Activity of novel non-amphipathic cationic antimicrobial peptides against Candida species

Abstract

Background and objectives: Candida species are problematic opportunistic pathogens in the hospital setting, where they are frequently associated with opportunistic infections of indwelling medical devices. There are only a few effective classes of antifungal agents currently available, and some species, such as Candida lusitaniae, Candida glabrata and Candida krusei, are intrinsically resistant to some of these drugs, further reducing existing therapeutic options. We have recently developed synthetic, non-amphipathic cationic antimicrobial peptides (CAPs) based on the structure of native hydrophobic membrane-spanning domains of integral membrane proteins. In this article, we report on the activity of these CAPs and new variants thereof against eight Candida species.

Methods and results: Using a combination of MIC, haemolysis, time–kill and biofilm killing assays, we demonstrate activity of CAPs in the micromolar range against eight Candida species, with little toxicity to mammalian cells. The synthetic peptides killed both the fluconazole-susceptible and fluconazole-resistant strains of Candida albicans, Candida tropicalis and C. glabrata by 4 logs or more within 3 h, and also killed pre-formed yeast biofilms on plastic surfaces.

Conclusions: These peptides show promise as a basis for development of novel, broad-spectrum antimicrobial agents.

biofilms, fungi, CAPs, kaxins

Introduction

Candida species are the most common opportunistic fungal pathogens of mammals. Nosocomial fungal infections are associated with high levels of mortality.¹ Incidence of Candida infection has risen markedly in the past few decades, in part due to the heavy use of antibacterial agents combined with the availability of only a limited number of safe, effective antifungal drugs; in part due to the emergence of HIV/AIDS; and in part due to the increased use of immunosuppressive chemotherapeutic regimens.^2–4 The most common agents used to treat Candida infections are the triazoles (i.e. fluconazole and itraconazole) and the polyenes (i.e. amphotericin B). While fluconazole is considered a safe drug, even at relatively high concentrations, polyenes and to a lesser extent ketoconazole demonstrate toxicity at high doses.^5,6 Another problem is that development of resistance to one compound often results in cross-resistance to other members of the same class.⁷ Some yeast species demonstrate inherently reduced susceptibility to these compounds, for example Candida lusitaniae to amphotericin B and Candida krusei to fluconazole.⁸ The widespread use of triazoles for therapy has led to increases in the incidence of these naturally less-susceptible species.^9,10 The propensity of yeast infections to be associated with medical devices, on which the fungi grow as highly drug-tolerant biofilms,^11,12 further exacerbates the therapeutic challenge.

We described recently the development of a new class of antimicrobial peptides that were originally designed to mimic transmembrane segments of integral membrane proteins and were tagged with lysine residues to facilitate solubilization in aqueous media.¹³ These peptides have a non-amphipathic hydrophobic core segment, which distinguishes them from many natural linear cationic antimicrobial peptides (CAPs) that typically form amphipathic α-helices upon interaction with a hydrophobic membrane environment. These peptides (designated kaxins) have the prototypical structure KKAAAXAAAAAXAAWAAXAAAKKKK-amide, where X is one of the 20 naturally occurring amino acids. Only those peptides with a core segment above a specific hydrophobicity threshold on the Liu–Deber scale¹⁴ (i.e. those peptides in which position X is occupied by F, W, L, I, M, V, C, Y or A) demonstrated the ability to insert spontaneously into lipid micelles and had antibacterial activity in the micromolar range without toxicity to mammalian cells. We have shown previously that placing all of the K residues on the N-terminus and generating all-d-enantiomeric versions, in combination with decreasing the length of the hydrophobic segment, resulted in shorter (and therefore less expensive) peptides that generally displayed increased antimicrobial activity.¹³

In the present work, we tested the antifungal activity of a subset of those peptides previously shown to have good antibacterial activity without mammalian cell cytotoxicity, and new derivatives thereof (Table 1), against clinical isolates of eight different pathogenic Candida species, specifically Candida albicans, Candida tropicalis, Candida parapsilosis, Candida dubliniensis, Candida glabrata, Candida guillermondii, C. krusei and C. lusitaniae, including strains resistant to fluconazole. The ability of the peptides to kill yeast biofilms was tested, and the time course of peptide killing was determined.

