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Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint 5 6
Novel mechanisms of surfactants against Candida albicans growth and morphogenesis
3 4 7 8 9 10 11 12 1 2 4 6 15 16 17 18 19 20 21 22 23 24 25
Q1
Qilin Yu a, Bing Zhang a, Feiyang Ma a, Chang Jia a, Chenpeng Xiao a, Biao Zhang b, Laijun Xing a, Mingchun Li a,⇑ a b
Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Department of Microbiology, College of Life Science, Nankai University, Tianjin 300071, PR China Tianjin Traditional Chinese Medicine University, Tianjin 300193, PR China
a r t i c l e
i n f o
Article history: Received 6 September 2014 Received in revised form 25 November 2014 Accepted 5 December 2014 Available online xxxx Keywords: Surfactant Candida albicans Morphogenesis Polarized growth
a b s t r a c t Candida albicans is a common opportunistic fungal pathogen, causing not only superficial mucosal infections but also life-threatening systemic candidiasis in immune-compromised individuals. Surfactants are a kind of amphiphilic compounds implemented in a wide range of applications. Although their antimicrobial activity has been characterized, their effect on C. albicans physiology remains to be elucidated. In this study, we investigated the inhibitory effect of two representative surfactants, cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), on C. albicans growth and morphogenesis. Both surfactants exhibited inhibitory effect on C. albicans growth. This effect was not attributed to plasma membrane (PM) damage, but was associated with mitochondrial dysfunction. Excitingly, the surfactants, especially CTAB, showed strong inhibitory effect on hyphal development (IC50 = 0.183 ppm for CTAB and 6.312 ppm for SDS) and biofilms (0.888 ppm for CTAB and 76.092 ppm for SDS). Actin staining and Hwp1-GFP localization further revealed that this inhibition is related to abnormal organization of actin skeleton and subsequent defect in polarized transport of hyphae-related factors. This study sheds a novel light on the antimicrobial mechanisms of surfactants, and suggests these agents as potential drugs against C. albicans hyphae-related infections in clinical practice. Ó 2014 Published by Elsevier Ireland Ltd.
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1. Introduction
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Candida albicans is a common opportunistic fungal pathogen, causing not only superficial mucosal infections but also life-threatening systemic candidiasis [1,2]. One of the most important properties of this pathogen is its ability of hyphal development under certain environmental stimuli, such as amino acids, low nitrogen source, serum and hypoxia [3]. Hyphal development is governed by a complicated signaling network that elaborately senses the stimuli and activates expression of hyphae-related genes, such as HWP1 [4,5]. Their products are then transported from the endoplasmic reticulum to the hyphal tip and provide materials for hyphal elongation [6]. Since the significance of hyphal development during invading host tissues, the system that mediates this process is a potential target against C. albicans infections [7,8]. Surfactants, also named surface active agents, are a kind of compounds containing both hydrophobic groups and hydrophilic groups. Due to the amphiphilic property, they are implemented
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in a wide range of applications [9–11]. Especially, these compounds are widely used as broad-spectrum antimicrobial agents [12,13]. Moreover, evidence suggests that these agents, in spite of their potential toxicity to mammalian cells, have potential use as anticancer agents [14]. Therefore, the surfactants may be developed as novel drugs in clinical practice. Cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), two representative surfactants, are widely used in biological researches. In this study, we investigated their effect on C. albicans growth and morphogenesis, and found that both agents had much stronger inhibitory effect on hyphal development than on growth. Furthermore, this inhibition is related to abnormal organization of actin skeleton and subsequent defect in transport of hyphae-related factors. Our findings suggested that the surfactants may be potential agents against C. albicans infections.
⇑ Corresponding author. E-mail address: [email protected] (M. Li). http://dx.doi.org/10.1016/j.cbi.2014.12.014 0009-2797/Ó 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Q. Yu et al., Novel mechanisms of surfactants against Candida albicans growth and morphogenesis, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.12.014
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2. Materials and methods
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2.1. Agents, strains and growth conditions
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The surfactants used in this study were purchased from Sigma, USA. The stock solutions of the agents were prepared in liquid RPMI-1640 medium (RPMI-1640 powder (Sigma, USA) 1%, 3-(Nmorpholino) propanesulfonic acid (Sigma, USA) 0.418%, uridine 80 ppm, pH 7.4) at the initial concentration of 1000 ppm, and diluted with the same medium to certain concentrations. Normally, the wild-type C. albicans strain BWP17 was used for growth inhibition tests, hyphal induction and biofilm assays. To investigate the effect of the surfactants on polarized transport of Hwp1, the strain NKF152 containing the Hwp1-GFP fusion gene (based on the plasmid pMG2082) was used [27]. The strains were overnight cultured in liquid YPD medium (yeast extract 1%, peptone 2%, glucose 2%) with shaking at 30 °C, and then used for further experiments.
