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. Author manuscript; available in PMC: 2011 Dec 1.
Published in final edited form as: Mol Oral Microbiol. 2010 Dec 1;25(6):418–425. doi: 10.1111/j.2041-1014.2010.00590.x

Activity of Antimicrobial Peptide Mimetics in the Oral Cavity: I. Activity Against Biofilms of Candida albicans

Jianyuan Hua 1, Radha Yamarthy 1, Shaina Felsenstein 1, Richard W Scott 2, Kenneth Markowitz 1, Gill Diamond 1,*
PMCID: PMC2992321  NIHMSID: NIHMS231206  PMID: 21040515

Summary

Naturally occurring antimicrobial peptides hold promise as therapeutic agents against oral pathogens such as Candida albicans, however numerous difficulties have slowed their development. Synthetic, non-peptidic analogs that mimic the properties of these peptides have many advantages and exhibit potent, selective antimicrobial activity. Several series of mimetics (MW <1,000) were developed and screened against oral Candida strains as a proof-of-principle for their antifungal properties. One phenylalkyne and several arylamide compounds with reduced mammalian cytotoxicities were found to be active against C. albicans. These compounds demonstrated rapid fungicidal activity in liquid culture even in the presence of saliva, and demonstrated synergy with standard antifungal agents. When assayed against biofilms grown on denture acrylic, the compounds exhibited potent fungicidal activity as measured by metabolic and fluorescent viability assays. Repeated passages in sub-MIC levels did not lead to resistant Candida in contrast to fluconazole. Our results demonstrate the proof-of principle for the use of these compounds as anti-Candida agents, and their further testing is warranted as novel anti-Candida therapies.

Keywords: antifungal, denture, fungicide, defensin, resistance

Introduction

Oral infections with Candida, oropharyngeal candidiasis (OPC), were observed in 90% of patients undergoing chemotherapy for acute leukemia (Rodu et al., 1988), and 95% of patients with HIV (Dupont et al., 1992). Although the introduction of highly active anti-retroviral therapy has reduced these numbers in HIV patients, the occurrence is still very high. In denture stomatitis, which commonly affects edentulous individuals (Webb et al., 1998), Candida grows as a biofilm on the bioprosthetic materials used to make dentures (Edgerton et al., 1993), and is highly resistant to standard antifungal agents (Chandra et al., 2001). Treatment of candidiasis is either with topical antifungal agents such as Nystatin, or with systemic agents, including azoles, such as fluconazole or itraconazole, or echinocanadins, such as capsofungin. With the recurrence of OPC in HIV patients, long-term treatments have led to a significant rise in antifungal-resistant organisms (for review, see (Ghannoum and Rice, 1999)).

With the initial discovery of naturally occurring, broad-spectrum antimicrobial peptides (AMPs) such as defensins and magainins (reviewed in (Diamond et al., 2009)), activity against C. albicans was examined as a potential alternative to standard antifungal treatments. AMPs exhibit potent in vitro anti-Candidal activities under normal conditions (Selsted et al., 1985, Benincasa et al., 2006, Zasloff, 1987). Examination of human saliva also identified naturally occurring peptides known as histatins with anti-Candidal activity in vitro, (Oppenheim et al., 1988), and synergy with antifungals such as azoles (Wakabayashi et al., 1996). Synthetic peptides also exhibit potent activity against Candida species (Burrows et al., 2006, Nikawa et al., 2004)), and using a mouse model of candidiasis, peptides derived from lactoferrin exhibited activity in vivo (Takakura et al., 2003), suggesting that molecules derived from AMPs would be useful drugs to treat these infections. However, Candida demonstrate an innate immune evasion strategy of proteolytic cleavage of AMPs (Meiller et al., 2009).

Given their very broad specificity, amphiphilic AMPs appear to be ideal therapeutic agents. However, significant pharmaceutical issues, including poor tissue distribution, systemic toxicity and difficulty and expense of manufacturing, have severely hampered clinical progress. To address this problem, researchers have developed different types of small molecule peptide mimetics. These include peptoids, β-peptides, arylamide oligomers and phenylene ethynylenes (reviewed in (Rotem and Mor, 2008)). We have recently described a series of non-peptidic analogues that have many advantages over peptides because of their small size, which increases stability and enhances tissue distribution, and ability to fine-tune their physical properties for optimization of potency and safety (reviewed in (Som et al., 2008)). These include the phenylethynylene derivative, mPE (PMX70004), which exhibits broad-spectrum antimicrobial activity against oral pathogens (Beckloff et al., 2007), including three different strains of C. albicans and five non-albicans species (C. glabrata, C. dubliniensis, C. parapsilosis. C. tropicalis and C. krusei). Two other classes, based on arylamide and arylurea scaffolds also demonstrate broad-spectrum antimicrobial activity (Liu et al., 2004, Tang et al., 2005, Tang et al., 2006). Together, they represent a class of AMP mimetics that exhibit the activity of AMPs, without the protease sensitivity and expense of the peptides. To assess their potential as antifungal therapies for oral Candida infections, we examined as a proof-of-principle the antifungal activities of representatives of the three classes of mimetics on C. albicans, both in planktonic states and in biofilms on denture material.

