Matrine reduces yeast-to-hypha transition and resistance of a fluconazole-resistant strain of Candida albicans

Journal of Applied MicrobiologyVolume 117, Issue 3 p. 618-626 Original ArticleFree Access Matrine reduces yeast-to-hypha transition and resistance of a fluconazole-resistant strain of Candida albicans J. Shao, J. Shao Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, ChinaSearch for more papers by this authorT. Wang, T. Wang Laboratory of Biochemistry and Molecular Biology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, ChinaSearch for more papers by this authorY. Yan, Y. Yan Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, ChinaSearch for more papers by this authorG. Shi, G. Shi Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, ChinaSearch for more papers by this authorH. Cheng, H. Cheng Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, ChinaSearch for more papers by this authorD. Wu, D. Wu Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, ChinaSearch for more papers by this authorC. Wang, Corresponding Author C. Wang Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, China Correspondence Changzhong Wang, Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei 230038, China. E-mail: wangchangzhong53@126.comSearch for more papers by this author J. Shao, J. Shao Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, ChinaSearch for more papers by this authorT. Wang, T. Wang Laboratory of Biochemistry and Molecular Biology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, ChinaSearch for more papers by this authorY. Yan, Y. Yan Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, ChinaSearch for more papers by this authorG. Shi, G. Shi Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, ChinaSearch for more papers by this authorH. Cheng, H. Cheng Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, ChinaSearch for more papers by this authorD. Wu, D. Wu Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, ChinaSearch for more papers by this authorC. Wang, Corresponding Author C. Wang Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei, China Correspondence Changzhong Wang, Laboratory of Microbiology and Immunology, School of Chinese and Western Integrative Medicine, Anhui University of Chinese Medicine, Hefei 230038, China. E-mail: wangchangzhong53@126.comSearch for more papers by this author First published: 26 May 2014 https://doi.org/10.1111/jam.12555Citations: 24AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Aims To evaluate the potential effect of matrine on reducing the growth of hypha and lowering the resistance of a fluconazole-resistant colony of Candida albicans. Methods and Results Candida albicans SC5314 and a fluconazole-resistant C. albicans 215 were used. As for C. albicans SC5314, minimal inhibitory concentration (MIC80) and effective concentration (EC50) were determined, 1 mg ml−1 matrine could inhibit nearly 80% of planktonic growth by inverted microscope, 2 mg ml−1 matrine suppressed 50% of metabolic activity of biofilm by XTT assay, vanishing hypha could be observed on spider agar containing 2 mg ml−1 matrine, the expressions of three hypha-related genes, namely ALS 3, SUN 41 and PBS 2, were suppressed by 29, 45 and 61% by 2 mg ml−1 matrine. Also, matrine could lower the resistance of C. albicans 215, in either the free-floating form or the biofilm phenotype. Conclusions Matrine had favourable antifungal potential and might be able to reverse the fluconazole resistance of clinical isolates at relatively high concentration. The anti-candidal performance of matrine could be tightly associated with yeast-to-hypha transition proved by spider agar test and qRT-PCR. Significance and Impact of Study More efforts are needed to find new antifungal agents. Matrine could be a potential candidate to fight against Candida-related infections by regulating yeast-to-hypha transition. Introduction Candida albicans is a commonly benign commensal and also an important opportunistic pathogen if its living loci and environment are changed or the host immunity is suppressed in immunocompromised individuals (Gow et al. 2012). Due to overuse of conventional antifungal agents, C. albicans has become more difficult to cure in clinical Candida infections, especially candidiasis and catheter-related disseminated candidemia, with higher morbidity and mortality. Current antifungal agents include azoles (such as fluconazole and itraconazole; Uppuluri et al. 2011; Nweze et al. 