The Effect of Novel Heterocyclic Compounds on Cryptococcal Biofilm

Abstract

Biofilm formation by microorganisms depends on their communication by quorum sensing, which is mediated by small diffusible signaling molecules that accumulate in the extracellular environment. During human infection, the pathogenic yeast Cryptococcus neoformans can form biofilm on medical devices, which protects the organism and increases its resistance to antifungal agents. The aim of this study was to test two novel heterocyclic compounds, S-8 (thiazolidinedione derivative, TZD) and NA-8 (succinimide derivative, SI), for their anti-biofilm activity against strains of Cryptococcus neoformans and Cryptococcus gattii. Biofilms were formed in a defined medium in 96-well polystyrene plates and 8-well micro-slides. The effect of sub-inhibitory concentrations of S-8 and NA-8 on biofilm formation was measured after 48 h by a metabolic reduction assay and by confocal laser microscopy analysis using fluorescent staining. The formation and development of cryptococcal biofilms was inhibited significantly by these compounds in concentrations below the minimum inhibitory concentration (MIC) values. These compounds may have a potential role in preventing fungal biofilm development on indwelling medical devices or even as a therapeutic measure after the establishment of biofilm.

Keywords: Cryptococcus; auto-inducer; biofilm; quorum sensing; succinimide derivative; thiazolidinedione derivative.

Figure 1

The effect of S-8 and NA-8 on biofilm metabolic activity. C. neoformans H-99 (a) and C. gattii R-272 (b) biofilms were grown in 96-well microtiter plates as described in the Methods section. The inhibition of biofilm formation by S-8 and NA-8 were examined by measuring metabolic activity (XTT assay). Mean ± standard error (SE).

Figure 2

Effect of S-8 and NA-8 on the viability of C. neoformans and C. gattii biofilms. (a) Treatment of C. neoformans H-99 with S-8 and NA-8 during biofilm formation reduces nucleic acid staining by SYTO9. Treatment with S-8 also causes yeast death in the biofilm; (b) Treatment of C. gattii R-272 with S-8 and NA-8 during biofilm formation reduces nucleic acid staining by Syto9. Medium with 0.5% DMSO was used as a control. ×400. MIC, minimum inhibitory concentration. DMSO, dimethyl sulfoxide.

Figure 3

Effect of sub-inhibitory concentrations of S-8 and NA-8 on the biofilm thickness of C. neoformans (a) Treatment of C. neoformans H-99 during biofilm formation with NA-8 but not S-8 reduced biofilm thickness; (b) Treatment of C. gattii R-272 during biofilm formation with S-8 and NA-8 reduced biofilm thickness. Medium with 0.5% DMSO was used as a control. Mean ± SE. ×40.

Figure 3

Effect of sub-inhibitory concentrations of S-8 and NA-8 on the biofilm thickness of C. neoformans (a) Treatment of C. neoformans H-99 during biofilm formation with NA-8 but not S-8 reduced biofilm thickness; (b) Treatment of C. gattii R-272 during biofilm formation with S-8 and NA-8 reduced biofilm thickness. Medium with 0.5% DMSO was used as a control. Mean ± SE. ×40.