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2.2. Growth inhibition assays To investigate the inhibitory effect of the surfactants on C. albicans growth, overnight cultured cells were washed twice with PBS, and suspended in RPMI-1640 medium to an initial optical density at 600 nm (OD600) of 0.2. 50 lL of the suspension were added into 96-well polystyrene microplate wells, and then 50 lL of surfactant solutions were mixed with the cell suspensions, obtaining the mixture containing the yeast cells with the OD600 of 0.1 and the surfactants with the following concentrations, 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 ppm for CTAB, and 0, 1.25, 2.5, 5, 10, 20, 40, 80, 160 ppm for SDS. The plates were covered with their lids, sealed with parafilm and incubated at 37 °C for 24 h. The OD600 of each well was then determined using a microplate reader (Bio-Rad, USA). The percent of growth for each treatment was calculated as the OD600 of each surfactant-treated group divided by the control (without the treatment of surfactants). The growth inhibition tests were also performed in liquid YPD medium (yeast extract 1%, peptone 2%, glucose 2%) and SC medium (yeast nitrogen base 0.67%, glucose 2%, amino acid mixture 0.2%) to evaluate the effect of media in CTAB activity.
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2.3. PM damage assays
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To evaluate the effect of CTAB on PM integrity, overnight cultured fungal cells were suspended in fresh YPD medium containing CTAB with the following concentrations, 0, 0.25, 0.5, 1, 2, 4, 8 ppm. The cells were cultured with shaking at 30 °C for 24 h and stained with PI (final concentration 5 ppm, Sigma, USA) for observation. At least 30 fields were observed. PI-positive and total cells in each group were quantified. The percent of PI-positive cells for each treatment was then calculated as the number of PI-positive cells divided by that of total cells. PM integrity was also evaluated by lactate dehydrogenase (LDH) assays. The CTAB-treated cultures were centrifuged at 12,000 rpm for 10 min, and the supernatant was used for determining LDH activity by an LDH Assay Kit (KEYGEN BIOTECH, China).
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2.4. Measurement of mitochondrial membrane potential (MMP)
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CTAB-treated cells were washed and suspended with PBS and stained with JC-1 (final concentration 1 ppm, Sigma, USA) for 30 min. Fluorescence intensity of JC-1 aggregates (red, FL-1) and monomers (green, FL-2) were recorded by a flow cytometer (FACSCalibur, BD, USA). The cells exhibiting decreased red fluorescence density than the control (untreated) cells were considered as the
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cells with decreased MMP, and the percent of cells with decreased MMP was recorded.
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2.5. Hyphal induction
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C. albicans hyphae were induced as described previously [15]. Briefly, overnight cultured cells were washed with PBS and suspended in RPMI-1640 medium containing the surfactants with the indicated concentrations. The suspensions were then incubated with shaking at 37 °C for 4 h. Cells were harvested, fixed with 4% formaldehyde, and observed by a light microscope (Olympus, Japan). The number of hyphal cells and total cells was counted in each field, and the percent of hyphal cells were calculated as the number of hyphal cells divided by the number of total cells. At least 30 fields were determined.
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2.6. Biofilm assays
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C. albicans biofilms were cultured in polystyrene microtiter plates (Corning Inc., USA). Overnight cultured cells were washed with PBS and suspended in RPMI-1640 medium containing the surfactants at different concentrations, with the OD600 of 0.1. 100 lL of the suspensions were then added into 96-well microtiter plates. The plates were covered with lids and incubated at 37 °C. After 24 h of incubation, the plates were washed 3 times with PBS, and the biofilm activity in each well was detected by XTT (2,3-bis(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assays [16]. To observe biofilm structures, the cultured biofilms were washed with PBS and fixed with 3% glutaraldehyde. The samples were then sectioned, dehydrated with ethanol, dried in vacuum desiccators, coated with gold and observed by a scanning electron microscope (SEM, QUANTA 200, Czech). To evaluate the effect of surfactants on biofilm maintenance, the biofilms were pre-formed as described above. The wells were then washed with PBS, and 100 lL RPMI-1640 medium containing the surfactants with different concentrations were added into the wells. The plates were covered with their lids and incubated at 37 °C for further 24 h. The metabolic activity of biofilms was also measured by the XTT reduction assay.
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2.7. Actin staining
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To observe actin skeleton, the C. albicans cells were washed twice with PBS, fixed with 4% formaldehyde for 10 min, permeabilized with 0.5% Triton 100 for 30 min and washed again with PBS. 5 lL of rhodamine phalloidin (final concentration 1 ppm, Cytoskeleton, USA) were then added into 100 lL of the cells. The cells were incubated at the room temperature for 30 min, and washed 3 times with PBS for observation.
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2.8. Fluorescence microscopy
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The PI-stained and rhodamine phalloidin-stained cells were observed by a fluorescence microscope (Olympus, USA) using the RFP filter set. To detect Hwp1 localization, the strain NKF152 was cultured in RPMI-1640 containing the surfactants as described above, and Hwp1-GFP was observed by the fluorescence microscope using the GFP filter set.
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2.9. Statistical analysis
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The experiments were performed with five replicates, and the values represent the means ± standard deviations (SD). Significant differences between the treatments were determined using oneway ANOVA (P < 0.05). Statistical analysis and IC50 calculation were performed by SPSS (Version 20, IBM, USA).
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3. Results
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3.1. The surfactants exhibit a distinct inhibitory effect on C. albicans growth
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Surfactants are well-known antimicrobial agents. To investigate whether the agents have antifungal activity, we first tested the growth biomass of C. albicans under treatment of two widely-used surfactants, CTAB and SDS. Liquid sensitivity tests showed that CTAB concentrations P0.5 ppm and SDS concentrations P40 ppm led to significant inhibition of fungal growth (Fig. 1A and B). Notably, high concentrations of CTAB (P1 ppm) resulted in >80% decrease in growth biomass (Fig. 1A). Although both surfactants showed expected inhibitory effect on C. albicans growth, CTAB (IC50 = 0.758 ppm) had much stronger inhibitory effect than SDS (IC50 = 45.961 ppm) (Table 1). Therefore, the inhibitory activity against C. albicans growth is distinct for these two agents. In addition, we also tested the effect of CTAB against C. albicans growth in other media, such as in YPD medium and SC medium, and found that this agent showed similar inhibitory effect (data not shown). Therefore, this inhibitory effect is not medium-dependent.