2012), echinocandins (such as caspofungin and anidulafungin; Katragkou et al. 2011; Wiederhold et al. 2011) and several ‘nonantibiotic agents’ (such as ethanol, EDTA, phytocompounds; Ramage et al. 2007; Khan and Ahmad 2012; Rane et al. 2012). However, many isolated clinical strains of C. albicans were found to be fluconazole-resistant, making conventional anti-candidal drugs futile. Therefore, more anti-candidal agents with low toxicity and high efficiency are urgently needed (Tobudic et al. 2012). Plant-originated compounds have been considered to be the huge antifungal bank with a broad spectrum of infection-inhibited functions (Dixon 2001). Some of those agents have been tested to be promising candidates for future clinical treatment against biofilm, including tetrandrine (Zhang et al. 2010), the derivative of andrographolide (Zeng et al. 2011), etc. Matrine is a quinolizidine alkaloid of such phytoanticipin, originating from the root of Chinese Sophora herb plants with multiple purposes, including antiviral (Yang et al. 2012), antitumor (Wang et al. 2012), anti-inflammatory (Zhang et al. 2011) functions, etc. Although matrine and its derivative oxymatrine were observed to be effective against pathogenic forest fungi (Yang and Zhao 2006), anti-Candidal potential of matrine has not been reported. The invasive hypha is one of the most virulent forms of C. albicans, because it can help the pathogen to penetrate epithelial and endothelial cells, colonize and disperse into the bloodstream (Dalle et al. 2010; Zhu and Filler 2010). Moreover, it can escape from the engulfment of macrophage (Lorenz et al. 2004). Yeast-to-hypha transition is controlled by quorum sensing (QS) molecule, which is closely related to biofilm formation. The biofilm resistant to most conventional antifungals is a self-protection architecture of sessile cells, enclosed by extracellular polymeric substances (Ramage et al. 2002). It is proposed that the suppression or disruption of morphological changes of C. albicans from yeast to hypha presents a unique antifungal strategy (Gauwerky et al. 2009). In this study, we demonstrated that matrine suppressed planktonic cells and biofilms of C. albicans by inhibiting yeast-to-hypha transition by XTT assay, qRT-PCR and inverted microscope. A fluconazole-resistant C. albicans isolate was treated with matrine and showed that matrine reversed the fluconazole resistance. Materials and methods Organism, medium and chemicals Candida albicans SC5314 was obtained from the Centers for Disease Control and Prevention (CDC) of Hefei (Anhui, China). Clinical isolate 215 was donated by Dr. Yuanying Jiang's laboratory, College of Pharmacy, Second Military Medical University, Shanghai, China. Sabouraud dextrose agar medium (10 g l−1 peptone, 20 g l−1 agar, 40 g l−1 glucose, 0·1 g l−1 chloramphenicol), RPMI-1640 liquid medium and spider agar (1% mannite, 1% nutrient broth, 0·2% K2HPO4, 1·35% agar) were all obtained from Gibco (Invitrogen, Carlsbad, CA). Matrine powder (only for experiments) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, Beijing, China). Sodium chloride, potassium chloride, calcium chloride, sodium bicarbonate, menadione and acetone were all analytical grade (AG, Alexis, Lausen, Switzerland). qRT-PCR package was got from ToyoBo (Tokyo, Japan). XTT {2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide} was purchased from BBI (Amherst, MA). Strain broth The isolates were subcultured on Sabouraud dextrose agar for 24 h at 37°C and stored at 4°C for use. A single colony was picked, inoculated into RPMI-1640 liquid medium and cultured in an orbital shaker (Fuma, Shanghai, China) at 200 rev min−1 37°C for 24 h. Then, an aliquot of 1 ml cultivated strain solution was added into fresh 100 ml RPMI-1640 liquid medium for propagation at 37°C for another 12–18 h. MIC80 and EC50 tests The MIC80 of fluconazole and matrine (Fig. 1) were determined by broth micro-dilution method according to CLSI M27-A2 with small modifications. Briefly, the original stocks of fluconazole and matrine were 32 and 24 mg ml−1, respectively. For MIC80 and EC50 of C. albicans SC5314, the stock solution was serially diluted to the final concentrations from 0·0000625 to 0·032 mg ml−1 for fluconazole, and 0·01563 to 8 mg ml−1 for matrine with 10 gradients. For MIC80 and EC50 of C. albicans 215, the stock solution was serially diluted to the final concentrations from 0·03125 to 6 mg ml−1 for fluconazole, and 0·02344 to 12 mg ml−1 for matrine with also 10 