Table 1.

Cationic peptides used in this study

Peptide	Sequencea	Haemolytic activity, human erythrocytesb (standard deviation)	MIC (mg/L) versus Pseudomonas aeruginosa ATCC 27853	MIC (mg/L) versus Staphylococcus aureus ATCC 25923	Source or reference
Phe peptides
F17-6K	KKKKKKAAFAAWAAFAA-NH2	1.7% (±0.3%)	32	64	13
dF17-6K	kkkkkkaafaawaafaa-NH2	2.1% (±0.9%)	16	64	13
F18 amphipathic	KAFKKWAKFAAKAAKAAA-NH2	7.0% (±1.3%)	16	>128	this work
dF21-10K	kkkkkkkkkkaafaawaafaa-NH2	1.0% (±0.6%)	32	32	this work
Trp peptides
W17-6K	KKKKKKAAWAAWAAWAA-NH2	3.3% (±2.5%)	32	128	13
dW17-6K	kkkkkkaawaawaawaa-NH2	0.8% (±0.2%)	16	128	this work
5W17-6K	KKKKKKAWWAAWAAWWA-NH2	52.4% (±11.0%)	64	32	this work
dW17-Nva-6K	kkkkkka(Nva)wa(Nva)w(Nva)aw(Nva)a-NH2c	44.0% (±2.1%)	64	32	this work
Reference peptides
Magainin II amide	GIGKFLHAAKKFAKAFVAEIMNS-NH2	106.7% (±1.2%)	64	16	25
Cecropin P1	SWLSKTAKKLENSAKKRISEGIAIQGGPR-NH2	1.6% (±1.3)	16	>128	26

a

Peptide sequences in uppercase are l-enantiomers; those in lowercase are d-enantiomers. Bold font indicates the ‘guest residue’ positions within the peptides.

b

Haemolytic activity compared with Triton X-100 (100%) and buffer alone (0%).

c

Nva = norvaline = NHCH(CH₂CH₂CH₃)-CO-.

Materials and methods

Strains and growth conditions

Yeast strains of the eight separate Candida species used in this study are listed in Table 2. These strains include American Type Culture Collection reference strains and clinical isolates obtained from the Clinical Microbiology Laboratory of the Hospital for Sick Children, kindly provided by Dr Susan Richardson. The susceptibility of each strain to fluconazole as determined previously by the Clinical Microbiology Laboratory using NCCLS methodology (standard M27-A2)¹⁵ is indicated in Table 2. The strains were maintained at −80°C in tryptic soy broth supplemented with 15% glycerol. For experiments (given below), each strain was streaked for single colonies on Mueller–Hinton agar plates and incubated at 37°C overnight.

Table 2.

Average MIC values and ranges (mg/L) for the Candida strains tested

	Species and strain
	C. albicans
Antifungal or peptide	CAS16635	CAA60315	CA4128	CA02045	CA00681
Fluconazole	16	0.125	1	0.25	64
Magainin II	64 (32–64)	64 (64)	64 (32–64)	32 (32)	64 (64)
Cecropin P1	>256 (>256)	>256 (256 to >256)	>256 (256)	>256 (256)	>256 (256)
F17-6K	32 (16–32)	32 (32)	16 (8–16)	32 (32–64)	64 (32–64)
dF17-6K	32 (16–32)	32 (16–32)	8 (8–16)	16 (16)	32 (32)
W17-6K	64 (64)	32 (32–64)	64 (64)	32 (32)	64 (64)
dW17-6K	64 (64)	32 (32)	16 (16)	16 (16)	32 (32)
F18 amphipathic	64 (64)	64 (32–64)	32 (32)	32 (16–32)	64 (64)
dF21-10K	32 (32)	16 (16)	32 (32)	32 (16–32)	32 (32)
5W17-6K	32 (32)	32 (32)	64 (32–64)	32 (32)	32 (32)
dW17-Nva-6K	64 (32–64)	32 (32)	32 (32)	32 (32)	32 (32–64)

