Assessing the potential of four cathelicidins for the management of mouse candidiasis and Candida albicans biofilms

Biochimie 121 (2016) 268e277

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Research paper

Assessing the potential of four cathelicidins for the management of mouse candidiasis and Candida albicans biofilms Haining Yu a, *, Xuelian Liu a, Chen Wang a, Xue Qiao a, Sijin Wu a, Hui Wang a, Lan Feng a, Yipeng Wang b, ** a b

Institute of Marine Biological Technology, School of Life Science and Biotechnology, Dalian University of Technology, Dalian, Liaoning, 116024, China College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, 215123, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 June 2015 Accepted 29 November 2015 Available online 2 December 2015

As the most common fungal pathogen of humans, severe drug resistance has emerged in the clinically isolated Candida albicans, which lead to the urgency to develop novel antifungal agents. Here, four our previously characterized cathelicidins (cathelicidin-BF, Pc-CATH1, Cc-CATH2, Cc-CATH3) were selected and their antifungal activities against C. albicans were evaluated in vitro and in vivo using amphotericin B and LL-37 as control. Results showed that all four cathelicidins could eradicate standard and clinically isolated C. albicans strains with most MIC values ranging from 1 to 16 mg/ml, in less than 0.5 h revealed by time-kill kinetic assay. Four peptides only exhibited slight hemolytic activity with most HC50 > 200 mg/ ml, and retained potent anti-C. albicans activity at salt concentrations below and beyond physiological level. In animal experiment, 50 mg/kg administration of the four cathelicidins could significantly reduce the fungal counts in a murine oral candidiasis model induced by clinically isolated C. albicans. The antibiofilm activity of cathelicidin-BF, the most potent among the five peptides was evaluated, and result showed that cathelicidin-BF strongly inhibited C. albicans biofilm formation at 20 mg/ml. Furthermore, cathelicidin-BF also exhibited potent anti-C. albicans activity in established biofilms as measured by metabolic and fluorescent viability assays. Structure-function analyses suggest that they mainly adopt an a-helical conformations, which enable them to act as a membrane-active molecule. Altogether, the four cathelicidins display great potential for antifungal agent development against candidiasis.
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Keywords: Cathelicidins Antifungal susceptibility Oral candidiasis Advanced structures Anti-biofilm

1. Introduction Candida albicans, the most common fungal pathogen of humans, colonizes asymptomatically in skin, gastrointestinal tract, oral and vaginal mucosa in about 30e70% of individuals, and its overgrowth often results in candidiasis [1]. A common form of candidiasis restricted to the mucosal membranes in mouth or vagina is thrush, which is usually easily cured in normal people, can develop into very severe mucosal and systemic infections in immunocompromised individuals, such as stomatitis, vaginitis, bloodstream infections and deep tissue infections [1]. So far, although a lot of antifungal agents have been developed and administered to the

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H. Yu), [email protected] (Y. Wang).

patients with candida infections, however, extensive drugresistance still has been widely observed in clinically isolated C. albicans strains and become a severe challenge for public health nowadays [2]. In most cases, C. albicans could attach to a tissue surface and encase themselves in a self-released slimy polysaccharide and protein layer, known as biofilms, which is hardly penetrated by traditional antibiotics and finally prevent fungus from being cleared [3]. Besides, biofilm forming on implant surfaces becomes a more and more serious problem and a major cause of chronic infections worldwide [4]. The antibiotic resistance of biofilm is also owing to the slow growth of biofilm cells, which are tolerant to the antibiotics capable of penetrating the biofilm matrix [5]. Therefore, tight surveillance and novel antifungal agents that could directly target cell membrane are in great needed to manage such pandrug-resistant C. albicans infections. Cathelicidins are a family of multifunctional antimicrobial peptides (AMPs) found in vertebrates, and most abundantly in

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H. Yu et al. / Biochimie 121 (2016) 268e277

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Table 1 Structural parameters of the five cathelicidins. Sample

Amino acid sequence (Length)

Mw/pI

Net charge

Origin

Helix content

cathelicidin-BF Pc-CATH1 Cc-CATH2 Cc-CATH3 LL-37

KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF (30) RIKRFWPVVIRTVVAGYNLYRAIKKK (26) LVQRGRFGRFLKKVRRFIPKVIIAAQIGSRFG (32) RVRRFWPLVPVAINTVAAGINLYKAIRRK (29) LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLV PRTES (37)

3637.50/11.79 3175.90/11.60 3715.54/12.70 3379.11/12.18 4493.32/10.61

11 8 9 7 6

Bungarus fasciatus Phasianus colchicus Coturnix coturnix Coturnix coturnix Homo sapiens