gradients. Both MIC80 and EC50 tests were performed in a 96-well polystyrene microtitre plate (Corning, NY). Each well had 100 μl RPMI-1640 medium containing drugs and 100 μl strain culture with the final inoculum size of 1 × 103 CFU ml−1 for MIC tests at 37°C for 48 h and 1 × 106 CFU ml−1 for EC50 tests at 37°C for 24 h. The negative control had only RPMI-1640 medium in each well, while the positive control had both the medium and strain without agents. The MIC80 was defined as the lowest concentrations of fluconazole or matrine to inhibit 80% growth of C. albicans compared with the positive control at the wavelength of 492 nm by a microtitre plate (Sanco Instrument Co., Shanghai, China; Arthington-Skaggs et al. 2002). The EC50 (50% effective concentration) was defined as the 50% reduction of OD492 compared to the positive control by XTT assay (Nett et al. 2008). Both MIC80 and EC50 were performed in triplicate and repeated for thrice. Figure 1Open in figure viewerPowerPoint The chemical structure of matrine. XTT assay XTT assay was employed to detect the metabolic activity of Candida biofilm (Pierce et al. 2008). After incubation with matrine for 24 h, the supernatant was discarded and each well was washed twice by pH 7·2 PBS. A total of 50 mg XTT was dissolved with 100 ml Ringer's solution, and mixed with freshly menadione solution dissolved into acetone to the final concentration of 1·72 mg ml−1 at 5000 : 1 (v/v) immediately before use. The prepared XTT solution was sterilized by 0·22 μm millipore filter (Millipore, Billerica, MA) and then added to pre-washed wells and incubated for 2 h at 37°C. Subsequently, 100 μl coloured solution was transferred to other 96-well plate, and the OD was measured at a wavelength of 492 nm by a 318-microplate reader (Sanco Instruments, Shanghai, China). Each test was performed in triplicate and repeated for thrice. Inverted microscope One hundred microlitre C. albicans (=1 × 103 CFU ml−1) treated separately by 100 μl matrine with the final concentrations of 0·01 mg ml−1, 0·1 mg ml−1, 1 mg ml−1 at 37°C for 48 h were observed by CKX41-32 inverted microscope (OLYMPUS, Tokyo, Japan) with ×400 magnitude. The photographs were processed by Photoshop CS3 (Adobe, San José, CA). Biofilm suppression of Candida albicans One hundred microlitre strain broth (approx. 1 × 106 CFU ml−1 formerly adjusted by hemocytometer) was added into 96-well polystyrene microtitre plate and co-incubated with 100 μl matrine solution to the final concentration of 0·1 mg ml-1, 0·5 mg ml−1 and 2 mg ml−1 for 24 h at 37°C. The metabolic activity was performed by XTT assay. Each assay was performed in triplicate and repeated for thrice. Hyphal inhibition of Candida albicans One hundred microlitre strain broth (approx. 1 × 102 CFU ml−1) was coated on sabouraud dextrose agar plate and spider agar plate with or without 2 mg ml−1 matrine for 4 days at 37°C, respectively. The morphological changes of Candida colony were recorded by digital camera with 8 million pixels (Olympus, Tokyo, Japan). Total RNA isolation and cDNA synthesis One millilitre strain broth (=1 × 106 CFU ml−1) was firstly transferred into a sterilized, flat-bottomed 24-well polystyrene microtitre plate (Corning, NY). After being mixed with 0·1 mg ml−1, 0·5 mg ml−1 and 2 mg ml−1 matrine at 37°C for 24 h, the cells were harvested by centrifuging at 1000 g for 5 min. The well without matrine treatment was set as control. Cell pellets were washed thrice by sterilized PBS and transferred into RNase-free screw-cap tubes. Total RNA was extracted using MagExtractor-RNA kit (ToyoBo, Tokyo, Japan). Six microlitre of the extracted total RNA was incubated with 2 μl 4× DNA Master: gDNA Remover and 2 μl 5RT-Master MixII and then reverse transcribed into cDNA by the following steps: initial RNA denaturation of 65°C for 5 min and 4°C for 1 min, then 50°C for 5 min, 98°C for 5 min and 4°C for 1 min according to ReverTra Ace qPCR RT Master Mix with gDNA Remover kit (ToyoBo). The prepared cDNA was diluted 10 × fold before use. All experiments were performed on ice. Primer design and quantitative real-time PCR analysis Primer Premier 5·0 was used for designing primers (ALS3, SUN41, PBS2, Table 1) which were synthesized by Sangon Biotech (Shanghai, China). Real-time PCR mixture (=25 μl) was composed of 12·5 μl 2 × SYBR Green Real-time PCR, 1 μl PCR Forward Primer, 1 μl PCR Reverse Primer, 0·5 μl cDNA and 10 μl ddH2O. The reaction was run on ABI7000 fluorescent quantitative PCR system (Applied Biosystem, Carlsbad, CA) with conditions as follows: initial step at 95°C for 60 s and then 40 cycles at 95°C for 15 s, 55°C for 15 s, 72°C for 45 s. All data were normalized to housekeeping gene ACT1 (inner reference), the internal reference gene. The relative target gene expression was calculated as a fold change of value, in which . Each sample was set in triplicate and repeated for thrice. Table 1. The primers of ALS 3, SUN 41, PBS 2 and ACT 1 for qRT-PCR Primer Sequence (5′-3′) ALS 3-f TAATGATGGTGGCAAGAA ALS 3-r TAGCGAATCCCATTGTAC SUN 41-f TGCTAAATCCGAAGAAAC SUN 41-r AACCACCAGTTGAAGATG PBS 2-f TACGCCAGAAGCAGATAC PBS 2-r TGAAGACCCAGACAGAAA ACT 1-f TTGATTTGGCTGGTAGAG ACT 1-r ATGGCAGAAGATTGAGAA The resistance reversion of a clinical isolate As for the free-floating form of C. albicans, 215, 100 μl strain culture and 100 μl RPMI-1640 medium with final concentrations of 0·25 mg ml−1 matrine + 0·06 mg ml−1 fluconazole, 0·5 mg ml−1 matrine + 0·06 mg ml−1 fluconazole, 1 mg ml−1 matrine + 0·06 mg ml−1 fluconazole, 2 mg ml−1 matrine + 0·06 mg ml−1 fluconazole, 3 mg ml−1 matrine + 0·06 mg ml−1 fluconazole were separately mixed with the final inoculum size of 1 × 103 CFU ml−1 for 48 h at 37°C in 96-well polystyrene microtitre plate. The well with only strain was set as negative control and that with only medium as positive control. The end point was determined as the lowest concentrations of fluconazole and matrine to induce 80% reduction of OD492 compared with the positive control by XTT assay. For biofilm of C. albicans, 215, 100 μl strain culture and 100 μl RPMI-1640 medium with final concentrations of 0·375 mg ml−1 matrine + 2 mg ml−1 fluconazole, 0·75 mg ml−1 matrine + 2 mg ml−1 fluconazole, 1·5 mg ml−1 matrine + 2 mg ml−1 fluconazole, 3 mg ml−1 matrine + 2 mg ml−1 fluconazole, 6 mg ml−1 matrine + 2 mg ml−1 fluconazole were separately mixed with the final inoculum size of 1 × 106 CFU ml−1 for 24 h at 37°C in a 96-well polystyrene microtitre plate. The end point was determined as the lowest concentrations of fluconazole and matrine to induce 50% reduction of metabolic activity compared with the positive control at the wavelength of 492 nm. The well with no drugs was set as positive control and that with only medium as negative control. Each test was performed in triplicate and repeated for thrice. Statistics All experimental data were analysed by spss 11.5 with expression of Mean ± standard deviation (SD). The comparison among groups adopted one-way analysis of variance (anova), and P < 0·05 was considered as significant. qRT-PCR results were graphed with GraphPad Prism (ver. 5.0). Results Matrine inhibits planktonic cell and biofilm phenotype of Candida albicans SC5314 The MIC80 and EC50 of matrine were 1 and 2 mg ml−1, respectively, as shown in Table 2. By inverted microscope, it was observed that the hyphal phase of C. albicans was shortened when matrine concentrations increased from 0·01 to 1 mg ml−1 (Fig. 2). In Fig. 2a, the visual field was filled with strip-type hyphal pathogens and round-shape yeasts when no matrine was added. After being incubated with 10 and 100 μg ml−1 matrines, respectively, the hyphal cells were under control, and yeast-state C. albicans was reduced considerably (Fig. 2b,c). Both yeast-state and hypha-state pathogens were suppressed to a large extent with relatively less hypha seen (Fig. 2d). Subsequently, antibiofilm potential of matrine was also performed. Compared with the negative control (no matrine), the metabolic activity of matrine slashed 50% when 2 mg ml−1 matrine was used (=EC50), the metabolic activities were reduced by approximately 12 and 16% when 0·1 and 0·5 mg ml−1 matrine were employed (Fig. 3), indicating the favourable inhibition of matrine on planktonic cells and biofilm phenotype of C. albicans at its relative high concentration. Table 2. Effects of matrine and fluconazole alone and in combination against Candida albicans SC5314 and 215 Agents MIC80 (mg ml−1) EC50 (mg ml−1) FICI for C. albicans 215 SC5314 215 SC5314 215 Planktonic cellaa FICI for planktonic cell is equal to (MICmatrine in combination/MICmatrine alone) + (MICfluconazole in combination/MICfluconazole alone), in which synergism was interpreted as FICI<0·5, indifference was defined as 0·5 < FICI ≤ 4·0, and antagonism was FICI > 4·0. Biofilmbb FICI for biofilm is equal to (ECmatrine in combination/ECmatrine alone) + (ECfluconazole in combination/ECfluconazole alone), in which the interpretations were determined