title: Signalling and regulation in Candida biofilms
Creators: name: Alem, Mohammed A.S.
Publisher: name: University of Glasgow
Year: 2006

description: Candida albicans and related Candida species are common members of the normal flora in humans; however, they are opportunistic pathogens and may cause superficial or systemic disease. Candidaemia is the most extensively studied nosocomial invasive fungal infection, and recent data show that Candida species are the fourth most commonly recovered blood culture isolates. It is now clear that this kind of infection is often due to the formation of Candida biofilms on catheters and other indwelling medical devices. This project investigates the role of quorum sensing in Candida biofilm formation, and the effect of prostaglandins, COX inhibitors and the combined effect of COX inhibitors with antifungal agents on biofilm development. Additional investigations monitor prostaglandin production by planktonic cells and biofilms of Candida albicans. Quorum sensing is known to be involved in bacterial biofilm formation, and in Candida, two signal molecules (farnesol and tyrosol) were recently identified. In this study the effects of farnesol on germ-tube and biofilm formation were investigated. Farnesol totally prevented germ-tube formation and inhibited biofilm formation by 30 % when 1mM farnesol was added at an early stage. Scanning electron microscopy revealed that only yeast cells were formed in biofilms grown in the presence of farnesol. Supernatants from biofilms formed after 24h and 48h inhibited germ-tube formation by 10 % and 29 %, respectively, indicating that they contained farnesol or farnesol-like activity. It has been reported that planktonic cultures of C. albicans produce tyrosol, another quorum-sensing molecule. In this study, HPLC confirmed that tyrosol was produced by both planktonic cells and biofilms of C. albicans, and biofilms produced significantly higher levels of tyrosol than planktonic cells. Overall, the results demonstrated that biofilm development in C. albicans is under the control of at least two quorum-sensing molecules. Farnesol acts as a negative signal and inhibits hyphal production. Tyrosol acts as a positive signal and promotes hyphal production. Prostaglandins are now known to be produced by C. albicans and may play an important role in fungal colonization. Their synthesis in mammalian cells is decreased by inhibitors of the cyclooxygenase isoenzymes required for prostaglandin formation, hi this study, the effects of nonsteroidal anti-inflammatory drugs (all cyclooxygenase inhibitors) on biofilm formation by three strains of C. albicans were investigated. Seven out of nine drugs tested at a concentration of 1 mM inhibited biofilm formation. Aspirin, etodolac and diclofenac produced the greatest effects, with aspirin causing up to 95% inhibition. Surviving cells had a wrinkled appearance, as judged by scanning electron microscopy, and consisted of both yeasts and hyphae. The effect of aspirin on viability of both biofilm and planktonic cells was pH dependent, with the greatest effect at pH 3 or in unbuffered yeast nitrogen base medium. The effect of aspirin on metabolic activity and viability of mature C. albicans biofilms was similarly pH dependent. At pH 3, aspirin inhibited metabolic activity of mature C. albicans biofilms more than fluconazole or a mixture of fluconazole and aspirin. This study also demonstrated that both biofilms and planktonic cells of C. albicans synthesize extracellular prostaglandin(s) during growth at 37°C, but biofilm cells secrete significantly more when production is determined on the basis of cell dry weight. Prostaglandin synthesis by both cell types was sensitive to the cyclooxygenase inhibitors aspirin, diclofenac and etodolac. A morphological mutant blocked in two signalling pathways (cph1/cph1 efg1/efg1) produced prostaglandin levels similar to those of the parent strain but formed yeast-only biofilms. Unicellular organisms such as fungi have been reported to interact with hormones, such as fungal sex hormones and mammalian hormones, hi this study the effect of several steroids including progesterone, corticosterone, dexamethasone, prednisolone, hydrocortisone and estradiol on C. albicans biofilm development was investigated. The results showed that biofilm formation was not affected by any of these compounds. However, at 1μM some steroids such as progesterone and dexamethasone inhibited germ-tube formation by more than 25%. At 100 to 1000 μM, all steroids inhibited germ-tube formation.