Materials and Methods

Yeast Strain

Candida albicans ATCC 90028 (obtained from the laboratory of Dr David Perlin, PHR/UMDNJ) were used for all assays and are cultured on YPD agar (1% yeast extract, 2% peptone, 2% dextrose, pH 5.7) at 37°C. For liquid assays, single colonies were dispersed in RPMI-1640 (Mediatech, Inc.) with MOPS, pH 7.0 at a concentration of 2.5×106 CFU/ml.

Peptide Mimetic Compounds

Twelve different peptide mimetic compounds developed by PolyMedix, Inc. were tested in antimicrobial screens (MIC assay). Further comprehensive assays were performed on the following four compounds, mPE (PMX70004), PMX30016, PMX000519, PMX10149 (for structures, see online supplemental figure 1), all of which exhibited MIC values below 8μg/ml. All compounds were dissolved in DMSO (Sigma) at the stock concentration of 20mg/ml, and stored at -20°C.

MIC assay

Assays were carried out in 96-well plates using the NCCLS method as previously described (Beckloff et al., 2007). Mimetic compounds were diluted in 50μl RPMI/MOPS in a 96-well plate (Tissue culture treated, Falcon). 50μl suspensions of Candida were added to each well, and the plate was then incubated at 37°C in a humidified chamber for a period of 24 hours. MIC is determined as the first well without the visibility of turbidity in the broth for the fungicide (mimetic compounds), or the first well without the increase of OD at 600nm for fluconazole. A sample (25μL) from the well defined as the MIC, in addition with the wells with three higher concentrations, are plated onto YPD agar. Colonies were counted after 24 hours. The MFC (Minimal fungicidal concentration) is defined as the lowest concentration at which no colonies are observed. All MIC/MFC assay were performed in duplicate.

Fungicidal Kinetics

Fresh cultures of Candida were resuspended in RPMI-MOPS at a concentration of 2.5×106 CFU/ml. Samples (500μL) were incubated in the presence of the mimetic at 37°C, and aliquots were removed at the indicated timepoints, diluted in PBS and plated on YPD agar for colony counts after 24 hour growth at 37°C.

Resistance Assays

Fresh cultures of Candida (2.5×106 CFU/ml) were grown in 3ml RPMI-1640-MOPS in the presence of 0.5× MIC of each compound at 37°C shaking incubator for 24 hours. 50μl of the culture were plated on YPD agar and incubated at 37°C overnight. Colonies were resuspended in RPMI-MOPS and cultured under the same conditions in 0.5×MIC. This culturing procedure was repeated serially for 20 passages. MIC assays were performed at every other passage.

Analysis of C. albicans Biofilms

The denture biofilm model is carried out as described (Chandra, et al., 2001, Chandra et al., 2009). Briefly, 8cm by 6cm double thickness strips of medium thickness pink dental base plate wax (Benco Dental, Wilkes-Barre, Penn. USA) were pressed onto a wet paper towel heated on a hot plate to 65°C to give the pressed side a slightly rough surface texture. The wax strips were packed, flasked and processed into denture base acrylic (Lucitone 199® Dentsply International, Milford, DE. USA). The resulting strips of denture acrylic had a smooth surface and a textured surface that resembled the tissue-facing aspect of a denture. These strips were then cut into 2cm squares using a low speed saw (Isomet®, Buehler Ltd., Lake Bluff, Ill. USA) and diamond blade with water lubricant. The cut surfaces were then polished using 600 grit silicon carbide paper (also from Buehler Ltd.) and water lubricant. The acrylic squares were stored in sterile water until use.

The denture squares were pre-coated with pooled (n=3), clarified human saliva for 30′ at 37°C. Saliva was removed, and C. albicans were diluted to a concentration of 106 cfu/ml, and 100μl was placed on the squares. The squares were incubated at 37°C in a humid chamber for 3 hours. Non-adherent cells were removed, and the squares were further incubated in 6-well plates in 2.5ml RPMI-MOPS medium for 72 hours at 37°C, 100% humidity. For treatment, medium was replaced with fresh RPMI-MOPS containing the peptide mimetics. To quantify viability, an XTT assay was carried out using the TOX-2 kit (Sigma) diluted according to the manufacturer's instructions in RPMI without Phenol red, and incubated for 3 hours at 37°C. Metabolic activity was quantified by measuring optical density at 450nm and 600nm. Parallel cultures were scraped with a rubber scraper, resuspended in PBS, and plated on YPD-agar to quantify viable colonies.