56.67% 38.46% 62.50% 65.52% 78.38%

Fig. 1. Helix-wheel plots of the five cathelicidins: cathelicidin-BF, Pc-CATH1, Cc-CATH2, Cc-CATH3 and LL37. The hydrophobic and hydrophilic residues are separated with dash dot line, with the hydrophobic residues being concentrated on upper side of the helix and hydrophilic amino acids on the lower.

circulating neutrophils, myeloid bone marrow cells, and also in mucosal epithelial cells and skin keratinocytes [6,7]. Cathelicidins serve a critical role in mammalian innate immune defense against invasive bacterial infection [6,7]. Most of them possess potent antimicrobial activities against a broad range of microorganisms including bacteria, fungi, protozoa and certain viruses [8]. More interestingly, some of them exhibit great efficacy against a large number of clinically isolated drug-resistant pathogens [7]. Nowadays, some cathelicidin-derived AMPs, such as Iseganan, Omiganan, and MBI 594AN, have been already in clinical trials, suggesting their good prospects for the development of novel peptide antibiotics [9]. In previous works, we have identified a lot of cathelicidins from different species of vertebrates, and some have been proved to possess potent and broad-spectrum antimicrobial activities. In the

present study, we selected four of them, cathelicidin-BF from Bungarus fasciatus [10], Pc-CATH1 from Phasianus colchicus [11], Cc-CATH2 and 3 from Coturnix coturnix [12], to examine their antiC. albicans activities in vitro and in vivo in a murine oral candidiasis model, using human LL-37 and amphotericin B as positive control. Besides, their bacterial killing kinetics, hemolysis, cytotoxicity and salt stability were also examined. The effect of cathelicidin-BF on the C. albicans biofilm formation was investigated by XTT assays, and the antifungal activity against C. albicans communities in an established biofilms was measured by metabolic and fluorescent viability assays. The current results would provide basic concepts to evaluate the potential of these cathelicidins as novel antifungal agents against superficial and systemic candidiasis caused by C. albicans, especially against oral candidiasis and biofilm cultures of oral pathogens.

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Fig. 2. Homology models of the four cathelicidins. The model was produced by MODELLER (version 9.10). Visualization of the structure was accomplished by Pymol (version 1.7.6) and represented in both the cartoon and surface forms. Residues of arginines and lysines are displayed in blue and red, respectively.

Table 2 Antifungal and hemolytic activities of the five cathelicidins. Microorganism

MIC (mg/ml) Pc-CATH1

Cc-CATH2

Cc-CATH3

C-BF

LL-37

Amphotericin B

C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans HC50 IC50

2e4 8e16 16 16 32 4e8 16e32 16e32 >100 >200

8e16 8e16 16 16 32 16e32 32 16e32 >200 >200

8 16 ND 16e32 ND 16 32 16 >200 >100

2e4 1 1 1 1e2 1e2 1 1 >200 >200

16e32 ND ND ND ND ND ND ND 75 >100

2 2 2 2 2 2 2 2 e -

ATCC2002 0804 (IS) 0401 (IS) 0102 (IS) 2710 (IS) 2815 (IS) 2821 (IS) 0802 (IS)

MIC: minimal inhibitory concentration. These concentrations represent mean values of three independent experiments performed in duplicates. HC50: concentrations of cathelicidins producing 50% hemolysis. IC50: concentrations of cathelicidins producing 50% cell growth inhibition. These concentrations represent mean values of three independent experiments. ND: no detectable activity in inhibition zone assay (2 mg/ml); -: no assay; IS: clinically isolated strain. C-BF: cathelicidin-BF.

2. Materials and methods

The conventional antifungal agent amphotericin B was used as positive control in the following experiments.

2.1. C. albicans strains and peptides A standard strain of C. albicans ATCC2002 conserved in glycerol at
80  C in our laboratory and seven clinically isolated C. albicans strains collected from local hospital were used in this study. C. albicans were cultured on Sabouraud Dextrose (SD) agar medium (SigmaeAldrich, St. Louis, MO.). Cathelicidin-BF, Pc-CATH1, Cc-CATH2, Cc-CATH3 and LL-37 were synthesized by the peptide synthesizer GL Biochem (Shanghai) Ltd. (Shanghai, China), and analyzed by HPLC and MALDI-TOF mass spectrometry to confirm that the purity was higher than 98%. The synthetic peptides were dissolved in sterile deionized water to an ultimate concentration of 2 mg/ml and stored at
20  C until used.

