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This study examined the direct interaction of serotonin (5-hydroxytryptamine (5-HT)) with Aspergillus species. Accumulation of 5-HT in aspergilli was investigated by immunofluorescence staining and laser confocal scanning microscopy. The influence of 5-HT on fungal ergosterol content, cell membrane integrity, fungal growth and hyphal elongation was determined. 5-HT was localised in the cytoplasm of Aspergillus spp., as 5-HT fluorescent signals appeared after 30 min at 4°C and in the presence of inhibitors of oxidative phosphorylation. 5-HT treatment of Aspergillus spp. significantly affected ergosterol synthesis, fungal cell membrane integrity and hyphal elongation (P < 0.05). 5-HT treatment for 4 h resulted in a lag of re-growth (post-antifungal effect). In conclusion, our findings suggest that 5-HT affects hyphal growth and diminishes fungal cell membrane integrity.
Invasive aspergillosis has emerged worldwide as an important cause of infection among patients undergoing cancer chemotherapy, haematopoietic stem cell transplantation or solid organ transplantation [1-4]. The crude mortality from invasive aspergillosis is ca. 85%, which falls to ca. 50% if treated [3,5]. Aspergillus fumigatus is the most prominent pathogen in the Aspergillus genus, accounting for >90% of human Aspergillus infections, followed by Aspergillus flavus .
Innate immunity plays an important role in defence against Aspergillus infections. Serotonin (5-hydroxytryptamine (5-HT)) is a monoamine neurotransmitter both in the central and peripheral nervous systems. Outside the central nervous system, 5-HT is present in platelets, pulmonary neuroendocrine cells and enterochromaffin cells of the gut . 5-HT contributes to many physiological functions and is involved in several interactions of the immune system [7,8], displaying antioxidative properties on the antibacterial function of polymorphonuclear neutrophils .
We observed that 5-HT is fungicidal against a wide range of Aspergillus spp.  and that platelets attenuate fungal virulence in vitro . 5-HT is stored in the dense granules of platelets at 65mM and there is a coincidence of low 5-HT with certain diseases, e.g. acquired immune deficiency syndrome (AIDS) and an increased rate of infections . Aspergilli induce platelet activation followed by 5-HT release from their granules .
These antifungal capabilities of 5-HT led us to examine the interaction of 5-HT and Aspergillus spp.
Fourteen clinical isolates each of Aspergillus fumigatus, A. terreus and A. flavus were studied. The isolates were maintained as conidial suspensions in sterile water at room temperature and subcultures were grown on Sabouraud-2% dextrose agar (SDA) (Merck, Darmstadt, Germany) and incubated at 35°C for 4 days.
Lyophilised serotonin hydrochloride (mol. wt. 212.7 g/L; Sigma–Aldrich, Vienna, Austria) was dissolved in 1mL of distilled water to a final concentration of 470 mM. 5-HT dilutions were prepared in RPMI 1640 (Sigma–Aldrich, Vienna, Austria) at concentrations ranging from 3.6 μM to 58mM.
Conidia (1×104 colony-forming units (CFU)/mL) of Aspergillus spp. were grown on coverslips for 18 h at 35°C and stained according to the protocol of Fischer and Timberlake  with slight modifications. In brief, prior to fixation with 3% formaldehyde (Merck, Vienna, Austria), hyphae were treated with 5-HT at concentrations ranging from 3.6μM to 58mM. Incubation times were 30 min, 60 min, 90 min, 6 h and 12 h at 4°C, 20°C and 35°C. Fungi were then incubated for 60 min at room temperature with the primary monoclonal mouse antihuman serotonin antibody (Dako, Copenhagen, Denmark), diluted 1:80 in blocking buffer and visualised with the secondary fluorescein isothiocyanate (FITC)-conjugated polyclonal rabbit antimouse immunoglobulin G (Dako, Copenhagen, Denmark), diluted 1:100 in blocking buffer. In addition, fungi were stained with Calcofluor white solution (Molecular Probes, Eugene, OR), diluted 1:2 with phosphate-buffered saline (PBS). The samples were visualised with an Inverse Axiovert 100M BP (Zeiss, Vienna, Austria) equipped with a laser scanning module 510. The images were processed by the same software. Z-series optical sections were recorded at 1 μm using a 63× (numeral aperture = 1.4) oil immersion lens. For controls, hyphae were prepared and treated as described above either without 5-HT or without monoclonal mouse antihuman serotonin antibody.