To visualize activity microscopically, Candida are cultured in YPD agar plate to log phase and resuspended in RPMI-MOPS at the density of 108 cells/ml. Pooled, clarified human saliva was added to chamber slides (Labtek) for three hours. 200μl of the resuspended Candida was added to the chamber after the removal of saliva. Cells were incubated for 3 hours, and non-adherent cells were removed, and the slides were washed with RPMI-MOPS. The slides were cultured for a period of 72 hours before the treatment. The biofilms were visualized using the LIVE/DEAD BacLight Bacterial Viability kit (Molecular Probes) as described (Jin et al., 2005). Stained slides were incubated at 30°C dark chamber for 20min and the pictures were captured using with a Zeiss LSM 510 on Zeiss Axiovert 100M Base.

Results

Fungicidal activity of mimetics against C. albicans

After assessing the MIC of 12 different compounds, the fungicidal kinetics of the four most active compounds were tested at 50μg/ml (figure 1A). Since no reduction in cfu was observed by 10 minutes with PMX3001016, only the three remaining compounds were also tested at 10μg/ml (B). Of the compounds tested, mPE exhibits the most rapid killing, with a three-log reduction in cfu after 1-5 minutes at 50μg/ml. When this compound was incubated with C. albicans hyphae, no viable cells were visible at concentrations higher than 32μg/ml after 24 hour incubation (data not shown), indicating that the compound could also kill cells in this form.

Fig. 1. Kinetics of activity against C. albicans.

Fig. 1

Fig. 1

Fig. 1

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5×105cfu C. albicans ATCC90028 were incubated with the indicated compounds at either 50μg/ml (A) or 10μg/ml (B) for the times indicated. Viable cfu were quantified by plating. Results shown are the mean fold reduction in cfu +/- standard deviation of three independent experiments.

Activity against C. albicans biofilms

To examine the ability of the compounds to kill C. albicans in a biofilm, cells were grown on saliva-coated squares of polymethylmethacrylate as described (Chandra et al., 2001). They were then treated with increasing concentrations of either mPE or PMX30016 for 24 hours, and cell viability was measured by XTT assay. The results in figure 2A clearly demonstrate that incubation with both compounds resulted in a significant reduction of viable C. albicans in this model of Candida infection. To confirm that the reduction in the metabolic activity measured by the XTT assay reflects Candidacidal activity, a parallel biofilm culture treated with PMX30016 was scraped from the denture material, dispersed by vortex and plated to determine viable blastoconidia. The results in figure 2B show a direct relationship between metabolic activity as measured by XTT assay and viable organisms.

Fig. 2. Activity of peptide mimetics against biofilms of C. albicans.

Fig. 2

Fig. 2

Fig. 2

Fig. 2

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Biofilms were grown on saliva-coated denture material (A, B) or in saliva-coated wells of a chamber slide (C, D) for three days. Compounds were added to the growth medium for 24 hours, and metabolic activity was measured by XTT assay (n=3; results are mean +/- standard deviation) (A). In panel B, activity was measured by incubating biofilms with PMX30016, followed by plating the fungi to quantify viable organisms (results shown are representative of three independent experiments). Visualization of killing was carried out by Live/Dead staining followed by confocal fluorescent microscopy (C). D, Time course of exposure to 100μg/ml mPE with Live/Dead staining.

To visualize the killing, we treated simple biofilms of C. albicans grown on saliva-coated plastic with 0, 50 or 100μg/ml mPE for 24 hours. The medium was removed, and the Candida were stained with SYTO9 and propidium iodide and visualized by fluorescence microscopy. While this stain is usually used to visualize bacterial viability, it has been demonstrated to be effective in studying Candida biofilms as well (Jin, et al., 2005). In this case, the green dye (SYTO9) stains cells regardless of viability, while the red dye (propidium iodide) stains only cells with compromised membranes. The results in Figure 2C demonstrate that mPE treatment results in increased membrane permeability, which allows the red fluorescent stain entrance into the fungi. Further examination of biofilms that were treated with 100μg/ml mPE for 0, 1 or 5 minutes showed the rapid development of membrane permeability, with visible staining within 1 minute of exposure (Figure 2D)

Development of resistance

To determine whether growth in sub-MIC concentrations of the drugs would lead to resistant organisms, we grew C. albicans 90028 in 0.5× MIC of the compounds (or fluconazole as a positive control) for 20 serial passages. After every second passage, the MIC was quantified on the growing organisms. The results shown in figure 3 demonstrate that no resistance developed over the passages, in contrast to fluconazole, which rapidly led to resistance.

Fig. 3. Development of resistance to mimetics.

Fig. 3

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C. albicans 90028 was grown in 0.5× MIC of the compounds listed, at 35°C. At every second passage, a sample was tested for MIC by standard methods.