2.2. Structure modeling Secondary structures of the cathelicidins were predicted using The PSIPRED Protein Structure Prediction Server provided by Bioinformatics Group of UCL Department of Computer Science (http:// bioinf.cs.ucl.ac.uk/psipred/). Helix-wheel plot was carried out by software package provided by The Expert Protein Analysis System (ExPASy) proteomics server (http://heliquest.ipmc.cnrs.fr/). The three dimensional structure of Pc-CATH1, Cc-CATH2 and CcCATH3 were modeled by homology. A BLAST search with default setting was performed in the Protein Data Bank (PDB) to obtain the suitable templates. Multi-templates were selected for each peptide

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Table 3 Salt dependence of the antifungal activity of the four cathelicidins. MIC (mg/ml)

Microorganism

Pc-CATH1 (0 mM NaCl)

Pc-CATH1 (100 mM NaCl)

Pc-CATH1 (200 mM NaCl)

Cc-CATH2 (0 mM NaCl)

Cc-CATH2 (100 mM NaCl)

Cc-CATH2 (200 mM NaCl)

2e4

4e8

1

8e16

16e32

16e32

8e16 16 16 32 4e8 16e32 16e32

16 16 32 32 4e8 16e32 16e32

32 32 32 32 16 16 16e32

8e16 16 16 32 16e32 32 16e32

16e32 16e32 32 32 16e32 32 16e32

16e32 32 32 32 32 32 16e32

Microorganism

Cc-CATH3 (0 mM NaCl)

Cc-CATH3 (100 mM NaCl)

Cc-CATH3 (200 mM NaCl)

C-BF (0 mM NaCl)

C-BF (100 mM NaCl)

C-BF (200 mM NaCl)

C. albicans ATCC2002 C. albicans 0804 (IS) C. albicans 0401 (IS) C. albicans 0102 (IS) C. albicans 2710 (IS) C. albicans 2815 (IS) C. albicans 2821 (IS) C. albicans 0802 (IS)

8 16 ND 16e32 ND 16 32 16

8 16e32 ND 16e32 ND 16e32 32 32

16e32 16e32 ND 32 ND 8e16 32 32

2e4 1 1 1 1e2 1e2 1 1

4 1e2 1e2 1 1e2 1e2 1e2 1

4e8 1e2 2e4 1e2 1e2 1 1e4 1e2

C. albicans ATCC2002 C. albicans 0804 C. albicans 0401 C. albicans 0102 C. albicans 2710 C. albicans 2815 C. albicans 2821 C. albicans 0802

(IS) (IS) (IS) (IS) (IS) (IS) (IS)

MIC: minimal inhibitory concentration. These concentrations represent mean values of three independent experiments performed in duplicates. C-BF: cathelicidin-BF; ND: no detectable activity in inhibition zone assay; IS: clinically isolated strain.

Table 4 Killing kinetics of the cathelicidins against C. albicans. Time

Colony forming units (103, CFUs/ml) 0h

Samples Pc-CATH1

0.1 h

50 ± 4.3

0.25 h

0.5 h

1h

3h

6h

6 ± 0.5

0 ± 0.0

0 ± 0.0

0 ± 0.0

0 ± 0.0

1 ± 1.0

0 ± 0.0

0 ± 0.0

0 ± 0.0

0 ± 0.0

1 ± 1.6

0 ± 0.0

0 ± 0.0

0 ± 0.0

0 ± 0.0

1 ± 0.6

0 ± 0.0

0 ± 0.0

0 ± 0.0

0 ± 0.0

45 ± 5.2

22 ± 8.9

2 ± 1.5

4 ± 2.5

13 ± 9.6

59 ± 10.3

78 ± 12.3

212 ± 82.6

1780 ± 185.2

21890 ± 3012.2

23 ± 3.2 Cc-CATH2

50 ± 4.3 20 ± 5.6

Cc-CATH3

50 ± 4.3 21 ± 6.5

Cathelicidin-BF

50 ± 4.3 3 ± 3.2

Amphotericin B

50 ± 4.3 43 ± 12.3

Deionized water

50 ± 4.3 48 ± 16.0

C. albicans 0102(IS) was mixed with the cathelicidins at concentration of 5  MIC for 0, 0.1, 0.25, 0.5, 1, 3 and 6 h. The results represent mean values of three independent experiments.

in order to generate the model as accurate as possible. Based on the similarities and coverage with Pc-CATH1, the crystal structures of PDB entry 2AMN (85% identity, query cover 100%), 3BBN (86% identity, query cover 26%) and 3U54 (78% identity, query cover 34%) by SOLUTION NMR were exploited as templates for homology modeling simultaneously. Similarly, the crystal structures of PDB entry 2GDL (71% identity, query cover 96%) and 3H0J (38% identity, query cover 87%) by SOLUTION NMR, and 2AMN (62% identity, query cover 100%) and 2HFR (100% identity, query cover 89%) by SOLUTION NMR were used as templates for Cc-CATH2 and CcCATH3, respectively. The tertiary structures of three peptides were generated and optimized using MODELLER (version 9.10). The three dimensional structure of cathelicidin-BF were modeled by Rosetta ab initio (version 3.5), since there was no suitable template obtained from blast search. All of the three dimensional models were visualized by Pymol software (version 1.7.6) without any other refinements.