as *. Matrine 1 3 2 6 0·197 0·5 Fluconazole 0·001 2 0·002 8 a FICI for planktonic cell is equal to (MICmatrine in combination/MICmatrine alone) + (MICfluconazole in combination/MICfluconazole alone), in which synergism was interpreted as FICI<0·5, indifference was defined as 0·5 < FICI ≤ 4·0, and antagonism was FICI > 4·0. b FICI for biofilm is equal to (ECmatrine in combination/ECmatrine alone) + (ECfluconazole in combination/ECfluconazole alone), in which the interpretations were determined as *. Figure 2Open in figure viewerPowerPoint Inverted microscope (×400) of Candida albicans treated by (a) 0 mg ml−1, (b) 0·01 mg ml−1, (c) 0·1 mg ml−1 and (d) 1 mg ml−1 matrine. The strain broth (=1 × 103 CFU ml−1) was co-incubated with different concentrations of matrine at 37°C for 48 h. Figure 3Open in figure viewerPowerPoint The influences of 0·1 mg ml−1, 0·5 mg ml−1 and 2 mg ml−1 matrine on Candida albicans by XTT reduction assay. The strain broth was adjusted to the final concentration of 1 × 106 CFU ml−1 and incubated with different concentrations of matrine for 24 h at 37°C. The control had only strain solution and medium but without matrine. **, P < 0·01, compared with the control. The inhibitory effect of matrine on hyphae formation in Candida albicans SC5314 From the morphology shown by inverted microscope (Fig. 2), it was inferred that matrine was likely to suppress hyphae formation, an essential existence responsible for biofilm development. Therefore, further studies were performed. Firstly, two mediums were used: one with the ordinary Sabouraud dextrose agar, the other with the spider agar in favour of hyphal growth. It was observed that C. albicans exhibited wrinkled appearances on both media when no matrine was added. Being compared Fig. 4b with Fig. 4a, it showed the developed colony on spider agar had much more notable wrinkles than that on Sabouraud dextrose agar, which appeared mainly in the central region of the colony. However, wrinkles of pathogenic colony disappeared when being co-incubated with 1 mg ml−1 matrine (Fig. 4c). Secondly, three hypha-related genes, that is, ALS 3, SUN 41 and PBS 2, were evaluated by qRT-PCR (Fig. 5). Under the same initial bacterial density (=106 CFU ml−1), 1 mg ml−1 matrine presented the strongest inhibitions on ALS 3 by 29% (Fig. 5a), SUN 41 by 45% (Fig. 5b) and PBS 2 by 61% (Fig. 5c) when using 1 mg ml−1 matrine. Both the morphological and qRT-PCR results all indicated that matrine presented anti-free-floating and antibiofilm forms of C. albicans by restricting hyphal development. Figure 4Open in figure viewerPowerPoint The photographs of (a) Sabouraud dextrose agar with no matrine, (b) spider agar with no matrine, (c) Sabouraud dextrose agar/spider agar with 2 mg ml−1 matrine to observe yeast-to-hypha transition of Candida albicans colony. The strain broth containing 1 × 102 CFU ml−1 strains was co-cultured with different agar mediums and 1 mg ml−1 matrine at 37°C for 4 days. Figure 5Open in figure viewerPowerPoint The fold changes of ALS3 (left), SUN41 (middle) and PBS2 (right) by qRT-PCR after the treatments of 0·1 mg ml−1, 0·5 mg ml−1 and 2 mg ml−1 matrine on Candida albicans (=1 × 106 CFU ml−1) co-incubated at 37°C for 24 h. Other experimental conditions could be seen in 2. *P < 0·05; **P < 0·01, compared with the control. Matrine reverses fluconazole resistance of Candida albicans 215 A clinical fluconazole-resistant isolate was chosen to further identify the antifungal potential of matrine. The metabolic activity of the clinical isolate 215 was reduced by above 77% when 0·5 mg ml−1 matrine was used in combination with 0·06 mg ml−1 fluconazole with FICI 0·197 (P < 0·05, Fig. 6a). Likewise, it was observed that the metabolic activity decreased by approximately 47% when 1·5 mg ml−1 matrine and 2 mg ml−1 fluconazole were combined against the biofilm with FICI 0·5 (P < 0·05, Fig. 6b). These results proved that matrine might be not only synergistic with fluconazole but also stimulative to change fluconazole sensitivity against clinical-resistant Candida isolates. Figure 6Open in figure viewerPowerPoint Potential of matrine to reduce fluconazole resistances of Candida albicans 215 (≥ 0·064 mg ml−1) for (a) planktonic cells and (b) biofilm by XTT metabolic activity assay. As for planktonic cells, the concentration of fluconazole was constantly set at 0·06 mg ml−1, while the concentrations of matrine were set as five gradients: 0·25, 0·5, 1, 2 and 3 mg ml−1; as for biofilm, the