	C. dubliniensis
	CDTGH43	CDTGH44	CDTGH60	CDTGH74	CDTGH127
Fluconazole	2	1	0.125	0.5	1
Magainin II	128 (128)	128 (64–128)	64 (64–128)	64 (64)	64 (64)
Cecropin P1	256 (256)	256 (256)	128 (128)	256 (256)	256 (256)
F17-6K	64 (64)	64 (32–64)	32 (32)	32 (32)	32 (32)
dF17-6K	32 (32)	32 (32)	32 (32)	32 (32)	32 (32)
W17-6K	64 (64)	32 (32)	32 (32)	32 (32)	32 (32)
dW17-6K	32 (32–64)	32 (32)	32 (32)	16 (16)	32 (32)
F18 amphipathic	64 (64)	64 (32–64)	32 (32)	32 (32)	32 (32)
dF21-10K	32 (32)	32 (32)	32 (32)	32 (32)	32 (32)
5W17-6K	64 (32–64)	32 (32)	32 (32–64)	32 (32)	32 (32)
dW17-Nva-6K	64 (32–64)	32 (32)	32 (32–64)	32 (32)	32 (32)

	C. glabrata
	CG5701142	CG12659	CG24482	CG72511	CG29927
Fluconazole	>64	4	8	>64	16
Magainin II	256 (128–256)	64 (64)	32 (32)	256 (256)	128 (128)
Cecropin P1	>256 (>256)	>256 (256 to >256)	128 (128)	>256 (256 to >256)	>256 (>256)
F17-6K	64 (64)	64 (64)	32 (32)	64 (64)	32 (32–64)
dF17-6K	32 (32–64)	64 (64)	16 (16–32)	32 (32)	32 (32)
W17-6K	64 (64)	128 (64–128)	32 (16–32)	128 (128)	128 (128)
dW17-6K	64 (32–64)	64 (64)	32 (32)	64 (32–64)	64 (64)
F18 amphipathic	128 (128)	128 (128)	32 (32)	128 (64–128)	64 (64)
dF21-10K	32 (32)	64 (32–64)	32 (16–32)	32 (32)	32 (32)
5W17-6K	32 (32)	64 (32–64)	32 (32)	64 (32–64)	64 (64)
dW17-Nva-6K	32 (32)	32 (32–64)	32 (32)	32 (16–32)	64 (64)

	C. guillermondii
	CGuB305	CGuB3001220	ATCC 6260	CGu4326	CGu43409
Fluconazole	2	4	0.5	16	16
Magainin II	64 (64)	32 (32)	32 (32)	64 (64)	32 (32)
Cecropin P1	>256 (256 to >256)	>256 (256 to >256)	128 (128)	>256 (>256)	>256 (>256)
F17-6K	16 (16)	16 (16)	8 (8)	32 (32)	16 (16)
dF17-6K	16 (16)	16 (8–16)	8 (4–8)	8 (8–16)	16 (16)
W17-6K	16 (16)	16 (16)	8 (8)	32 (32)	16 (16)
dW17-6K	16 (16)	16 (16)	8 (4–8)	16 (16)	16 (16)
F18 amphipathic	16 (16)	16 (16)	8 (4–8)	32 (32)	16 (16)
dF21-10K	16 (8–16)	8 (8)	4 (4)	16 (8–16)	8 (8)
5W17-6K	16 (16)	16 (16)	8 (8–16)	32 (32)	16 (16)
dW17-Nva-6K	16 (16)	16 (16)	4 (4)	32 (16–32)	16 (8–16)