2.3. Antifungal assays A two-fold broth microdilution method was used for MIC determination. The experiment was conducted based on CLSI methodology [13]. Briefly, totally eight C. albicans strains were cultured on SD agar medium at 35  C for 24 h. Then five colonies were picked and suspended in 5 ml of sterile saline solution, and adjusted to a 0.5 McFarland density standard (1e5  106 CFU/ml). Afterwards, the inocula were diluted with RPMI-1640 medium (Gibco BRL, Gaithersburg, MD, USA) to a final concentration of 1e5  103 CFU/ml and mixed with equal volumes of two-fold serial dilutions of cathelicidins (100 ml) in 96-well microtiter plates. The plates were incubated at 35  C for 48 h and the minimal concentrations at which no visible growth of fungi occurred were recorded as MIC values, which is also applied to the one treated by amphotericin B.

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5  105 CFU/ml in RPMI-1640 medium. Cathelicidin was added to the C. albicans suspensions to a final concentration of 5  MICs, and incubated at 35  C for 0, 0.1, 0.25, 0.5, 1, 3 and 6 h. At each time point, a 10 ml fungal suspensions were removed and serially diluted with PBS (phosphate buffered saline). Finally, 100 ml dilutions were coated on SD agar plates at 35  C for 36e48 h and the CFUs were determined. Amphotericin B and sterile deionized were used as positive and negative controls, respectively. 2.6. Murine oral candidiasis model

Fig. 3. Therapeutic efficacies of the five cathelicidins for oral candidiasis in a murine model. After C. albicans 0102 (IS) infection for three days, 50 mg/ml cathelicidin samples were applied three times daily for three days (n ¼ 8). Vehicle (PG-NaP buffer) was used as negative control (n ¼ 8) and amphotericin B was used as positive control (n ¼ 8). Each point represents the determination from a single animal, and horizontal bars represent the mean values of each group. The values of cathelicidins and amphotericin B-treated groups were significant different from the value of vehicletreated group (P < 0.05). C-BF: cathelicidin-BF.

2.4. Hemolytic, cytotoxic and salt tolerant assays Hemolytic assay of the cathelicidins was conducted as previously reported [11]. Briefly, human fresh erythrocytes were washed with 0.9% (w/v) saline for two times and re-suspended to an ultimate concentration of 2% (v/v). The erythrocyte solutions (90 ml) were incubated with serial dilutions of cathelicidins (200, 100, 50 and 25 mg/ml) in ddH2O (10 ml) at 37  C for 30 min and then centrifuged at 2000 rpm for 5 min. The supernatants were removed and the absorbance at 540 nm was measured. 1% Triton X-100 (v/v) was used to determine the 100% hemolysis and sterile deionized water was used as negative control. The HC50 values, concentrations of cathelicidins producing 50% hemolysis were recorded. In vitro cytotoxic activity of the peptides was tested using a normal cell line HUVEC (human endothelial cells) in vitro via MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) method [12]. IC50 was defined as the concentration of peptides at which the absorbance at 490 nm was reduced by 50%. The salt tolerance of cathelicidins was examined as previously described with minor modifications [14]. C. albicans strains were incubated at 35  C for 48 h and then diluted to 1e5  103 CFU/ml in RPMI-1640 medium, supplemented with sodium chloride to final concentrations of 0, 100 and 200 mM, respectively. Two-fold serial dilutions of cathelicidins were added to equal volumes of C. albicans suspension (100 ml) in 96-well microtiter plates, and incubated at 35  C for 48 h before the MICs were determined.

2.5. Time-kill kinetic assay A quantitative killing assay over time was carried out to examine the antifungal rate of cathelicidins against C. albicans. The clinically isolated strain C. albicans 0102 (IS), the most sensitive one to the sample peptides among all strains tested, was used for the assay. The experiment was carried out according to the method described previously with minor modifications [10]. C. albicans 0102(IS) was incubated at 35  C to the exponential phase and diluted to