3.2. The inhibitory effect of surfactants is associated with mitochondrial depolarization rather than PM damage The antimicrobial activity of surfactants is proposed to be related to PM damage [17,18]. However, PI staining revealed that 0–0.5 ppm CTAB had no impact on PM integrity, since few PI-positive cells were observed under these concentrations. Even the concentration reached to 8 ppm, less than 4% cells showed damaged PM (Fig. 2A). LDH assays further demonstrated that there was no significant difference in LDH activity between the supernatant of CTAB-treated cultures and that of the control culture when the concentrations of CTAB were 62 ppm (Fig. 2B), suggesting that CTAB at these concentrations did not lead to PM damage and enhanced LDH release from the fungal cells. Hence, PM damage seems not to be involved in the inhibitory action of CTAB on the growth of this pathogen. Due to the significance of the mitochondria on energy metabolism and growth in fungal cells, we next investigated the effect of CTAB on mitochondrial function. Consistent with the results in mammalian cells [14], CTAB caused severe mitochondrial depolarization in C. albicans. The percent of cells with decreased mitochondrial membrane potential (MMP) rose to 18.45% when the concentration of CTAB reached to 2 ppm (Fig. 2B). Similarly, treatment of SDS also enhanced mitochondrial depolarization (data not shown). Together, these results implied that mitochondrial mem-
Table 1 IC50 values of the surfactants against growth and morphogenesis of C. albicans. Type
CTAB IC50 (ppm)
SDS IC50 (ppm)
Growth Hyphal formation Biofilm formation Biofilm maintenance
0.758 ± 0.140 0.183 ± 0.031 0.888 ± 0.129 4.061 ± 0.775
45.961 ± 3.128 6.312 ± 2.335 76.092 ± 10.426 >160
brane depolarization is responsible for surfactant-induced cytotoxicity in C. albicans.
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3.3. The surfactants strongly inhibit C. albicans morphogenesis
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Hyphal development is an important determinant for C. albicans to successfully invade host tissues [3,4]. We further tested the effect of the surfactants on this process. Excitingly, both surfactants exhibited stronger activity against hyphal development than against growth (Fig. 3A). The IC50 against hyphal development were only 0.183 ppm for CTAB and 6.312 ppm for SDS (Table 1). Hence, hyphal development is much more sensitive to the surfactants than growth, indicating high efficiency of the surfactants against C. albicans hyphal development. Biofilm is another morphogenetic type of C. albicans, which is associated with implant-related infections and resistance to antifungal drugs [19,20]. Since hyphal development is involved in biofilm formation [21], we hypothesized that the surfactants may also affect biofilm development. As expected, both CTAB and SDS strongly inhibited biofilm formation, and abundant yeast cells were observed under these treatments (Fig. 3B), suggesting that the defect in hyphal development caused by the surfactants was involved in the inhibition of biofilm formation. Moreover, these two surfactants also drastically reduced the activity of pre-formed biofilms. The IC50 against biofilm formation and biofilm maintenance were 0.888 ppm and 4.061 ppm for CTAB, 76.092 ppm and >160 ppm for SDS (Table 1). Therefore, both surfactants, especially CTAB, exerted inhibitory effect on C. albicans morphogenesis.
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3.4. The surfactants inhibit polarized organization of actin skeleton
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Next we explored possible mechanisms by which the surfactants inhibited C. albicans morphogenesis. Polarized organization of actin skeleton is essential for transport of hyphae-related factors to the tip and consequent hyphal development [4,22]. This organization requires the localization of cortical markers at the tip membrane and subsequent formation of polarisome adhering to the PM [23]. Due to the possible interaction between the surfactants and PM components, we hypothesized that the surfactants may disrupt
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Fig. 1. Effect of the surfactants CTAB (A) and SDS (B) on growth of C. albicans. Cells were cultured in RPMI-1640 medium containing different concentrations of the surfactants in 96-well polystyrene microplate wells. After 24 h of incubation, OD600 of the cultures in each well was then determined. The values represent mean ± SD from five replications. ⁄Significant difference between the control group (0) and the surfactant-treated groups (P < 0.05).
Please cite this article in press as: Q. Yu et al., Novel mechanisms of surfactants against Candida albicans growth and morphogenesis, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.12.014
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Fig. 2. Effect of CTAB on C. albicans PM integrity (A, B) and MMP (C). (A) The fungal cells were treated by CTAB for 24 h, stained with PI and observed. PI-positive and total cells in each group were quantified, and the percent of PI-positive cells for each treatment was calculated. The values represent mean ± SD from five replications. (B) LDH activity of the supernatant of the CTAB-treated cultures. (C) The CTAB-treated cells were stained by JC-1 and detected using flow cytometry. Fluorescence intensity of JC-1 aggregates (red, FL-1) and monomers (green, FL-2) were recorded. The percent of cells with decreased MMP was indicated in the right-down parts. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Effect of the surfactants on hyphal development (A) and biofilm formation (B). (A) C. albicans cells were cultured in RPMI-1640 medium containing the surfactants or not (Control) at 37 °C for 4 h. Cells were harvested and observed by a light microscope. Bar = 20 lm. (B) C. albicans biofilms were cultured in polystyrene microtiter plates containing the surfactants or not (Control) at 37 °C for 24 h. The samples were then dried, coated with gold and observed using SEM. Bar = 20 lm.
