) Effects of MH on biofilm formation and mature biofilms. MH inhibited C. albicans biofilm formation in vitro. Biofilm inhibition was evaluated using an XTT reduction assay and the results are presented as the percentage of MH-treated biofilms relative to control biofilms that were formed without drug treatment. ^****P<0.0001 and ^***P<0.001 vs. control. MH, minocycline; C. albicans, Candida albicans.

Figure 5

Gene expression levels in the C. albicans strain ATCC 90028. Fold change values of the expression of each target gene are shown relative to the values of the control group containing untreated C. albicans. The housekeeping gene ACT1 was used as an internal reference gene. The expression level of each target gene in the MH-free sample was set as 1.^**P<0.01; ^***P<0.001. C. albicans, Candida albicans; MH, minocycline.