Murine oral candidiasis model was established using Kunming mice according to previously described method with minor modifications [15]. Briefly, 48 Kunming mice of both sexes weighing between 18 and 20 g were randomly assigned into six groups (vehicle, Pc-CATH1, Cc-CATH2, Cc-CATH3, cathelicidin-BF and amphotericin B), with each group including 4 males and 4 females. Cyclophosphamide monohydrate (200 mg/kg of body weight) was subcutaneously injected into mice at 1 and 3 days before C. albicans infection to maintain neutropenia for at least 72 h. Teicoplanin (Sanofi, China) and ceftriaxone sodium (Qilu Pharm, China) were each given in drinking water (1 g/L) throughout the experiment to eradicate potential bacterial competitors. C. albicans 0102 (IS) was incubated at 35  C to 109 CFU/ml. Mice were anaesthetized by intramuscular injection of ketamine hydrochloride (Xi An Libang Pharm, China) and then infected by topical inoculation of C. albicans dilutions (0.1 ml) on the oral mucosal surfaces. After three days of infection, white patches on the mice tongue were observed. Cathelicidins were dissolved in PG-NaP buffer (45% polyethylene glycol 400, 45% glycerol, 10% 10 mM sodium phosphate buffer, pH 7.4) to prepare 50 mg/ml samples. 0.02 ml samples were applied for oral and gavage syringe feeding 3 times per day for 3 days. PG-NaP buffer and 0.02 ml amphotericin B (50 mg/ml) were used as vehicle and positive control, respectively. After the final administration, mice were sacrificed and tongues were cut off and weighed. The tongues were homogenized in 0.15 M NaCl solution (1 ml per tongue). Homogenates were serial diluted and plated on SD agar plates. After incubated at 35  C for 24 h, the C. albicans clones were counted and CFU/mg of tongue was calculated. All the animal experimental protocols were approved by the Animal Care and Use Ethics Committee of Dalian University of Technology. 2.7. Analysis of C. albicans biofilms Cathelicidin-BF that has the best antifungal activity among the five was chosen to examine the possible effect on the C. albicans biofilm formation and the established biofilm using previously described method with minor modifications [16]. In the biofilm inhibition assay, C. albicans 0102 (IS) was incubated at 35  C and diluted to 1  106 CFU/ml in RPMI-1640 medium. 2 ml culture was added to each well of 6-well plates with coverslips at the bottom, and incubated at 35  C in a humid chamber for 3 h. Non-adherent cells were removed by washing twice with PBS, and series concentrations of cathelicidin-BF (0, 0.5, 1, 2 mg/ml) were added into the plate, with amphotericin B (2 mg/ml) as control. After a 36-h incubation, the formation of the biofilm was evaluated using the XTT Cell Proliferation and Cytotoxicity Kit (KeyGEN BioTECH, Nanjing, China). In the biofilm disruption assay, the biofilm was formed as above but with a longer incubation time up to 72 h. Then cathelicidin-BF and amphotericin B of series concentrations (0, 2.5, 5, 10, 25, 50 mg/ ml) was added after non-adherent cells were washed out. The measurement of remaining biofilm was determined using the same kit as above. In order to further confirm the antifungal effect of cathelicidin-

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BF against C. albicans encased in biofilms, the established biofilm was treated with cathelicidin-BF (0, 25, 50 mg/ml), washed three times with PBS, and finally stained with 1 ml 1000-fold diluted PI/ SYBR Green I at 30  C in the dark for 20 min. After washing once with PBS, the plate was immediately photographed with fluorescence microscope. 2.8. Statistical analysis MIC values, HC50 values and data of time-kill kinetic assay were the geometric means of three independent experiments. Total 8 repeats per plate have been done for the murine oral candidiasis model experiment. The chi-square test for normality has been used, and the data was analyzed by one-way analysis of variance (ANOVA) test with a Bonferroni post test using GraphPad Prism5 (GraphPad Software, San Diego, CA). P < 0.05 was considered statistically significant. 3. Results 3.1. Characterizations of cathelicidins The sequences and chemical/physical parameters of the current five cathelicidins are shown in Table 1. Their secondary structures were predicted to mainly adopt the helical structures with different percentages in the presence of microbial membranes (Table 1 & Fig. 1). Additionally, cathelicidin-BF and Cc-CATH2 also exhibited partial strand structures of 16.7% and 25%, respectively (data not shown). The helical wheel diagrams of cathelicidins were subsequently plotted to estimate their amphipathicity (Fig. 1), which revealed apparent amphipathic structures for cathelicidin-BF, CcCATH2 and LL-37, with the hydrophobic residues being concentrated on upper side of the helix and hydrophilic amino acids on the lower. Whilst Cc-CATH3 and Pc-CATH1 are mostly comprised of hydrophobic residues, and do not show the amphipathicity. This amphipathic alpha-helix structural feature is commonly adopted by most of small cationic antimicrobial peptides and is believed to be important for their disrupting the microbial membrane integrity [17]. The homology modeled three dimensional structures of the four cathelicidins are shown in Fig. 2. All of them exhibit basically the wedge shapes made up of the a-helix, with Cc-CATH2 having the least helix percentage. Considering the membrane permeabilization mechanism via which cathelicidins exert their antimicrobial activities, the more the helix percentage the peptide contains, the easier it penetrates through the membrane structure. This is consistent with the result of antimicrobial assay, which showed that Cc-CATH2 had the relatively weakest antifugal activity. Furthermore, the cationic residues, including arginine (blue) and lysine (red) are distributed throughout the entire five sequences (Fig. 2). This arrangement is common in alpha-helical structures, where one face of the helix is oriented toward the hydrophobic core and one face is oriented toward the solvent-exposed surface. 3.2. Antifungal activities The MICs of the five cathelicidins against the eight C. albicans strains are shown in Table 2. The conventional antifungal agent, amphotericin B (2 mg/ml) was inactive to most of the C. albicans strains, except the standard strain C. albicans ATCC2002 and C. albicans 0102(IS). However, Pc-CATH1, Cc-CATH2 and cathelicidin-BF exhibited potent antifungal activities against all of the eight C. albicans strains, with most MIC values of 2e16 mg/ml. Among them, cathelicidin-BF showed the greatest potency, with most MICs ranging in 1e2 mg/ml. Cc-CATH3 also showed