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the normal structure and function of the tip membrane, causing mislocation of cortical markers and polarisome, followed by defect in polarized organization of actin skeleton. To verify this, we detected actin skeleton distribution in both the control cells and those treated by the surfactants. As expected, while the control cells showed normal polarized distribution of actin at the hyphal tip, the cells treated by CTAB or SDS failed in tip localization of actin (Fig. 4A). Therefore, the surfactants disrupted the polarized organization of actin skeleton, which may be associated with their
inhibitory effect on hyphal development and consequent biofilm formation.
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3.5. The surfactants inhibit polarized transport of Hwp1
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Hwp1 is a well-characterized hyphal cell wall protein of C. albicans, which is highly expressed and transported to the site of polarized growth in an actin-dependent manner during hyphal development [6]. To further confirm the inhibitory effect of the
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Please cite this article in press as: Q. Yu et al., Novel mechanisms of surfactants against Candida albicans growth and morphogenesis, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.12.014
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Fig. 4. Effect of the surfactants on polarized organization of actin skeleton (A) and polarized transport of Hwp1 (B). (A) C. albicans cells were cultured in RPMI-1640 medium containing the supernatants or not (Control) for 4 h, stained with rhodamine phalloidin and observed by fluorescence microscopy. Bar = 5 lm. (B) C. albicans cells containing Hwp1-GFP were cultured in RPMI-1640 medium containing the supernatant or not (Control) for 4 h and observed. Bar = 50 lm.
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surfactants on polarized growth, we examined the localization of Hwp1 in the strains expressing Hwp1-GFP fusion protein. In the control cells, Hwp1-GFP was regularly distributed at the hyphal cell wall and accumulated at the hyphal tip, indicating a normal polarized transport of this protein during hyphal development. In contrast, both CTAB and SDS severely disrupted the distribution of Hwp1-GFP at both the tip and the cell wall (Fig. 4B). Hence, the surfactants strongly inhibit the transport of Hwp1 to the tip and the cell wall. In addition, the surfactant-treated cells showed attenuated fluorescence density of Hwp1-GFP (Fig. 4B), suggesting that the surfactants not only inhibit polarized transport, but also have an impact on expression of hyphae-related genes.
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4. Discussion
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The surfactants are broad-spectrum antimicrobial agents widely used in agriculture, medicine and industry. However, the mechanisms how these agents fight against microbes remain to be elucidated. Traditionally, the activity of disrupting biological membrane structure is proposed to be involved in their toxicity to these organisms [17,18]. Further study revealed that these agents inhibit the activity of antioxidant enzymes, resulting intracellular accumulation of superoxide and hydrogen peroxide. This suggested that reactive oxygene species (ROS) may be associated with the antimicrobial action of surfactants [24]. In this study, we demonstrated that the tested surfactants, CTAB and SDS, showed inhibitory effect on C. albicans growth, and found that these agents caused mitochondrial depolarization rather than PM damage. Hence, mitochondrial dysfunction may also be responsible for the activity of these agents to inhibit eukaryotic microorganisms. As demonstrated above, the tested surfactants have a drastic impact on MMP. Since the possible action of the surfactants to disrupt biological membranes, we speculate that these agents may enter into fungal cells and interact with the mitochondrial membrane, leading to dissipation of proton gradient across the inner
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membrane and consequent decrease of MMP. This speculation is supported by Ito’s reports that CATB may prompt apoptosis of cancer cells by disrupting mitochondrial membranes [14]. However, we cannot rule out the possible role of ROS in reduction of MMP. Evidence has demonstrated that the mitochondria is one of the most important target of intracellular ROS [25,26]. The surfactants may cause accumulation of ROS, similar to Nakata1’s findings [24], followed by decline of MMP. Further studies in the possible relationship between surfactant treatment and oxidative stress would yield definitive conclusions. In C. albicans, morphogenetic processes, such as hyphal development and biofilm formation, play important roles in proliferation, invasion of host tissues and resistance to environmental stress. Therefore, inhibition of morphogenesis would be an effective option for treating C. albicans-related infections [7]. This study clearly revealed that the surfactant CTAB, even at low concentrations, has strong inhibitory effect on C. albicans morphogenesis, and this effect is associated with disturbance of polarized growth. Hence, the polarized system of this pathogen may be an attractive target to inhibit its morphogenetic processes and reduce the risk of related infections. Although the strong inhibitory activity of the tested surfactants (especially CTAB) against C. albicans growth and morphogenesis sheds novel light on antifungal strategies, it should be paid attention to the side effects of these agents on human bodies. For example, these agents have demonstrated to cause contact dermatitis and mucous irritation in vulnerable individuals [17,27]. The effective concentrations of surfactants against C. albicans may be not endurable for these patients. Hence, there is a need to explore other effective and safe surfactants to inhibit C. albicans-related infections. Nevertheless, CTAB showed a strong inhibitory effect against normal mammalian cells only at relative high concentrations (EC50 = 4.0–6.5 ppm) [14]. Hence, CTAB at the concentrations
