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Antimicrobial Agents and Chemotherapy, March 1999, p. 702-704, Vol. 43, No. 3
Academic Centre for Dentistry (ACTA),
Received 20 August 1998/Returned for modification 3 November
1998/Accepted 17 December 1998
The present study shows that a number of basic antifungal peptides,
including human salivary histatin 5, a designed histatin analog
designated dhvar4, and a peptide from frog skin, PGLa, are active
against amphotericin B-resistant Candida albicans, Candida krusei, and Aspergillus fumigatus
strains and against a fluconazole-resistant Candida
glabrata isolate.
The AIDS epidemic, improved
life-sustaining therapy, and aggressive anticancer therapy have
contributed to the rise in the number of severely immunocompromised
patients. This has led to an increase in mucosal and systemic
fungal infections, and the concomitant increased usage of antifungal
agents for prophylaxis is most likely the main cause of the
development of antifungal drug resistance (21).
As most of the currently available drugs are directed against the
ergosterol moiety in the fungal membrane (polyene antimycotics) or
against enzymes involved in the biosynthesis of ergosterol (azole
antimycotics) (19) there is a threat of cross-resistance (7, 21) and a clear demand for a new class of antimycotics with a different cellular target. Peptide antibiotics, which are believed to interact with the microbial membrane leading to the disruption of cellular integrity and cell death, may be a promising new
class of antifungal agents (3).
Histatins are natural human salivary peptides with strong fungicidal
activities in vitro and have proven to be valuable design templates for
the development of analogs with improved antifungal activities
(5). The aim of the present study was to test the antifungal
activities of a number of natural and designed basic antifungal
peptides against yeasts and fungi that are isolated from clinical
specimens with increasing frequency, such as Candida glabrata, Cryptococcus neoformans, and
Aspergillus fumigatus (4, 8), and against
fluconazole- or amphotericin B-resistant mutants. Fungicidal activities
were compared to the activity of the amphipathic macrolide antimycotic
drug amphotericin B, which is considered the "gold standard" in the
treatment of disseminated candidosis (21).
Human salivary histatin 5 (sequence,
DSHAKRHHGYKRKFHEKHHSHRGY), an amphipathic analog of the
C-14 terminus designated dhvar4 (KRLFKKLLFSLRKY), and a negative
control peptide, dcysS, derived from cystatin S
(SSSKEENRIIPGGI) were chemically synthesized as described
previously (5, 20). An antifungal peptide of amphibian origin, PGLa (15), was chemically synthesized and kindly
provided by H. V. Westerhoff (Department of Microbial Physiology,
Vrije Universiteit, Amsterdam, The Netherlands). The peptides
were dissolved to a concentration of 2 mg/ml in 10 mM potassium
phosphate buffer (PPB), pH 7.0, and stored at The fungicidal activities of these peptides were tested by incubating
an inoculum of 2 × 107 yeast blastoconidia with a
dilution series of peptide in 1 mM PPB, pH 7.0. After 1.5 h of
incubation, 50-µl samples were diluted in 9 ml of phosphate-buffered
saline, and viability was determined by plating 25 µl on Sabouraud
dextrose agar (Oxoid, Hants, United Kingdom), as previously described
(5). From the obtained killing curves, the concentrations of
peptide giving 50% reduction in viable counts (IC50s) were
determined (Table 1). Candida pseudotropicalis 311 (RIVM
4135), Candida albicans 315 (ATCC 10231), Candida
krusei 355 (ATCC 6258), and Candida parapsilosis 356 (ATCC 90018) were all highly susceptible to histatin 5, dhvar4, and
PGLa, with IC50s ranging from 0.3 to 2.4 µM. Also
C. neoformans 316 (RIVM 39231), the causative agent of
severe cryptococcosis in AIDS patients, was very susceptible to these
peptides. For histatin 5, similar results have been reported for three
clinically isolated C. neoformans strains
(17). C. glabrata 359 was markedly less
susceptible to histatin 5 and PGLa than the other strains tested;
IC50s of 29 and 8.5 µM, respectively, were
observed. The susceptibilities to amphotericin B differed among the
strains tested. Most strikingly, C. krusei 355 was
resistant (IC50s > 70 µM). This and previous findings (10) might point to an intrinsically reduced
susceptibility of C. krusei to amphotericin B.