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comparatively potent antifungal activities against most C. albicans strains, except C. albicans 0401 (IS) and C. albicans 2710 (IS). In contrast, LL-37 did not exhibit apparent antifungal activity, and was only active to C. albicans ATCC2002 with MIC of 16e32 mg/L. The current cathelicidins are effective in killing amphotericin-B resistant clinical isolates, which further proved that their antifungal mechanism is distinct from that of conventional antifungal agents. The synergy effect of the cathelicidins with amphotericin B was also studied. However, there was no difference of in antifungal effects between cathelicidins only and cathelicidins in combination with amphotericin B (data not shown). 3.3. Hemolysis, cytotoxicity and salt tolerance of the cathelicidins As listed in Table 2, all of them showed slight hemolysis to human fresh erythrocytes, with most HC50 values > 200 mg/ml, which was approximately ten times higher than MICs. Pc-CATH1, CcCATH2 and cathelicidin-BF have an IC50 to HUVEC higher than 200 mg/ml, whilst Cc-CATH3 and LL-37 have IC50 > 100 mg/ml, suggestive of extra low cytotoxicity and potential for therapeutic applications. Generally, cathelicidins induced neither lethality nor obvious toxicity even in a relatively high dose, and in vivo model via route of administration including i.v. (intravenous injection) [18], SC (subcutaneous injection) [19] and oral [20], indictive of their great potential of clinical application. The influence of sodium upon the antifungal activities of the cathelicidins was also examined. Unlike many AMPs whose activities are inhibited by sodium at physiological concentrations [21], the four cathelicidins showed salt-independent activities with or without the presence of 100 mM NaCl (Table 3). When the salt concentration reached 200 mM, the four peptides still maintained potent antifungal activities, but some MICs were slightly increased, suggesting their suitability for both local and systemic therapeutic applications. 3.4. Fungi killing kinetics of cathelicidins against C. albicans Using amphotericin B as positive control, killing kinetics of the four cathelicidins (Pc-CATH1, Cc-CATH2, Cc-CATH3 and cathelicidin-BF) against C. albicans 0102(IS) were investigated by a colony counting method. As illustrated in Table 4, at concentration of 5  MIC, under which all microorganisms can be completely killed without resuming growth, all the four cathelicidins could rapidly exert their activities, taking less than 0.5 h to kill all the C. albicans cells, which may impossibly allow the strain's evolution and resistance. More importantly, the colony forming units (CFUs) remained zero when the incubation time extended to 6 h, implying that the antifungal activities of the four peptides are lethal. Such fast killing kinetics can make it possible to develop topical applications, because the microorganisms could be quickly killed before the peptide is metabolized or mechanically cleared. In contrast, at the same concentration of 5  MIC, amphotericin B could not completely kill the C. albicans cells in 1 h. Furthermore, C. albicans 0102(IS) treated by amphotericin B was capable of resuming growth after 3 h of treatment. 3.5. In vivo therapeutic efficacy of cathelicidins for candida infections In vivo antifungal efficacy of the four cathelicidins was further investigated in a murine oral candidiasis model. After C. albicans infection for three days, 50 mg/kg of cathelicidin samples were administered 3 times per day and lasted for 3 days. As illustrated in (Fig. 3), all four cathelicidins could significantly reduce the C. albicans counts compared to vehicle, with the mean C. albicans