No. of Pages 6, Model 5G
16 December 2014 Chemico-Biological Interactions xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint 5 6
Novel mechanisms of surfactants against Candida albicans growth and morphogenesis
3 4 7 8 9 10 11 12 1 2 4 6 15 16 17 18 19 20 21 22 23 24 25
Q1
Qilin Yu a, Bing Zhang a, Feiyang Ma a, Chang Jia a, Chenpeng Xiao a, Biao Zhang b, Laijun Xing a, Mingchun Li a,⇑ a b
Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Department of Microbiology, College of Life Science, Nankai University, Tianjin 300071, PR China Tianjin Traditional Chinese Medicine University, Tianjin 300193, PR China
a r t i c l e
i n f o
Article history: Received 6 September 2014 Received in revised form 25 November 2014 Accepted 5 December 2014 Available online xxxx Keywords: Surfactant Candida albicans Morphogenesis Polarized growth
a b s t r a c t Candida albicans is a common opportunistic fungal pathogen, causing not only superficial mucosal infections but also life-threatening systemic candidiasis in immune-compromised individuals. Surfactants are a kind of amphiphilic compounds implemented in a wide range of applications. Although their antimicrobial activity has been characterized, their effect on C. albicans physiology remains to be elucidated. In this study, we investigated the inhibitory effect of two representative surfactants, cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), on C. albicans growth and morphogenesis. Both surfactants exhibited inhibitory effect on C. albicans growth. This effect was not attributed to plasma membrane (PM) damage, but was associated with mitochondrial dysfunction. Excitingly, the surfactants, especially CTAB, showed strong inhibitory effect on hyphal development (IC50 = 0.183 ppm for CTAB and 6.312 ppm for SDS) and biofilms (0.888 ppm for CTAB and 76.092 ppm for SDS). Actin staining and Hwp1-GFP localization further revealed that this inhibition is related to abnormal organization of actin skeleton and subsequent defect in polarized transport of hyphae-related factors. This study sheds a novel light on the antimicrobial mechanisms of surfactants, and suggests these agents as potential drugs against C. albicans hyphae-related infections in clinical practice. Ó 2014 Published by Elsevier Ireland Ltd.
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1. Introduction
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Candida albicans is a common opportunistic fungal pathogen, causing not only superficial mucosal infections but also life-threatening systemic candidiasis [1,2]. One of the most important properties of this pathogen is its ability of hyphal development under certain environmental stimuli, such as amino acids, low nitrogen source, serum and hypoxia [3]. Hyphal development is governed by a complicated signaling network that elaborately senses the stimuli and activates expression of hyphae-related genes, such as HWP1 [4,5]. Their products are then transported from the endoplasmic reticulum to the hyphal tip and provide materials for hyphal elongation [6]. Since the significance of hyphal development during invading host tissues, the system that mediates this process is a potential target against C. albicans infections [7,8]. Surfactants, also named surface active agents, are a kind of compounds containing both hydrophobic groups and hydrophilic groups. Due to the amphiphilic property, they are implemented
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in a wide range of applications [9–11]. Especially, these compounds are widely used as broad-spectrum antimicrobial agents [12,13]. Moreover, evidence suggests that these agents, in spite of their potential toxicity to mammalian cells, have potential use as anticancer agents [14]. Therefore, the surfactants may be developed as novel drugs in clinical practice. Cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), two representative surfactants, are widely used in biological researches. In this study, we investigated their effect on C. albicans growth and morphogenesis, and found that both agents had much stronger inhibitory effect on hyphal development than on growth. Furthermore, this inhibition is related to abnormal organization of actin skeleton and subsequent defect in transport of hyphae-related factors. Our findings suggested that the surfactants may be potential agents against C. albicans infections.
⇑ Corresponding author. E-mail address: [email protected] (M. Li). http://dx.doi.org/10.1016/j.cbi.2014.12.014 0009-2797/Ó 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Q. Yu et al., Novel mechanisms of surfactants against Candida albicans growth and morphogenesis, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.12.014
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2.1. Agents, strains and growth conditions
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The surfactants used in this study were purchased from Sigma, USA. The stock solutions of the agents were prepared in liquid RPMI-1640 medium (RPMI-1640 powder (Sigma, USA) 1%, 3-(Nmorpholino) propanesulfonic acid (Sigma, USA) 0.418%, uridine 80 ppm, pH 7.4) at the initial concentration of 1000 ppm, and diluted with the same medium to certain concentrations. Normally, the wild-type C. albicans strain BWP17 was used for growth inhibition tests, hyphal induction and biofilm assays. To investigate the effect of the surfactants on polarized transport of Hwp1, the strain NKF152 containing the Hwp1-GFP fusion gene (based on the plasmid pMG2082) was used [27]. The strains were overnight cultured in liquid YPD medium (yeast extract 1%, peptone 2%, glucose 2%) with shaking at 30 °C, and then used for further experiments.