	C. kruzei
	ATCC 6258	CK99250874
Fluconazole	32	64
Magainin II	64 (64)	128 (64–128)
Cecropin P1	>256 (>256)	>256 (>256)
F17-6K	64 (32–64)	64 (64)
dF17-6K	16 (16)	32 (32)
W17-6K	64 (64)	128 (128–256)
dW17-6K	32 (32)	64 (64)
F18 amphipathic	64 (64)	128 (128–256)
dF21-10K	32 (32)	32 (32)
5W17-6K	64 (64)	128 (128)
dW17-Nva-6K	64 (32–64)	64 (64)

	C. lusitaniae
	CL6133550	CL5127558	CLW14898	ATCC 200951	ATCC 42720
Fluconazole	0.5	0.25	0.5	0.125	0.25
Magainin II	64 (64–128)	64 (64)	64 (64)	64 (64)	64 (64)
Cecropin P1	>256 (>256)	>256 (>256)	>256 (>256)	>256 (>256)	>256 (>256)
F17-6K	64 (64)	32 (32)	32 (32)	32 (32)	32 (32)
dF17-6K	32 (32)	32 (32)	32 (16–32)	32 (32–64)	16 (16)
W17-6K	64 (64)	32 (32)	32 (32–64)	32 (32–64)	64 (64)
dW17-6K	64 (32–64)	32 (16–32)	32 (32)	32 (32)	64 (64)
F18 amphipathic	64 (64–128)	32 (32–64)	32 (32)	32 (32)	32 (32)
dF21-10K	64 (64)	32 (32)	32 (32)	32 (16–32)	16 (16)
5W17-6K	64 (64)	32 (32)	32 (32)	32 (32–64)	32 (32)
dW17-Nva-6K	64 (64)	32 (32)	32 (32)	32 (32)	32 (32)

	C. parapsilosis
	CPA0220586	CPA5070333	ATCC 22019	CPA5051563	CPS20911
Fluconazole	0.5	1	2	0.5	2
Magainin II	>256 (>256)	>256 (256 to >256)	64 (32–64)	>256 (>256)	>256 (256 to >256)
Cecropin P1	>256 (>256)	>256 (>256)	>256 (>256)	>256 (>256)	>256 (>256)
F17-6K	32 (32–64)	32 (32–64)	16 (16–32)	32 (32)	32 (32–64)
dF17-6K	32 (32)	32 (32)	16 (16)	32 (16–32)	32 (32)
W17-6K	128 (64–128)	64 (64)	32 (32–64)	32 (32)	64 (32–64)
dW17-6K	64 (64)	64 (32–64)	32 (16–32)	32 (32)	32 (32)
F18 amphipathic	128 (64–128)	64 (64–128)	32 (16–32)	64 (32–64)	64 (64)
dF21-10K	16 (16–32)	32 (32)	16 (16–32)	16 (16)	32 (32)
5W17-6K	64 (64)	64 (64)	32 (32)	64 (64)	64 (64)
dW17-Nva-6K	64 (64)	64 (64)	32 (32)	64 (64)	64 (64)

	C. tropicalis
	CT72241607	CT3038192	CTA5300325	CTA2190735	CTT197865
Fluconazole	0.5	>64	1	0.25	0.125
Magainin II	64 (32–64)	64 (64)	64 (64)	32 (32–64)	64 (64)
Cecropin P1	128 (128–256)	>256 (>256)	>256 (>256)	128 (128–256)	>256 (>256)
F17-6K	64 (64)	64 (32–64)	128 (64–128)	32 (32)	256 (128–256)
dF17-6K	64 (64)	32 (16–32)	64 (64)	16 (16)	128 (64–128)
W17-6K	128 (64–128)	16 (16)	128 (128)	32 (16–32)	>256 (256–>256)
dW17-6K	64 (64)	16 (16)	128 (64–128)	16 (16–32)	128 (128)
F18 amphipathic	64 (64–128)	128 (128)	128 (64–128)	16 (16–32)	>256 (256–>256)
dF21-10K	64 (64)	16 (16)	32 (32)	16 (16)	32 (32)
5W17-6K	64 (64)	32 (32)	64 (64)	32 (32)	64 (16–64)
dW17-Nva-6K	128 (64–128)	32 (32)	64 (32–64)	32 (16–32)	64 (64)