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CFUs 10 e 100 times lower than that of vehicle group (P < 0.05). Among all, cathelicidin-BF exhibited the highest efficacy even better than amphotericin B, consistent with in vitro antifungal results. 3.6. Effect of cathelicidin-BF against C. albicans biofilm The effect of cathelicidin-BF, the most potent peptide among the five, upon the C. albicans ATCC2002 biofilm formation was tested by measuring the percentage of biofilm formation. The biofilm of C. albicans 0102 (IS) treated with cathelicidin-BF was obviously less than that of untreated and amphotericin B-treated (2 mg/ml) strains (Fig. 4A), and this inhibitory activity is dose-dependent with the maximal inhibition of 90% observed under 2 mg/ml cathelicidin-BF treatment, while amphotericin B of 2 mg/ml only exerting 10% inhibition (Fig. 4C). The current result clearly shows that cathelicidinBF could significantly inhibit the formation of C. albicans biofilm at sub-MIC. To examine the activity of cathelicidin-BF killing C. albicans in an established biofilm, both cathelicidin-BF and amphotericin B of serious concentrations were loaded on the biofilm, and metabolic activity was determined by XTT assay. The biofilm was notably ruptured under treatment of 50 mg/ml cathelicidin-BF, whilst the biofilm processed with 50 mg/ml amphotericin B appeared intact (Fig. 4B). This result is consistent with fungi metabolic activity measurement, which demonstrated that treatment with cathelicidin-BF and amphotericin B at 50 mg/ml resulted in significant decline of metabolic activities of C. albicans by 96% and 70%, respectively, and cathelicidin-BF in a very low dose as 2.5 mg/ml also showed marked antifungal activity against C. albicans in a biofilm (Fig. 4D). In order to visually confirm this effect, the preformed biofilms treated with cathelicidin-BF of increasing concentrations were next stained with PI/SYBR Green I and photographed using fluorescence microscopy. As peptide's concentration increased, the green dye representing cell viability was reduced significantly, while the red dye that stains only dead cells with broken membranes was increased a lot (Fig. 4E). Current results suggested that cathelicidin-BF can effectively kill the C. albicans in a preformed biofilm, and under concentration as high as 50 mg/ml cathelicidinBF almost destroyed all of the cell membrane, whereby the red fluorescent stain nearly occupied all vision (Fig. 4E). 4. Discussion For the last two decade, extensive use of antifungal agents has fostered the emergence and expansion of drug-resistant C. albicans strains, which seriously threatens the public health. The resistance of C. albicans is dependent upon diverse mechanisms during its adaptive evolution. On the genetic level, single nucleotide polymorphism, loss-of-heterozygosity (LOH) and gross chromosomal rearrangements count for most important processes in the development of drug resistance [2,22,23]. On the protein level, mutations in drug targets [24], efflux pumps [25], membrane composition [26], and ergosterol biosynthesis pathway are proved related with C. albicans drug resistance. Nowadays, the development of biofilms by C. albicans has become increasingly important

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drug-resistance mechanism, and attracted more and more attentions worldwide. C. albicans forms a biofilm that allows for an aggregate cell colony to aid their retention, and continually reinfects the mucosa in candidiasis. Therefore, the development of novel antibiotics against biofilm is a major strategy to cope with candida infections, especially candidiasis. Cathelicidins, as one of the most important AMP family, have already been heralded as a promising alternative to conventional antibiotics, due to their simplicity in structures, potent activity and specific mechanisms of action. Several cathelicidin derived analogs have already undergone clinical trials, such as MBI-226 (Idolicidin analogue, Micrologix) against catheter-related blood stream infection [27], MX-594AN (Idolicidin analogue, Migenix) against acne vulgaris and Iseganan (Protegrin analogue, IntraBiotics) against oral mucositis [28,29]. At the same time, several cathelicidins have also been reported possessing antifungal activity against C. albicans [6,30]. Previously, we identified several cathelicidins and some of them exhibited strong activities to C. albicans [10e12,31]. In order to further investigate their potential as novel antifungal agents for the management of candida infections, we selected four of them (cathelicidin-BF, Pc-CATH1, Cc-CATH2, Cc-CATH3) and human cathelicidin, LL-37 to intently examine their in vitro and in vivo antiC. albicans activities and anti-biofilm activities. Antimicrobial assay indicated that Pc-CATH1, Cc-CATH2, CcCATH3 and cathelicidin-BF possess potent antifungal activities against the eight tested C. albicans strains, including standard and clinically isolated amphotericin B-resistant strains, with most MIC values ranging from 1 to 16 mg/ml. Several other cathelicidins have also been reported possessing anti-C. albicans activities, but MIC values were to some extent weaker than current four cathelicidins [32,33]. Moreover, almost no reports ever involved the antifungal assay against clinically isolated C. albicans strains. The cBDs (bdefensins), cBD103 and cCath (canine cathelicidin) reported by Santoro et al. killed C. albicans with MICs of 50, 25, and >200 mg/ml, respectively [34]. Chen et al. studied the anti-C. albicans ATCC activities of BF15 (a 15-mer peptide derived from Cathelicidin-BF) and series analogs, with most MICs of 64e128 mg/ml [35]. Mouse mCRAMP and porcine PR-39 are two of the most extensively studied cathelicidins, and mCRAMP was reported to inhibit C. albicans growth with MICs of 15e20 mM, whereas PR-39 was inactive [36]. In contrast, four cathelicidins in current study showed apparent advantage respecting C. albicans killing capacity, especially in killing clinically isolated strains. A big problem commonly associated with clinical applications of AMPs is their significant hemolytic activities against mammalian cells. Fortunately, no significant hemolytic activity to human fresh erythrocytes (most HC50 > 200 mM) was observed for all current cathelicidins. In addition, their extra low cytotoxicity implies a promising therapeutic potential. The antimicrobial activities of AMPs usually are different in the absence or presence of salt [37]. In current study, all of the four peptides retained their potent antiC. albicans activities at a low salt concentration below physiological level (100 mM) and a high salt concentration beyond physiological level (200 mM). To further investigate four cathelicidins' activities against biofilms of C. albicans, cathelicidin-BF with the most potent antifungal