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2.2. Growth inhibition assays To investigate the inhibitory effect of the surfactants on C. albicans growth, overnight cultured cells were washed twice with PBS, and suspended in RPMI-1640 medium to an initial optical density at 600 nm (OD600) of 0.2. 50 lL of the suspension were added into 96-well polystyrene microplate wells, and then 50 lL of surfactant solutions were mixed with the cell suspensions, obtaining the mixture containing the yeast cells with the OD600 of 0.1 and the surfactants with the following concentrations, 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 ppm for CTAB, and 0, 1.25, 2.5, 5, 10, 20, 40, 80, 160 ppm for SDS. The plates were covered with their lids, sealed with parafilm and incubated at 37 °C for 24 h. The OD600 of each well was then determined using a microplate reader (Bio-Rad, USA). The percent of growth for each treatment was calculated as the OD600 of each surfactant-treated group divided by the control (without the treatment of surfactants). The growth inhibition tests were also performed in liquid YPD medium (yeast extract 1%, peptone 2%, glucose 2%) and SC medium (yeast nitrogen base 0.67%, glucose 2%, amino acid mixture 0.2%) to evaluate the effect of media in CTAB activity.
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2.3. PM damage assays
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To evaluate the effect of CTAB on PM integrity, overnight cultured fungal cells were suspended in fresh YPD medium containing CTAB with the following concentrations, 0, 0.25, 0.5, 1, 2, 4, 8 ppm. The cells were cultured with shaking at 30 °C for 24 h and stained with PI (final concentration 5 ppm, Sigma, USA) for observation. At least 30 fields were observed. PI-positive and total cells in each group were quantified. The percent of PI-positive cells for each treatment was then calculated as the number of PI-positive cells divided by that of total cells. PM integrity was also evaluated by lactate dehydrogenase (LDH) assays. The CTAB-treated cultures were centrifuged at 12,000 rpm for 10 min, and the supernatant was used for determining LDH activity by an LDH Assay Kit (KEYGEN BIOTECH, China).
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2.4. Measurement of mitochondrial membrane potential (MMP)
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CTAB-treated cells were washed and suspended with PBS and stained with JC-1 (final concentration 1 ppm, Sigma, USA) for 30 min. Fluorescence intensity of JC-1 aggregates (red, FL-1) and monomers (green, FL-2) were recorded by a flow cytometer (FACSCalibur, BD, USA). The cells exhibiting decreased red fluorescence density than the control (untreated) cells were considered as the
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cells with decreased MMP, and the percent of cells with decreased MMP was recorded.
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2.5. Hyphal induction
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C. albicans hyphae were induced as described previously [15]. Briefly, overnight cultured cells were washed with PBS and suspended in RPMI-1640 medium containing the surfactants with the indicated concentrations. The suspensions were then incubated with shaking at 37 °C for 4 h. Cells were harvested, fixed with 4% formaldehyde, and observed by a light microscope (Olympus, Japan). The number of hyphal cells and total cells was counted in each field, and the percent of hyphal cells were calculated as the number of hyphal cells divided by the number of total cells. At least 30 fields were determined.
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2.6. Biofilm assays
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C. albicans biofilms were cultured in polystyrene microtiter plates (Corning Inc., USA). Overnight cultured cells were washed with PBS and suspended in RPMI-1640 medium containing the surfactants at different concentrations, with the OD600 of 0.1. 100 lL of the suspensions were then added into 96-well microtiter plates. The plates were covered with lids and incubated at 37 °C. After 24 h of incubation, the plates were washed 3 times with PBS, and the biofilm activity in each well was detected by XTT (2,3-bis(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assays [16]. To observe biofilm structures, the cultured biofilms were washed with PBS and fixed with 3% glutaraldehyde. The samples were then sectioned, dehydrated with ethanol, dried in vacuum desiccators, coated with gold and observed by a scanning electron microscope (SEM, QUANTA 200, Czech). To evaluate the effect of surfactants on biofilm maintenance, the biofilms were pre-formed as described above. The wells were then washed with PBS, and 100 lL RPMI-1640 medium containing the surfactants with different concentrations were added into the wells. The plates were covered with their lids and incubated at 37 °C for further 24 h. The metabolic activity of biofilms was also measured by the XTT reduction assay.
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2.7. Actin staining
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To observe actin skeleton, the C. albicans cells were washed twice with PBS, fixed with 4% formaldehyde for 10 min, permeabilized with 0.5% Triton 100 for 30 min and washed again with PBS. 5 lL of rhodamine phalloidin (final concentration 1 ppm, Cytoskeleton, USA) were then added into 100 lL of the cells. The cells were incubated at the room temperature for 30 min, and washed 3 times with PBS for observation.
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2.8. Fluorescence microscopy
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The PI-stained and rhodamine phalloidin-stained cells were observed by a fluorescence microscope (Olympus, USA) using the RFP filter set. To detect Hwp1 localization, the strain NKF152 was cultured in RPMI-1640 containing the surfactants as described above, and Hwp1-GFP was observed by the fluorescence microscope using the GFP filter set.
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2.9. Statistical analysis
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The experiments were performed with five replicates, and the values represent the means ± standard deviations (SD). Significant differences between the treatments were determined using oneway ANOVA (P < 0.05). Statistical analysis and IC50 calculation were performed by SPSS (Version 20, IBM, USA).