Peptide synthesis

Peptides were synthesized as described previously¹³ using standard Fmoc technology and were purified subsequently using HPLC. The purity of each peptide was verified using mass spectroscopy analysis. Peptides were diluted in stock solutions to 2.6 mg/mL, divided into 500 µL aliquots and stored at −20°C.

Determination of MICs

MICs were determined twice, each time in triplicate, in 96-well polypropylene plates as described previously.¹³ The methodology that was used previously to determine bacterial MICs with the same peptides was used to facilitate direct comparisons of peptide efficacy across a broad range of cell types. Briefly, yeast strains were inoculated from fresh plates into Mueller–Hinton broth (MHB) and grown overnight at 37°C with 150 rpm shaking. The cells were harvested by centrifugation and resuspended in fresh MHB to a final concentration of 10⁷ cfu/mL, as determined by optical density at 600 nm and viable plate counts. Peptides were serially diluted from 256 to 0.25 mg/L in MHB (190 µL), and 10 µL of standardized yeast suspension was added to each well. Positive controls contained no peptide. Plates were incubated for 18 h at 37°C and the lowest concentration of the peptide preventing growth (indicated by lack of visible turbidity) was recorded. Positive growth controls (no peptide) demonstrated visible turbidity after 18 h at 37°C.

Erythrocyte lysis assay

The propensity of the peptides to cause lysis of erythrocytes was measured as described previously.¹³ Each experiment was repeated twice in duplicate. Briefly, freshly collected human erythrocytes were washed and resuspended in sterile PBS (14.4 g NaCl, 0.612 g NaH₂PO₄.H₂O and 2.178 g Na₂H₂PO₄.7H₂O per litre, pH 7.1) to 4% (v/v). Peptides were diluted in sterile PBS to 1300 mg/L, and 100 µL of the erythrocyte suspension and 100 µL of the peptide solution were added to the wells of a 96-well round-bottomed polypropylene microtitre plate. PBS and 0.1% Triton X-100 were used as agents for 0 and 100% haemolysis, respectively. Plates were incubated for 1 h at 37°C and centrifuged at 1000 g for 5 min. The supernatant (100 µL) was transferred to a 96-well flat-bottomed polystyrene plate, and the release of haemoglobin was monitored by measuring the absorbance at 540 nm using a microplate reader.

Time course of killing

To determine the kinetics of peptide killing, selected peptides were diluted in MHB to a final concentration that was twice the previously determined MIC for the strain to be tested. Using 96-well polypropylene plates, 190 µL aliquots of the peptide solution were inoculated with 10 µL of a standardized yeast suspension (10⁷ cfu/mL in MHB) of the test organism and the plate was incubated at 37°C with 100 rpm shaking for up to 4 h. Triplicate aliquots were removed at 30 min intervals, serially diluted and plated for the determination of viable counts.