To investigate the role of inhibitors of oxidative phosphorylation on 5-HT uptake, the method of Oberparleiter et al.  was used with slight modifications. In short, fungi were prepared as described above and hyphae of Aspergillus spp. were treated with 1.25 mM, 2.5 mM, 5mM and 10mM sodium azide (Sigma–Aldrich, Vienna, Austria), 20 mM, 40mM and 80mM potassium cyanide (KCN) (Merck, Vienna, Austria) and 200μM and 400μM carbonyl cyanide m-chlorophenylhydrazone (Sigma, Vienna, Austria) for 30 min and then co-incubated with 3.6μM, 3.6 mM, 14mM and 58mM 5-HT for 30 min, 60 min and 90 min at 35°C. Hyphae were then washed with PBS before immunofluorescence staining.
Intracellular sterols were extracted as described previously with slight modifications [15,16]. In short, conidial suspensions of Aspergillus spp. were prepared as described above and inoculated into 25mL of fluid SDA (BD, Vienna, Austria), supplemented with different concentrations of 5-HT and incubated for 48 h at 37°C under rotation. The ergosterol content was calculated as a percentage of cell weight by the following equations: %ergosterol + %24(28)dehydroergosterol (DHE) = [(A281.5/290)×F]/pellet weight, %24(28)DHE = [(A230/518)×F]/pellet weight and %ergosterol = [%ergosterol + %24(28)DHE] − %24(28)DHE, where F is the factor for dilution in ethanol and 290 and 518 are the E values determined for crystalline ergosterol and 24(28) DHE, respectively. Tests were performed in duplicate and were repeated four times.
5-HT-induced fungal cell membrane injury was assessed using two dyes, fluorescein diacetate (FDA) (Merck, Vienna, Austria) and propidium iodide (PI) (Molecular Probes, Eugene, OR) as reported previously [17-19]. Briefly, 1×104 CFU/mL of Aspergillus spp. were grown on coverslips at 35°C for 18 h to form hyphae. Fungi were then treated with 5-HT ranging from 3.6μM to 29mM for another 60 min. Samples were washed and then FDA and PI were added simultaneously at 0.01% and incubated for 10 min at room temperature. All samples were embedded in 50% glycerol solution mounting medium and visualised with a Zeiss Axioplan fluorescence microscope (Zeiss, Vienna, Austria) and appropriate filter combinations. For controls, hyphae were treated either with 70% ethanol or PBS (Sigma–Aldrich, Vienna, Austria). Experiments were performed twice in duplicate.
The morphology of Aspergillus spp. treated with or without 5-HT was investigated by assessing germination and hyphal elongation . Therefore, 100μL of 2×105 CFU/mL in RPMI 1640 were inoculated with 100μL each of the drug solution into microwell plates (Greiner, Kremsmünster, Austria) and incubated for 16 h. For controls, 100μL of the fungal suspensions were incubated with 100μL of RPMI 1640 only. The morphology of the organisms was determined microscopically at the time points indicated; a micrometer was used for length measurement. Each sample was assessed in triplicate, measuring 50 organisms per sample and repeated three times.
Conidial suspensions were prepared as described above and incubated with 5-HT for 1 h and 4 h at 35°C. Lag of re-growth was assessed using a modification of the procedure of Nagl et al. . Concentrations equipotent to, one dilution above and one dilution below the minimal fungicidal concentration (MFC) for each isolate were investigated. Afterwards, fungi were washed twice with sterile water, centrifuged at 4000×g for 2 min and refilled with RPMI 1640. Quantitative cultures of non-diluted samples and 1:100 and 1:1000 dilutions in distilled water were spread on SDA, incubated at 35°C and examined visually for growth every 12 h. The times required for colony count of untreated and treated isolates were compared and the cultures were examined for lag of re-growth. Each experiment was performed twice in triplicate.
All data are presented as mean±standard deviation of three measurements (each sample tested in triplicate). Statistical significance was determined using Student's t-test. All comparisons were two-sided and P < 0.05 was considered significant.
Green fluorescent signals representing 5-HT appeared in the whole cytoplasm of Aspergillus spp. after 30 min of incubation at 3.6μM 5-HT. The distribution of the fluorescence excludes compartmentalisation of 5-HT but supports cytoplasmic localisation. The fungal cell wall stained with Calcofluor white encircled the green fluorescent signals in the cytoplasm (Fig. 1A). Immunostaining was specific, since fluorescence was not detected in the controls (Fig. 1B). Similar results were observed when Aspergillus spp. was treated simultaneously with 5-HT and inhibitors of oxidative phosphorylation, as green fluorescent signals were detected (data not shown).