concentration of fluconazole was constantly set at 2 mg ml−1, while the concentrations of matrine were set as five gradients: 0·375, 0·75, 1·5, 3 and 6 mg ml−1; the control had only strain solution and medium but without matrine. *P < 0·05, compared with the control. Discussion Candida albicans has become one of the most severe nosocomial challenges in immune-deficient patients. The potential pharmaceutic use of antifungals from plant extractions and derivatives is promising in preparing novel antifungal agents to combat clinical Candidal problems. Most agents originated from plant have been applied to fight against nonfungal infections in vitro (Zeng et al. 2011; Selles et al. 2013). Matrine is a phytoanticipin that is one of the native components in Sophora herb plants. As tested previously, 1 mg ml−1 matrine only caused about 14·5% haemolysis, compared with nearly complete haemolysis by 0·125 mg ml−1 Amphotericin B (Wang et al. 2008). In spite of its renowned activities on virus, tumour, etc., there have been few reports concerning matrine against C. albicans. This study showed the antifungal effect of matrine on planktonic cells and biofilm phenotype, which was achieved by inhibiting yeast-to-hypha transition. To study the critical role of matrine on hyphal growth, the expressions of ALS 3, SUN 41 and PBS 2 were measured by qRT-PCR and observed to be down-regulated. ALS 3 was a member of agglutinin-like family (ALS) located in the cell wall and expressed higher in hypha than in yeast (Nailis et al. 2009). SUN 41 was also a cell wall protein and responsible to control hyphal morphogenesis (Norice et al. 2007). PBS 2 was essential in cell wall construction and played a role in response to osmotic and oxidative stress (Arana et al. 2005). In C. albicans, yeast-to-hypha transition is under strict control of regulatory circuits. The three selected hyphae-specific genes (HSGs) could promote hyphal growth and biofilm development through activating at least two intracellular signal pathway, that is, cyclic AMP-protein kinase A (cAMP-PKA) pathway and the Cek1 mitogen-activated protein kinase (MAPK) cascade (Sudbery 2011; Gow et al. 2012). More HSGs would be analysed to corroborate the relationship between down-regulation of HSGs by matrine and cAMP-PKA- and Cek1-MAPK-dependent pathways. Clinically, long-range contact with fluconazole, the most frequently used antifungals, induces plenty of appearances of fluconazole-resistant isolates of C. albicans, threating the conventional treatment (Slavin et al. 2010). In this study, matrine was found to tremendously lower MIC80 and EC50 of fluconazole in a fluconazole-resistant strain, namely C. albicans 215, which was selected by screening the clinical C. albicans collected in our laboratory due to its highest MIC80 and EC50. Generally, the fluconazole resistance of C. albicans is largely associated with the mutation or overexpression of ERG11 encoding lanosterol 14α-demethylase, leading to the decrease of binding affinity of fluconazole or the increase of ergosterol. Therefore, matrine might act in a mode to compensate the changes to be synergistic with fluconazole. In addition, there has been an observation of fluconazole to inhibit the biofilm formation of fluconazole-resistant C. albicans, inferring that the efflux determinants might only exert main influence in the initiation of biofilm formation or in the existence of low concentration of fluconazole or in the absence of fluconazole (Bruzual et al. 2007). As a result, matrine might lessen efflux pump-mediated resistance. However, it might not rule out the possibility that positive-charged matrine could absorb onto and penetrate into the cell wall or extracellular matrix, both of which exhibited electronegativity, like the mode of action of defensins (Vylkova et al. 2007), to facilitate the penetration of fluconazole by certain unknown mechanisms. More efforts on antiresistant mechanism of matrine against clinical Candida isolates are needed. Also from the results of fluconazole-resistant reversion, it was inferred that matrine could be a suitable alternative for clinical combination treatment with azoles against C. albicans, or even other non-C. albicans Candida (NCAC) species. However, as matrine exhibited antimicrobial potential at quite high concentration, oral administration could be employed instead of commonly intravenous injection, which should be under rigorous consideration. 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