Biofilm killing assay

C. albicans and C. tropicalis biofilms were tested for susceptibility to peptide killing using methods adapted from Brooun et al.¹⁶ Each experiment was repeated at least twice in triplicate. Washed overnight cultures of C. albicans (strain CA02045) or C. tropicalis (strain CT72241607) were suspended at a concentration of 10⁶ cfu/mL in MHB, and 200 µL aliquots were dispensed into a 96-well polystyrene plate. A corresponding 96-peg lid (Nunc) was immersed in the yeast suspensions to allow the formation of biofilms for 24 h at 37°C, with 100 rpm shaking to provide shear forces. The colonized pegs were rinsed in sterile PBS, pH 7.1, to remove non-adherent cells and were immersed in a second plate containing triplicate aliquots of 200 µL MHB without the peptide (control), dF17-6K or dF21-10K at 5 or 10 times the MIC determined previously for the test strain (Table 2). This plate was incubated as described above for 18 h. The pegs were rinsed in sterile PBS to remove non-adherent cells and then immersed in a third plate containing 200 µL of sterile PBS per well. The plate was subjected to 10 min of sonication in a sonicating water bath to dislodge biofilm organisms.¹⁷ Each aliquot was then vortexed (2 min) to minimize cell clumping and serially diluted for viable plate counts.

Results

Modification of peptide sequences

The sequences of peptides tested against the pathogenic yeast strains are shown in Table 1. In our previous study,¹³ the kaxin demonstrating the highest overall activity against both Gram-negative and Gram-positive bacteria was dF17-6K, a 17 amino acid d-enantiomeric peptide with six Lys residues at the N-terminus. A similar peptide containing Arg instead of Lys, dF17-6R, had essentially identical antibacterial activity but a marked increase in mammalian cell cytotoxicity, as measured by increased erythrocyte lysis. The variations introduced for this work were: (i) an increase in the number of Lys residues present on the N-terminus from 6 to 10, to raise the overall positive charge; (ii) substitution of Trp for Phe in the enantiomeric 17mers; and (iii) creation of a peptide containing four residues of the non-natural amino acid norvaline (Nva) in place of Ala. Similar to Val, Nva has a side chain containing three carbons, but they are arranged in a linear, rather than branched, configuration, in effect making the side chain longer.

Activity of optimized peptides against pathogenic yeast species

The MICs of the kaxins against a panel of 37 strains of eight Candida species were determined (Table 2) as described in the Materials and methods section. The MICs were determined in MHB for direct comparison with MICs of these peptides for representative Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria, which are shown in Table 1. The well-characterized CAPs magainin II amide and cecropin P1 (Table 1) were included as controls. On average, the peptides dF21-10K and dW17-Nva-6K showed the best antifungal activity of this group (MIC values ranging from 4 to 64 mg/L for both), with dF17-6K also showing good activity (MICs ranging from 8 to 64 mg/L), better than magainin II amide (MICs ranging from 32 to >256 mg/L). Cecropin P1 demonstrated relatively poor activity against the Candida strains tested (MICs of 128 to >256 mg/L). There were only minor differences in the activities of the peptides when tested against strains of Candida exhibiting low fluconazole MICs (<1 mg/L) versus high fluconazole MICs (>16 mg/L; Table 2), and the peptides showed activity against C. glabrata and C. krusei, which exhibit intrinsic resistance to fluconazole. With the exception of C. tropicalis, there was little intra-species variation in MIC of the peptides tested. To ensure that alteration of peptide sequences did not result in increased toxicity to mammalian cells, lysis of erythrocytes exposed to the peptides at 650 mg/L was determined as described previously¹³ (Table 1). Most peptides demonstrated very low levels of haemolysis, but magainin and kaxins containing five Trp residues (5W17-6K) or norvaline (dW17-Nva-6K) demonstrated significant haemolytic activity.