Factors that modify in vitro activity

Since a variety of physiological factors, including salt, serum proteins and factors found in saliva, are known to inhibit antimicrobial activity, we determined the effect of these factors on the activity of the compounds against C. albicans. Our results shown in table 1 demonstrate that there was no inhibition observed by saliva (and actually some increase in activity). These results support the further examination of the compounds as oral anti-fungal drugs. While other antimicrobial peptides exhibit reduced anti-candidal activity in the presence of salt (Vylkova et al., 2007), our assays are carried out in physiological salt (154mM), demonstrating that there is no detrimental effect of salt on this activity.

Table 1. IN VITRO ACTIVITY OF MIMETICS IN SALIVA.

Fold change in MIC
Saliva concentration (%) mPE PMX30016 PMX519 PMX10149
0 0 0 0 0
10 0.5 0 0 0
25 0.5 0.5-0 1 0
50 0.5 0.5 1 0
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To examine the potential for combination therapy with other antifungals, we determined the activity of mPE and PMX30016 in the presence of the common oral antiseptic agent, chlorhexidine and the standard antifungal agent, itraconazole. The results in table 2 demonstrate that the compound exhibits potent synergistic activity (i.e., an FIC index<1) with itraconazole, suggesting that it could be used in conjunction with this compound

Table 2. SYNERGISTIC ACTIVITY OF MIMETICS.

Compound 1 Compound 2 FIC Index
mPE Itraconazole 0.5
PMX30016 Itraconazole 0.2
PMX30016 Chlorhexidine 1
mPE Chlorhexidine 1
mPE PMX30016 1
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Discussion

Oral candidiasis is routinely treated with topical and systemic antifungal agents including azoles such as fluconazole and itraconazole (Vazquez, 2000). However, resistance to these agents is a common occurrence, and together with the immunodeficiency often found in these patients, failure of standard therapies suggests novel strategies. Since their discovery, AMPs have been suggested as a useful source of antimicrobial therapeutic agents, especially due to the low development of resistance (Diamond, et al., 2009)). Histatins are naturally occurring antifungal peptides found in human saliva, that exhibit potent activity against C. albicans (Oppenheim et al., 1988) (Helmerhorst et al., 1997). Anti-candidal activity of other AMPs such as β-defensins (Vylkova et al., 2007), cathelicidins (Benincasa et al., 2006) and piscidins (Sung et al., 2008) has also been described. Indeed, the normal expression of β-defensins may be necessary for natural defense against Candida growth (Conti et al., 2009). However, numerous technical and biological issues have made their development into useful drugs difficult. We have previously demonstrated that small mimetics based on AMP structures are highly active against a wide variety of bacterial species, including biofilms of Streptococcus mutans (Beckloff et al., 2007, Tew et al., 2006). Here we show proof-of-principle for these compounds exhibiting potent antifungal activity against Candida species, in both planktonic and biofilm forms, and are active in the presence of saliva.

The rapidity with which killing is observed, together with the evidence that membrane permeability occurs with a short exposure suggests that these mimetics, like their AMP models, act on membranes, although this might not be the primary target of some peptides (for review see (Brogden, 2005)). Indeed, the phenylethynylene derivative we use here, mPE (Nusslein et al., 2006, Arnt et al., 2006), is able to cause leakage of dyes from phospholipid vesicles, at concentrations similar to those required to kill bacteria. It also promotes the loss of the membrane potential in bacteria at concentrations and over a time course consistent with its antimicrobial activity. Furthermore, the lack of resistance development after 20 passages at sub-MIC levels, similar to what is found with many peptides, is suggestive of physical action on membranes, as opposed to intracellular activity against a metabolic target such as is seen with histatins (Kavanagh and Dowd, 2004). This is supported by recent work using short antimicrobial peptides, demonstrating membrane activity as determined by dye-leakage and membrane depolarization experiments, as well as visualization by electron microscopy (Zhou et al.). Further studies on the mechanism of action of these mimetics, both against single organisms and on the biofilms, will allow a direct comparison with peptides, and will lead to a greater understanding of their activity in order to design the optimal structures.

Together, our results support the development of these compounds as therapies against oral infections such as candidiasis. The low toxicities against mammalian cells (Tew et al., 2006), activity against biofilms grown on denture materials, and the lack of resistance found with the mimetics are important features for new anti-Candida agents.

Supplementary Material

Supp fig S1

Acknowledgments

The authors thank Dr. Isaac Rodriguez-Chavez, Director, NIDCR AIDS and Immunosuppression Program, for his scientific input in this manuscript. The authors thank Dr. David Perlin and Dr. Steven Park of the Public Health Research Institute for their helpful advice. This work was supported by US Public Health Service grant R43 DE18371 to RS and GD.

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Supplementary Materials

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