A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro

Abstract

The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm infections.

Figure 1. Relative expression of genes related to ACH metabolism.

(A) A schematic model of central carbon metabolism incorporated with genes of interest within ethanol metabolism (abbreviations as follows: G6P =  glucose-6-phosphate, KICA =  α-ketoisocaproic acid). (B) Heat map panel of gene expression in C. albicans biofilms at three different stages of growth at pH 5.2 and 7.4 after 24 h exposure to PBS, caspofungin, HICA, leucine, cysteine or ethanol. Fold changes are expressed relative to control (RPMI) at the corresponding time point. Black represents no change in expression, green is up-regulation, and red is down-regulation. A brighter color indicates greater degree of change in expression. Relative gene expressions were calculated by the Pfaffl method using the REST 2009 software provided by Qiagen .

Figure 2. Summary of study design.

Figure 3. Changes in metabolic activity and biomass of C. albicans biofilms.

Metabolic activities measured by XTT-assay (panel A, B) and dsDNA levels (panel C, D) reflecting the biomass of C. albicans biofilms at pH 5.2 and 7.4. Biofilms were grown for 4, 24 or 48 h in RPMI at pH 7.4 and then exposed to PBS, caspofungin, HICA, leucine, cysteine or ethanol for another 24 h. Values were measured twice in triplicate and expressed as mean (±SEM). Means were compared to the control treatment (RPMI). Statistically significant differences were calculated using a GEE-model and were marked (**p<0.001,* 0.001<p<0.05).

Figure 4. ACH production by C. albicans biofilms.

Mean (±SEM) ACH production by C. albicans biofilms. Biofilms at three different stages of growth (4 h, 24 h and 48 h), were incubated for 30 min at 37°C with 100 mM D-glucose (A,B) or 0.05% ethanol (C,D) after 24 h exposure to RPMI, ethanol, caspofungin or HICA at pH 5.2 or 7.4. Values were measured twice in triplicate and means were compared at each time point to the control treatment (RPMI). Statistically significant differences were calculated using a GEE-model and were marked (**p<0.001, * 0.001<p<0.05).

Figure 5. Microscopic examination of C. albicans biofilms exposed to HICA.

SEM images were taken of biofilms which were grown for 24% HICA for another 24 h at pH 5.2 (A) or pH 7.4 (B). Control images were taken of biofilms grown in RPMI-medium without HICA at pH 5.2 (C) or pH 7.4 (D). Insets show hyphal structures in detail. Scale bars indicate 10 µm in main images and 5 µm in insets.