Time course of killing of pathogenic yeast

One of the important advantages of antimicrobial peptides over traditional antifungal agents is their ability to cause rapid killing via physical mechanisms,^18,19 which precludes development of resistance. We tested the time course of killing of fluconazole-susceptible and fluconazole-resistant strains of C. glabrata, C. tropicalis and C. albicans using the most effective non-haemolytic peptides dF21-10K and dF17-6K. Magainin II amide was used as the control peptide. Representative Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria were tested for comparison (data not shown). Figure 1 shows that the kaxins kill more rapidly than magainin II amide. Only C. tropicalis strains were killed (>6 log decrease) by magainin II amide within the 4 h test period (Figure 1a), whereas a proportion of the C. albicans and C. glabrata cells survived. In contrast, dF21-10K (Figure 1c) killed >6 logs each of C. tropicalis, C. albicans (both fluconazole-susceptible and fluconazole-resistant strains) and the fluconazole-susceptible strain of C. glabrata within the 4 h test period. Therefore, the kaxins are capable of more rapid and complete killing of yeast than the magainin II control, but there is some strain specificity, since the fluconazole-resistant C. glabrata strain was less susceptible to killing. Helmerhorst et al.²⁰ showed recently that unlike other yeast and fungal species, C. glabrata was insensitive to killing by the salivary antimicrobial peptides histatin 1, 3, 5 and P-113, and much less susceptible to killing by the amphibian-derived CAPs magainin II and PGLa. Extension of the exposure period or an increase in dose may be required to effect complete killing of more recalcitrant strains.

Figure 1.

Time course of killing. Replicate standardized suspensions of yeast cells were mixed with 2× MIC of the test peptide, determined previously for the strain of interest, and incubated using the procedure given in the text. Samples were serially diluted at 30 min intervals for viable plate counts. Standard error bars are too small to be visible in this format. Strains used were as follows: C. albicans susceptible, strain CA02045; C. albicans resistant, strain CA00681, resistant to fluconazole (MIC > 64 mg/L); C. glabrata susceptible, strain CG12659; C. glabrata resistant, strain CG5701142, resistant to fluconazole (MIC > 64 mg/L); C. tropicalis susceptible, strain CT72241607; and C. tropicalis resistant, strain CT3038192, resistant to fluconazole (MIC > 64 mg/L). (a) Magainin II amide; (b) dF17-6K; (c) dF21-10K.

Effect of antimicrobial peptides on yeast biofilms

Nosocomial fungal infections are frequently associated with medical devices, such as central lines, peritoneal dialysis catheters and urinary catheters, on which the fungi grow as adherent biofilms.^11,12 Previous studies have shown that growth in biofilms significantly increases resistance of yeast to killing with antifungal compounds. For example, Hawser and Douglas showed that, depending on the drug tested, from 30 to 2000 times the MIC for isogenic planktonic cells was required to reduce the metabolic activity of C. albicans biofilms by 50%.²¹ Numerous studies have confirmed the increased antifungal resistance of biofilms formed by several yeast species.¹¹

Peptides showing the best antifungal activity, dF17-6K and dF21-10K, were tested for their ability to kill pre-formed yeast biofilms. C. albicans and C. tropicalis, two species frequently associated with clinically-significant biofilm infections, were tested for biofilm susceptibility to the peptides. For both peptides, we chose to test the efficacy of 5 and 10 times the previously determined MIC for the strains to be tested, based on previous reports showing increased tolerance of fungal biofilms to antifungal agents. Figure 2 shows that at 5× MIC both peptides effected only small decreases in the numbers of viable cells recovered from the biofilms. At 10× MIC, dF17-6K reduced the number of viable biofilm cells by 2–3 logs, but dF21-10K was more effective, completely eradicating pre-formed yeast biofilms containing >10⁵ viable cells.

Figure 2.

Killing of biofilms by peptides. Biofilms of either C. albicans (strain CA02045) or C. tropicalis (strain CT72241607) were permitted to form on polystyrene pegs for 24 h as described in the Materials and methods section, prior to 18 h of exposure to 5× or 10× the previously determined MIC of the test peptide for each strain (see Table 2). Results are the average of two separate experiments performed in triplicate. Asterisks indicate no growth at any dilution on viable plate counts.