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3. Results
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3.1. The surfactants exhibit a distinct inhibitory effect on C. albicans growth
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Surfactants are well-known antimicrobial agents. To investigate whether the agents have antifungal activity, we first tested the growth biomass of C. albicans under treatment of two widely-used surfactants, CTAB and SDS. Liquid sensitivity tests showed that CTAB concentrations P0.5 ppm and SDS concentrations P40 ppm led to significant inhibition of fungal growth (Fig. 1A and B). Notably, high concentrations of CTAB (P1 ppm) resulted in >80% decrease in growth biomass (Fig. 1A). Although both surfactants showed expected inhibitory effect on C. albicans growth, CTAB (IC50 = 0.758 ppm) had much stronger inhibitory effect than SDS (IC50 = 45.961 ppm) (Table 1). Therefore, the inhibitory activity against C. albicans growth is distinct for these two agents. In addition, we also tested the effect of CTAB against C. albicans growth in other media, such as in YPD medium and SC medium, and found that this agent showed similar inhibitory effect (data not shown). Therefore, this inhibitory effect is not medium-dependent.
3.2. The inhibitory effect of surfactants is associated with mitochondrial depolarization rather than PM damage The antimicrobial activity of surfactants is proposed to be related to PM damage [17,18]. However, PI staining revealed that 0–0.5 ppm CTAB had no impact on PM integrity, since few PI-positive cells were observed under these concentrations. Even the concentration reached to 8 ppm, less than 4% cells showed damaged PM (Fig. 2A). LDH assays further demonstrated that there was no significant difference in LDH activity between the supernatant of CTAB-treated cultures and that of the control culture when the concentrations of CTAB were 62 ppm (Fig. 2B), suggesting that CTAB at these concentrations did not lead to PM damage and enhanced LDH release from the fungal cells. Hence, PM damage seems not to be involved in the inhibitory action of CTAB on the growth of this pathogen. Due to the significance of the mitochondria on energy metabolism and growth in fungal cells, we next investigated the effect of CTAB on mitochondrial function. Consistent with the results in mammalian cells [14], CTAB caused severe mitochondrial depolarization in C. albicans. The percent of cells with decreased mitochondrial membrane potential (MMP) rose to 18.45% when the concentration of CTAB reached to 2 ppm (Fig. 2B). Similarly, treatment of SDS also enhanced mitochondrial depolarization (data not shown). Together, these results implied that mitochondrial mem-
Table 1 IC50 values of the surfactants against growth and morphogenesis of C. albicans. Type
CTAB IC50 (ppm)
SDS IC50 (ppm)
Growth Hyphal formation Biofilm formation Biofilm maintenance
0.758 ± 0.140 0.183 ± 0.031 0.888 ± 0.129 4.061 ± 0.775
45.961 ± 3.128 6.312 ± 2.335 76.092 ± 10.426 >160
brane depolarization is responsible for surfactant-induced cytotoxicity in C. albicans.
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3.3. The surfactants strongly inhibit C. albicans morphogenesis
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Hyphal development is an important determinant for C. albicans to successfully invade host tissues [3,4]. We further tested the effect of the surfactants on this process. Excitingly, both surfactants exhibited stronger activity against hyphal development than against growth (Fig. 3A). The IC50 against hyphal development were only 0.183 ppm for CTAB and 6.312 ppm for SDS (Table 1). Hence, hyphal development is much more sensitive to the surfactants than growth, indicating high efficiency of the surfactants against C. albicans hyphal development. Biofilm is another morphogenetic type of C. albicans, which is associated with implant-related infections and resistance to antifungal drugs [19,20]. Since hyphal development is involved in biofilm formation [21], we hypothesized that the surfactants may also affect biofilm development. As expected, both CTAB and SDS strongly inhibited biofilm formation, and abundant yeast cells were observed under these treatments (Fig. 3B), suggesting that the defect in hyphal development caused by the surfactants was involved in the inhibition of biofilm formation. Moreover, these two surfactants also drastically reduced the activity of pre-formed biofilms. The IC50 against biofilm formation and biofilm maintenance were 0.888 ppm and 4.061 ppm for CTAB, 76.092 ppm and >160 ppm for SDS (Table 1). Therefore, both surfactants, especially CTAB, exerted inhibitory effect on C. albicans morphogenesis.
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3.4. The surfactants inhibit polarized organization of actin skeleton
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Next we explored possible mechanisms by which the surfactants inhibited C. albicans morphogenesis. Polarized organization of actin skeleton is essential for transport of hyphae-related factors to the tip and consequent hyphal development [4,22]. This organization requires the localization of cortical markers at the tip membrane and subsequent formation of polarisome adhering to the PM [23]. Due to the possible interaction between the surfactants and PM components, we hypothesized that the surfactants may disrupt
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Fig. 1. Effect of the surfactants CTAB (A) and SDS (B) on growth of C. albicans. Cells were cultured in RPMI-1640 medium containing different concentrations of the surfactants in 96-well polystyrene microplate wells. After 24 h of incubation, OD600 of the cultures in each well was then determined. The values represent mean ± SD from five replications. ⁄Significant difference between the control group (0) and the surfactant-treated groups (P < 0.05).
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Fig. 2. Effect of CTAB on C. albicans PM integrity (A, B) and MMP (C). (A) The fungal cells were treated by CTAB for 24 h, stained with PI and observed. PI-positive and total cells in each group were quantified, and the percent of PI-positive cells for each treatment was calculated. The values represent mean ± SD from five replications. (B) LDH activity of the supernatant of the CTAB-treated cultures. (C) The CTAB-treated cells were stained by JC-1 and detected using flow cytometry. Fluorescence intensity of JC-1 aggregates (red, FL-1) and monomers (green, FL-2) were recorded. The percent of cells with decreased MMP was indicated in the right-down parts. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Effect of the surfactants on hyphal development (A) and biofilm formation (B). (A) C. albicans cells were cultured in RPMI-1640 medium containing the surfactants or not (Control) at 37 °C for 4 h. Cells were harvested and observed by a light microscope. Bar = 20 lm. (B) C. albicans biofilms were cultured in polystyrene microtiter plates containing the surfactants or not (Control) at 37 °C for 24 h. The samples were then dried, coated with gold and observed using SEM. Bar = 20 lm.
