Materials and methods
Reagents
Four chelators were examined in this study: TPEN (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), deferoxamine (DFO; Abcam, Cambridge, UK), and triethylenetetramine (TETA; Nacalai Tesque, Kyoto, Japan), which predominately chelate Zn2+, Fe2+ and Cu2+, respectively, and ethylenediaminetetraacetic acid (EDTA; Nippon Gene Co., Ltd, Tokyo, Japan), which predominantly chelates divalent cations such as Mg2+ and Ca2+. Tested final concentrations were [0, 0.01, 0.1, 1, 10, 100 μM] for TPEN, [0, 50, 500, 5000, 50,000 μM] for DFO and [0, 5, 50, 500, 5000 μM] for TETA and EDTA. TPEN was dissolved in DMSO. DFO, TETA and EDTA were dissolved in sterile water. All stock solutions were stored at − 30 °C until use.
T. asahii culture
The stock strain, T. asahii MPU 105, which is highly able to form biofilms, was selected from the MPU culture collection, and the strain was isolated from the blood of patient. The strain was routinely grown on Sabouraud dextrose agar plates at 27 °C.
Biofilm formation
Biofilms formed from cells grown in flat-bottomed, 96-well microtiter plates. Standardized cell suspensions (A630 = 0.1 in RPMI 1640-plus-MOPS medium, pH 7.0) were seeded into each well. The plates were incubated without shaking at 37 °C for 1 h, and then the supernatants were removed and the cells were washed with PBS. Next, fresh medium was added, followed by incubation without shaking at 37 °C, and then the supernatants were removed after 24 h and replaced with fresh medium. Planktonic cells were removed after an additional 24 h incubation and the wells were washed with PBS. Semiquantitative evaluation of biofilm formation was performed using the XTT reduction assay. Absorbance was measured at wavelengths of 492 and 630 nm.
Microscopic analysis
T. asahii cells (A630 = 0.1) were added to RPMI 1640-plus-MOPS medium with each concentration of TPEN and ZnSO4 in 96-well plates and incubated without shaking at 37 °C for 48 h. Controls (no TPEN or ZnSO4) included the same amount of solvent as the other samples. Morphological changes were microscopically evaluated.
Statistical analysis
The significance of differences between groups was calculated using a Student’s t test. P < 0.05 was considered a statistically significant difference.
Results
We tested whether biofilm formation by T. asahii was inhibited by the cation chelators TPEN, DFO, TETA, and EDTA. TPEN, EDTA, and TETA reduced biofilm formation in a concentration-dependent manner (Fig. 1). DFO did not obviously inhibit biofilm growth at 50 mM (Fig. 1). IC50 values (the concentration of a compound required for the half inhibition of biofilm formation) of TPEN, TETA, and EDTA were 0.17 µM, 117 µM, and 47 µM, respectively. TPEN was the most effective cation chelator to inhibit biofilm formation by T. asahii.
TPEN has been used as a zinc chelator in several studies [15,16,17]. Therefore, we tested the effect of zinc on biofilm formation by T. asahii. Adding 1 to 10 µM of zinc increased biofilm formation, while high concentration of zinc decreased biofilm formation (Additional file 1: Figure S1). Zinc suppressed the inhibitory effects of TPEN on biofilm formation by T. asahii (Fig. 2). Moreover, the addition of excess amounts of zinc led to an increase in biofilm formation by T. asahii (Fig. 2). These results suggest that zinc is an important metal for biofilm formation by T. asahii. We examined whether the inhibitory effect of TPEN was reversible by removing TPEN from the medium. After 1 h or 24 h treatment with TPEN, TPEN was removed by changing the medium (Additional file 2: Figure S2a). Biofilm formation by T. asahii was increased by removing TPEN (Additional file 2: Figure S2b). The effect of zinc addition was also observed in this assay (Additional file 2: Figure S2b). These results indicate that TPEN treatment did not kill all T. asahii cells and that the inhibitory effect of TPEN was reversible.
We next examined morphological changes induced by TPEN. Control cells formed hyphae and arthroconidia. In the presence of 0.1 μM TPEN, hyphal formation decreased, whereas zinc addition induced hyphal elongation (Fig. 3). These results suggest that zinc regulated hyphal formation of T. asahii.
Discussion
In this study, the cation chelator TPEN inhibited biofilm formation and hyphal elongation in T. asahii. Zinc triggered hyphal elongation and enhanced biofilm formation. Only morphological transition in response to TPEN or zinc was observed in planktonic cells in this study. Direct observation of biofilm structure changes instigated by TPEN or zinc is an area for future research. Our findings suggest that T. asahii uses zinc as an environmental indicator.
In C. albicans, zinc is important in terms of both biofilm development and maintenance. The expression of genes related to zinc acquisition increases in biofilm cells of C. albicans [14, 18]. Our previous report suggested that C. albicans biofilm formation is regulated by zinc and that TPEN inhibits biofilm formation [14]. Depletion of divalent cations, such as iron, calcium, and magnesium, inhibit biofilm formation by Aspergillus fumigatus and Cryptococcus neoformans [19, 20]. In a murine model of invasive pulmonary aspergillosis caused by pathogenic fungus Aspergillus fumigatus, TPEN improved survival [16]. In vivo experiments to evaluate the inhibitory effect of TPEN on biofilm formation by T. asahii are areas for future research.
Zinc also promotes biofilm formation by the pathogenic bacterium Streptococcus pneumoniae [21]. Derivatives of 2-aminobenzimidazole inhibit biofilm development in methicillin-resistant S. aureus, vancomycin-resistant Enterococcus faecium and S. epidermidis, by chelating zinc [22]. In S. aureus, zinc activates intercellular adhesion during biofilm formation [23]. Thus, zinc may play a pivotal role in biofilm formation in both eukaryotes and prokaryotes. Therefore, cation chelators effectively prevent and treat biofilm-related infections.
In conclusion, zinc chelating has the potential to inhibit biofilm formation by T. asahii. Although further research is essential, TPEN could potentially be applied to catheters to prevent biofilm formation. The information may contribute to the prevention of biofilm formation by T. asahii on the surface of medical devices.