Discussion

In this work, we have shown that kaxins, as non-amphipathic cationic peptides, can kill clinical isolates of a wide range of pathogenic yeast species when applied at micromolar concentrations. Importantly, there was little difference in the MIC required to kill fluconazole-resistant strains compared with fluconazole-susceptible strains of the same species, suggesting that these peptides can be useful in the treatment of infections caused by strains that are less effectively treated with triazoles. Although in their current form this class of peptides has modest potency compared with small molecule drugs, with average MIC values in the 16–64 mg/L range for most strains, they may offer a basis for development of alternatives to traditional antifungal agents. Another important consideration is the potential for cytotoxicity to mammalian cells. Peptides containing five Trp residues (5W17-6K) or norvaline (dW17-Nva-6K) were haemolytic, as was the well-studied control peptide magainin II amide, making them less suitable for use as therapeutics even though the norvaline-containing peptide had good MICs of 4–64 mg/L for the Candida strains tested.

The best non-haemolytic peptide in the group tested, dF21-10K, demonstrated good killing kinetics, eradicating five of the six yeast strains tested in 4 h, including a highly fluconazole-resistant strain of C. albicans. Notably, this peptide had good activity against C. glabrata, a species known to exhibit intrinsic resistance to both triazoles and host antimicrobial peptides,^20,22 although, for reasons that are currently unknown, it was less effective against a strain with documented high-level fluconazole resistance. At 10× MIC, this peptide also killed pre-formed biofilms of C. albicans and C. tropicalis, two yeast species commonly isolated in the hospital setting. This represents true killing of the biofilm organisms, rather than dispersal, as the surrounding liquid medium was also sterilized (not shown). While consistent with the requirement for higher doses of antimicrobial agents due to the increased tolerance of biofilm organisms, the increased amount of peptide required for eradication was less than the 30–2000× MIC required for small molecule antifungals to kill biofilms.¹¹ In the future, it may be possible to reduce the amount of peptide required by attaching the peptides directly to the surface of biomaterials to prevent biofilm formation at the stage of initial microorganism attachment. This strategy has been shown to be successful for long-chain hydrophobic polycations, which are chemical mimics of antimicrobial peptides.²³ The technology for attachment of functional peptides to surfaces already exists,²⁴ and we are currently adapting it for use with antimicrobial peptides.

With regard to peptide composition, increasing the number of K residues on the N-terminus from 6 to 10 (compare dF17-6K with dF21-10K) appears to improve the ability of the peptides to kill Gram-positive bacteria and yeast while slightly decreasing the activity against Gram-negative bacteria, without altering the cytotoxicity of the peptide towards eukaryotic cells, based on a lack of increase in erythrocyte lysis. This result suggests that the increase in positive charge may improve interaction with—and/or promote penetration of—thick yeast cell walls. This improved dF21-10K peptide also shows good activity against important antibiotic-resistant Gram-positive bacteria, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci (VRE). MICs were 32–64 mg/L for MRSA and 32 mg/L for VRE (five strains of each tested, data not shown), the same concentration range that is effective against yeast. Therefore, these peptides show promise as the basis for development of novel, broad-spectrum antimicrobial agents.

Transparency declarations

The authors of this submission have no conflicts of interest to declare. The peptides described herein are the subject of a patent application filed by the Hospital for Sick Children Research Institute's Intellectual Property and Commercialization Development Office.

We thank Dr Yatika Kohli for assistance in the procurement of yeast strains and for provision of fluconazole MIC data. This work was supported, in part, by grants to L. L. B. from the Canadian Infectious Diseases Society and to C. M. D. from the Cystic Fibrosis Foundation of Canada and the National Institutes of Health. M. S. held a Sweden–America Foundation Award in 2001–2002. C. C. held a University of Toronto Open Fellowship. E. G. holds a CIHR Training Fellowship in Membrane Proteins Associated with Disease.

Author notes

1Infection, Immunity, Injury and Repair, Hospital for Sick Children Research Institute, Toronto, ON, Canada; 2Department of Surgery, University of Toronto, Toronto, ON, Canada; 3Structural Biology and Biochemistry Programs, Hospital for Sick Children Research Institute, Toronto, ON, Canada; 4Department of Biochemistry, University of Toronto, Toronto, ON, Canada