Transcriptional response of Candida albicans biofilms following exposure to 2-amino-nonyl-6-methoxyl-tetralin muriate

Abstract

Aim: To identify changes in the gene expression profile of Candida albicans (C albicans) biofilms following exposed to 2-amino-nonyl-6-methoxyl-tetralin muriate(10b) and clarify the mechanism of 10b against C albicans biofilms.

Methods: Anti-biofilm activity of 10b was assessed by tetrazolium (XTT) reduction assay and the action mechanism against biofilms was investigated by cDNA microarray analysis and real-time RT-PCR assay.

Results: Ten differentially expressed genes were directly linked to biofilm formation and filamentous or hyphal growth (eg, NRG1, ECE1 and CSA1). Decreased gene expression was involved in glycolysis (eg, HXK2 and PFK1) and antioxidant defense (eg, SOD5), while increased gene expression was associated with enzymes that specifically hydrolyzed beta-1,3 glucan (XOG1), and with lipid, fatty acid and sterol metabolism (eg, SLD1, ERG6 and ERG2). Functional analysis indicated that addition of anti-oxidant ascorbic acid reduced inhibitory efficiency of 10b on mature biofilm.

Conclusion: Inhibition of 10b on biofilm formation possibly depends on impairing the ability of C albicans to change its morphology via altering the expression of biofilm formation genes. Mitochondrial aerobic respiration shift and endogenous ROS augmentation might be a major contribution to reduce mature biofilm metabolic activity. The data may be useful for the development of new strategies to reduce the incidence of device-associated infections.

Figure 1

Chemical structure of 2-amino-nonyl-6-methoxyl-tetralin muriate (10b).

Figure 2

Morphology alteration of C albicans strain after treated with 10b. Confocal laser scanning microscopy of C albicans biofilms: (A) normal biofilm; (B) cells treated with 10 μmol/L farnesol at 0 h of the incubation time; (C) cells treated with 0.1 μmol/L 10b at 0 h of the incubation time; (D) cells treated with 1.0 μmol/L 10b at 0 h of the incubation time.

Figure 3

Inhibition of biofilm formation by different concentrations of 10b (A) and farnesol (B). biofilm formation (as measured by XTT reduction) is expressed as a percentage of control biofilms. The data are expressed as mean±standard deviation (SD) of three independent experiments. The mean±SD of the mean control values (A₄₉₂) was 1.083±0.056. Statistically significant differences as determined by Student's t test. ^bP<0.05, ^cP<0.01 vs control.

Figure 4

Gene expression changes in 25 genes of interest in C albicans SC5314 biofilms treated with 10b (10 μmol/L) relative to untreatment: (A) genes related to the specific action mechanism of 10b; (B) genes independent of the action mechanism of 10b. All the genes were examined by real-time RT-PCR with gene-specific primers.

Figure 5

Central carbon metabolism in C albicans biofilms during aerobic growth on glucose. The gray ellipses indicate low expression of genes encoded metabolic enzymes participated in aerobic oxidation process after treatment with 10b. The black ellipses indicate augmented tricarboxylic acid cycling process and endogenous reactive oxygen species generation after treatment with 10b. G-6-P, glucose-6-phosphate; F-1,6-2P, fructose-1,6-bisphosphate; G3P, glyceraldehyde-3-phosphate; 1,3-DPGA, 1,3-bisphosphoglycerate; PEP, phosphoenolpyruvate; TCA cycle, tricarboxylic acid cycle.

Figure 6

Effect of MCZ incubated for 6 h on mature (48 h) biofilms. MCZ was added to mature (48 h) biofilms and incubation was continued for 6 h at 37 °C. XTT reduction is expressed as a percentage of that of control mature biofilms (48 h) further incubated for 6 h. The results are mean±standard deviations of three independent experiments. Statistically significant differences as determined by Student's t test. ^bP<0.05, ^cP<0.01 vs control.