Fig. 4. Activities of cathelicidin-BF inhibiting C. albicans biofilms formation and killing C. albicans in established biofilms. A&B. The photographs of inhibition of biofilm formation and anti-biofilms activity of cathelicidin-BF (CATH-BF) in the 6-well plates with coverslip, AmpB: amphotericin B. Blank: blank wells washed with PBS. C. The inhibitory effect of cathelicidin-BF against C. albicans biofilm formation. C. albicans was grown in a 6-well plates with coverslips at 35  C for 36 h in the presence of different concentrations of cathelicidin-BF. Growth in control is set to 100% and percent of biofilm formed is indicated in gray bars and diagonal bar for cathelicidin-BF and amphotericin B, respectively. Data are presented as the mean ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; by unpaired t test). D. The killing effect of cathelicidin-BF against C. albicans in the biofilms. Cells treated with cathelicidin-BF and amphotericin B of series concentrations, negative control set to a 100%. Data are presented as the mean ± SD from three independent experiments. E. Visualization of cathelicidin-BF killing C. albicans in established biofilms photographed by fluorescence microscope. Living cells stained by SYBR Green I are green and dead cells stained by PI are red.

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activity was tested via XTT and fluorescent viability assays. It is indicated that cathelicidin-BF significantly inhibited the formation of C. albicans biofilms at sub-antimicrobial concentrations, and also exhibited potent activity of killing C. albicans in preformed biofilms. After treatment with cathelicidin-BF, the C. albicans encased in a biofilm formed at the bottom of a plate were permeable to PI and turned red, suggestive of cathelicidin-BF's physical action on membranes. In contrast, C. albicans organized in biofilm were much more resistant to amphotericin B. The rapidity of cathelicidin-BF's killing kinetics plus its ability to kill most C. albicans enclosed in biofilm at very low concentration, suggests that cathelicidin-BF, like other cationic and helical structured AMPs, may induce membrane permeability by acting on membranes, and adequately penetrate into the biofilms. This is further confirmed by their structural features. The current five cathelicidins all exhibit amphipathic a-helical structure. Interestingly, their activities are not proportional to the helical percentage in their overall structures. LL-37 with the poorest activity indeed has high percent of helical structure. This might be explained by their three dimensional structures. The differences of five cathelicidins in both the key residue positions and the overall molecular shapes suitable for piercing the membrane would help explaining this contradiction. Although membrane permeabilization drives the antifungal activity of most cationic and amphipathic peptides including cathelicidins, it's still not the sole mechanism for cell killing elucidated so far. Other factors involving interference of DNA and protein synthesis, inhibition of the proteasome function and regulation of immune system may also provide additional mechanisms, and modulate the activity and specificity of these peptides [38]. Our results also demonstrated that oral administration of the four cathelicidins was an effective treatment for oral candidiasis in the murine model. They could significantly reduce the C. albicans counts in the tongues of mice. Our results support the development of four cathelicidins as novel strategy against oral candidiasis, especially chronic infections involving biofilms. However, detailed pharmacodynamics-pharmacokinetics studies would be needed to determine the time/concentration relationship of the drug at the infection sites and, thus, to generate the dosing regimens that produce the best treatment outcome in vivo. 5. Conclusion Taken together, four our previously characterized cathelicidins, Pc-CATH1, Cc-CATH2, Cc-CATH3 and cathelicidin-BF have been suggested as potential novel antifungal agents for treating C. albicans infections including oral candidiasis. Acknowledgments This work was supported by the grants from Chinese National Natural Science Foundation (41206153), Growth Project for Liaoning Distinguished Young Scientists of Colleges and Universities (LJQ2013010), Dalian Distinguished Young Scientists Funding (2013J21DW013), Suzhou Science and Technology Development Project (SYN201407), Dalian University of Technology Research Funding (DUT13JB10). References [1] N.A. Gow, F.L. van de Veerdonk, A.J. Brown, M.G. Netea, Candida albicans morphogenesis and host defence: discriminating invasion from colonization, Nat. Rev. Microbiol. 10 (2012) 112e122. [2] M. Huang, K.C. Kao, Population dynamics and the evolution of antifungal drug resistance in Candida albicans, FEMS Microbiol. Lett. 333 (2012) 85e93. [3] P.S. Stewart, J.W. Costerton, Antibiotic resistance of bacteria in biofilms, Lancet 358 (2001) 135e138.

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