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the normal structure and function of the tip membrane, causing mislocation of cortical markers and polarisome, followed by defect in polarized organization of actin skeleton. To verify this, we detected actin skeleton distribution in both the control cells and those treated by the surfactants. As expected, while the control cells showed normal polarized distribution of actin at the hyphal tip, the cells treated by CTAB or SDS failed in tip localization of actin (Fig. 4A). Therefore, the surfactants disrupted the polarized organization of actin skeleton, which may be associated with their
inhibitory effect on hyphal development and consequent biofilm formation.
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3.5. The surfactants inhibit polarized transport of Hwp1
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Hwp1 is a well-characterized hyphal cell wall protein of C. albicans, which is highly expressed and transported to the site of polarized growth in an actin-dependent manner during hyphal development [6]. To further confirm the inhibitory effect of the
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Fig. 4. Effect of the surfactants on polarized organization of actin skeleton (A) and polarized transport of Hwp1 (B). (A) C. albicans cells were cultured in RPMI-1640 medium containing the supernatants or not (Control) for 4 h, stained with rhodamine phalloidin and observed by fluorescence microscopy. Bar = 5 lm. (B) C. albicans cells containing Hwp1-GFP were cultured in RPMI-1640 medium containing the supernatant or not (Control) for 4 h and observed. Bar = 50 lm.
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surfactants on polarized growth, we examined the localization of Hwp1 in the strains expressing Hwp1-GFP fusion protein. In the control cells, Hwp1-GFP was regularly distributed at the hyphal cell wall and accumulated at the hyphal tip, indicating a normal polarized transport of this protein during hyphal development. In contrast, both CTAB and SDS severely disrupted the distribution of Hwp1-GFP at both the tip and the cell wall (Fig. 4B). Hence, the surfactants strongly inhibit the transport of Hwp1 to the tip and the cell wall. In addition, the surfactant-treated cells showed attenuated fluorescence density of Hwp1-GFP (Fig. 4B), suggesting that the surfactants not only inhibit polarized transport, but also have an impact on expression of hyphae-related genes.
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4. Discussion
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The surfactants are broad-spectrum antimicrobial agents widely used in agriculture, medicine and industry. However, the mechanisms how these agents fight against microbes remain to be elucidated. Traditionally, the activity of disrupting biological membrane structure is proposed to be involved in their toxicity to these organisms [17,18]. Further study revealed that these agents inhibit the activity of antioxidant enzymes, resulting intracellular accumulation of superoxide and hydrogen peroxide. This suggested that reactive oxygene species (ROS) may be associated with the antimicrobial action of surfactants [24]. In this study, we demonstrated that the tested surfactants, CTAB and SDS, showed inhibitory effect on C. albicans growth, and found that these agents caused mitochondrial depolarization rather than PM damage. Hence, mitochondrial dysfunction may also be responsible for the activity of these agents to inhibit eukaryotic microorganisms. As demonstrated above, the tested surfactants have a drastic impact on MMP. Since the possible action of the surfactants to disrupt biological membranes, we speculate that these agents may enter into fungal cells and interact with the mitochondrial membrane, leading to dissipation of proton gradient across the inner
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membrane and consequent decrease of MMP. This speculation is supported by Ito’s reports that CATB may prompt apoptosis of cancer cells by disrupting mitochondrial membranes [14]. However, we cannot rule out the possible role of ROS in reduction of MMP. Evidence has demonstrated that the mitochondria is one of the most important target of intracellular ROS [25,26]. The surfactants may cause accumulation of ROS, similar to Nakata1’s findings [24], followed by decline of MMP. Further studies in the possible relationship between surfactant treatment and oxidative stress would yield definitive conclusions. In C. albicans, morphogenetic processes, such as hyphal development and biofilm formation, play important roles in proliferation, invasion of host tissues and resistance to environmental stress. Therefore, inhibition of morphogenesis would be an effective option for treating C. albicans-related infections [7]. This study clearly revealed that the surfactant CTAB, even at low concentrations, has strong inhibitory effect on C. albicans morphogenesis, and this effect is associated with disturbance of polarized growth. Hence, the polarized system of this pathogen may be an attractive target to inhibit its morphogenetic processes and reduce the risk of related infections. Although the strong inhibitory activity of the tested surfactants (especially CTAB) against C. albicans growth and morphogenesis sheds novel light on antifungal strategies, it should be paid attention to the side effects of these agents on human bodies. For example, these agents have demonstrated to cause contact dermatitis and mucous irritation in vulnerable individuals [17,27]. The effective concentrations of surfactants against C. albicans may be not endurable for these patients. Hence, there is a need to explore other effective and safe surfactants to inhibit C. albicans-related infections. Nevertheless, CTAB showed a strong inhibitory effect against normal mammalian cells only at relative high concentrations (EC50 = 4.0–6.5 ppm) [14]. Hence, CTAB at the concentrations