A dose-dependent decrease of ergosterol (P < 0.05) was observed in 5-HT-treated Aspergillus spp. compared with the control (Table 1). The degree of ergosterol decreased with increasing 5-HT concentrations.
A dose-dependent loss of fungal cell membrane integrity was observed in 5-HT-treated Aspergillus spp. (data not shown).
5-HT was found to be a strong inhibitor of fungal growth, as hyphal elongation of Aspergillus spp. was significantly decreased (P < 0.05) when treated with 5-HT (Table 2). No differences between the tested Aspergillus spp. were detected.
The lag of re-growth depended on the 5-HT concentration tested for all Aspergillus spp., as shown in Table 3. Treatment for 4 h with 5-HT at concentrations below and equipotent to the MFCs showed a lag of re-growth of 12–24 h for all isolates. Concentrations higher than the MFC resulted in a lag of re-growth and/or a decrease in CFU count. No effects were seen after an exposure time of 1 h (data not shown).
The present study shows that 5-HT exerts a direct influence on Aspergillus spp., as ergosterol synthesis, fungal membrane integrity, germination and hyphal elongation (P < 0.05) were significantly decreased. Moreover, 5-HT was found to accumulate intracellularly, independent of incubation time, temperature and metabolic energy. Short exposure of 5-HT resulted in a lag of re-growth in several fungi.
Many potential targets have been identified in fungi, and antimycotic drugs can act via extracellular and intracellular pathways . Most currently available drugs inhibit the synthesis of or interact with ergosterol, or directly damage the fungal cell membrane . In our study, 5-HT exerted a dose-dependent decrease of ergosterol synthesis (P < 0.05) followed by loss of cell membrane integrity in Aspergillus spp. The decrease in fungal viability is in parallel with an increase in intracellular fluorescence due to 5-HT accumulation; 5-HT internalisation may therefore result from cell membrane leakage in fungi.
The plasma membrane is a dynamic structure that segregates the intracellular milieu from the extracellular environment by regulating the entry and exit of small and large molecules . It has been shown that active transport of macromolecules, peptides or proteins into hyphae of various other filamentous fungi can be stopped by inhibitors of oxidative phosphorylation . Our data show that 5-HT internalisation was not prevented in the presence of inhibitors of oxidative phosphorylation. Neither KCN nor temperature treatment prevented the entry of 5-HT into the hyphal cytoplasm. These data refute active 5-HT transport into the fungal cytoplasm.
The ability of Aspergillus spp. to undergo morphological changes is an important virulence factor , as the onset of infection is associated with the appearance of hyphae. 5-HT altered the morphology of Aspergillus spp., since hyphal elongation was significantly decreased and a lack of germination was seen in some isolates. Moreover, a lag of re-growth was observed after exposure to 5-HT and the extent of these effects was dependent on the concentration and incubation time. The maximum duration of lag of re-growth was observed at incubation times shorter than those required for previous studies [25,26]. A similar result was found for sertraline, a selective serotonin reuptake inhibitor .
Currently, the role of 5-HT in antifungal host defence is unclear. There is a coincidence of low 5-HT in certain diseases, e.g. AIDS and an increased rate of infections . In vivo, the brain and enterochromaffin cells are the main producers of 5-HT, with 5-HT release from enterochromaffin cells taken up by and stored predominantly in platelets and mast cells . 5-HT is stored in dense granules of platelets at 65mM and is released by platelet degranulation . We identified fungal killing at concentrations between 14.6mM and 58mM  as well as alterations in candidal virulence at 3μM . Base levels of 5-HT in serum are ca. 3.5μM  and arise in multiple pathological situations. Our data indicate that 5-HT had only a weak influence on aspergilli at low concentrations but strong effects at pathological levels. The role of 5-HT release from platelets in defence against Aspergillus spp. needs to be further investigated, as we and others observed that platelets can damage hyphae of Aspergillus spp. [11,30].
In conclusion, our data indicate that 5-HT accumulates intracellularly, independent of time, temperature and energy. The interaction of 5-HT with Aspergillus spp. affects hyphal growth and diminishes fungal cell membrane integrity in vitro.
This project was funded by the Austrian Fond zur Förderung der wissenschaftlichen Forschung (P17484-B05).