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In this study we investigated whether the direct interaction between Candida albicans CBS 5982 and 5-hydroxytryptamine (5-HT) alters candidial virulence. Hyphae elongation, phospholipase activity and the production of secreted aspartyl proteinases (Saps) following 5-HT treatment were investigated. 5-HT treatment of C. albicans significantly (P < 0.05) affected hyphal extension, phospholipase activity and the production of Saps at concentrations of 118–0.46 mM. In conclusion, our findings suggest that the interaction between 5-HT and C. albicans may diminish the virulence properties of this fungal pathogen.
5-Hydroxytryptamine (5-HT; serotonin) is a biogenic amine belonging to the most common neurotransmitters in nature. In humans, 5-HT modifies body temperature, blood pressure, sexual behaviour and mood, and is also involved in several interactions of the immune system [1,2]. 5-HT is present in the brain, platelets, pulmonary neuroendocrine cells, enterochromaffin cells of the gut, and mast cells . The brain and enterochromaffin cells are the main producers of 5-HT in vivo . Platelets do not synthesise 5-HT but absorb the amine from plasma and store it in their dense granules. Holmsen and Weiss  found that the concentrations of 5-HT within the dense granules are up to 65 mM.
Recently, we observed that 5-HT has antifungal activity against Candida spp. in vitro . Schuff-Werner and Splettstoesser  observed antioxidative properties of platelet-released 5-HT on the bacterial function of polymor-phonuclear neutrophils, and there is a surprising coincidence of low 5-HT levels and an increased rate of infections. In this study, the direct influence of 5-HT against Candida albicans CBS 5982 with regard to virulence properties (hyphae elongation, extracellular phospholipases and secreted aspartyl proteinases (Saps)) following 5-HT treatment were evaluated in vitro.
Candida albicans CBS 5982 (Centraalbureau voor Schimmelcultures, Baarn, The Netherlands) was used for this in vitro study. The strain was initially grown on Sabouraud Dextrose Agar (Oxoid, Basingstoke, UK) at 30 °C for 48 h.
A stock solution of 5-HT (Sigma–Aldrich, Vienna, Austria) was prepared following the manufacturer’s instructions. Serial two-fold dilutions from 235 to 0.92 mM 5-HT were prepared and concentrations from 118 to 0.46 mM 5-HT were used for in vitro tests.
The isolate was tested using the microbroth dilution method according to the guidelines of the National Committee for Clinical Laboratory Standards .
An inoculum size of 1.3 × 105 blastoconidia/mL was transferred into microwell plates × (Greiner, Kremsmünster, Austria), incubated at 30 °C or 37 °C and evaluated for growth after 3 h, 6 h, 9 h and 12 h incubation. Each sample was performed in duplicate, measuring 50 organisms per sample; a micrometer was used for length measurement.
The extracellular phospholipase activity of C. albicans was measured by the egg yolk agar method .
Sap activity of C. albicans was assessed by a version of the protocol of Ollert et al. .
Statistical significance was determined using the Student’s t-test and f-test analysis. All comparisons were two-sided, and P < 0.05 was considered significant.
The minimum inhibitory concentration (MIC) for C. albicans was 14.7 mM and the minimum fungicidal concentration (MFC) was 29.4–14.7 mM. Differences in hyphal elongation were observed for C. albicans treated with 5-HT at concentrations between 14.7 mM and 3.7 mM (P < 0.05;Fig. 1).
Treatment with 5-HT decreased phospholipase activity compared with the positive control. Phospholipase activity was clearly impaired between 14.7 mM and 1.8 mM 5-HT, concentrations that had no effect on fungal growth (Fig. 1).
The production of Saps by C. albicans was decreased with 5-HT. The highest reduction in Saps was found at 7.3–1.8 mM 5-HT, concentrations that did not affect the growth of C. albicans (Fig. 1).
The present study indicates that the direct interaction between 5-HT and C. albicans affects fungal virulence properties such as hyphal elongation, extracellular phospholipase activity and Sap production.
Hyphal elongation of C. albicans was altered significantly (P < 0.05) at 14.7–3.7 mM 5-HT. The phenomenon of dimorphism by C. albicans is an important virulence factor . It is widely recognised that formation of hyphae enhances adherence and tissue invasion, and the transition from conidia to hyphae supports invasive fungal diseases . Hyphal elongation of 5-HT-treated fungus was more prominent at 37 °C compared with 30 °C.
In addition, concentrations of 14.7–1.8 mM 5-HT decreased phospholipase activity without showing an impairment of fungal growth. The role of extracellular phospholipase as a potential virulence factor in pathogenic fungi such as C. albicans has recently gained credence. Extracellular phospholipase activity has been shown to be predictive for mortality in a murine mouse model of disseminated candidiasis, and increased phospholipase activity has been correlated with increased mucosal pathogenicity in this opportunistic yeast . Phospholipase secretion is also not limited to one fungal genus [12,13]. The virulence of C. albicans is defined by the production of proteinases such as Saps and phospholipases . These hydrolytic enzymes destroy or derange constituents of host cell membranes, leading to physical disruption and/or membrane dysfunction .
The highest reduction in Saps was found at 7.3–1.8 mM 5-HT, concentrations that did not affect the growth of C. albicans. The majority of Sap proteins are secreted by those C. albicans cells that directly adhere to the epithelial surface . A dominant Sap isoenzyme in vitro and possibly in vivo is Sap2, which is the predominant isoenzyme investigated here [9,16].
In addition to being fungicidal at higher concentrations, 5-HT exerts an inhibitory effect on C. albicans even at concentrations that are up to one magnitude lower, by inhibiting phospholipase activity and Sap2 production.
The mechanism by which 5-HT acts on C. albicans is not yet known. The role of 5-HT in antifungal host defence needs to be clarified in more detail as there is a surprising coincidence of an increased rate of infections and low 5-HT concentrations such as in Down’s syndrome  and AIDS . Thrombocytes are able to store up to 65 mM 5-HT within the dense granules .
In conclusion, our findings show that 5-HT treatment of C. albicans affected hyphal elongation, phospholipase activity and production of Saps.
The data encourage us to define the role of 5-HT in antifungal host defence. It could be of great help to identify the mode of action in order to develop and research new antifungal agents.
The project was funded by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (P17484-B05).