A. fumigatus is a mold which causes life-threatening
infections in granulocytopenic patients. A clinical isolate from
sputum, A. fumigatus X 807219, was grown on Sabouraud
dextrose agar for 4 days at 30°C. To separate the conidia from
the conidiophores, the agar was covered with 20 ml of sterile 1 mM PPB,
pH 7.0, followed by filtration of the mycelial suspension over a
sterile cotton plug fitted as described previously (11). The
filtrate, containing 2 × 107 colony-forming
conidia/ml, was used in a killing assay. Conidia of A. fumigatus were resistant to the killing activity of amphotericin B
(Fig. 1). In contrast, the
IC50s of histatin 5, dhvar4, and PGLa for A. fumigatus conidia could be determined (IC50s, 18, 18, and 9 µM, respectively).
Beside the selection of fungi with lower intrinsic susceptibility
during therapy, resistant mutants may also arise as a result of
selection during treatment. A fluconazole-resistant C. glabrata strain (B 57149), isolated from a patient receiving
a combination therapy of fluconazole and ciprofloxacin
(12), was kindly provided by M. Marichal (Janssen Research
Foundation, Beerse, Belgium) and included in our study. The molecular
characterization of this strain has revealed that the acquired
resistance was due to an increased copy number of the CYP51 (ERG11)
gene encoding 14
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Amphotericin B- and Fluconazole-Resistant
Candida spp., Aspergillus fumigatus, and Other
Newly Emerging Pathogenic Fungi Are Susceptible to Basic
Antifungal Peptides
ABSTRACT
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FIG. 1.
Killing activities of histatin 5, dhvar4, PGLa, and
amphotericin B against A. fumigatus conidia. Error bars
indicate standard deviations.
-lanosterol demethylase, which is involved in the
ergosterol biosynthesis route (12). In a killing assay it
was found that this strain was very susceptible to basic peptides and
even more susceptible than fluconazole-sensitive C. glabrata 359 (IC50s, 1.5 and 29 µM, respectively;
Table 1). Interestingly, for recombinant
histatin 5, a similar result has been reported: an azole-resistant
C. glabrata strain overexpressing 14-lanosterol
demethylase was more susceptible to histatin 5 than an azole-sensitive
C. glabrata strain (18).
TABLE 1.
Killing activities of basic antifungal peptides and
amphotericin B against pathogenic yeasts
With increasing frequency case studies report on azole-resistant
Candida spp. that are cross resistant to amphotericin B
(1, 2, 9, 13, 16). Kelly et al. (6, 7) recently
described two clinical fluconazole-resistant C. albicans isolates defective in sterol
5,6-desaturation. As a result, these mutants synthesized
14-methylfecosterol instead of ergosterol and in consequence were
cross resistant to amphotericin B. The emergence of this kind of
cross-resistant strain is a threatening development and underlines the
requirement for new antifungals with a different target. We tested
whether a mutant of C. albicans (ATCC 32354) lacking
ergosterol was susceptible to basic peptides. This strain was a
generous gift from J. Brajtburg (Washington University School of
Medicine, St. Louis, Mo.) and was raised by random mutagenesis and
selected by subculturing on amphotericin B-containing plates
(14). In Fig. 2A and B it is
shown that the ergosterol-deficient mutant, in contrast to the
wild-type strain, was resistant to amphotericin B (IC50 > 70 µM) but susceptible to histatin 5, dhvar4, and PGLa
(IC50s, 2.5, 0.9, and 3.5 µM, respectively). These
results indicate that the antifungal target of the antibiotic peptides
tested is not the ergosterol moiety in the yeast membrane.
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In conclusion, this study demonstrates that newly emerging strains that are untreatable with currently available antimycotics are susceptible to natural and designed basic antimicrobial peptides. These peptides could possibly provide an attractive alternative for or a supplement to classic antimycotics in the treatment of persistent fungal infections.
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ACKNOWLEDGMENTS |
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This project was financially supported by the Dutch Technology Foundation (STW project VTH44.3302) and by Unilever Research, Oral Care, Bebington, United Kingdom.
The generous gifts of the mutant Candida strains from J. Brajtburg and P. Marichal are gratefully acknowledged.
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FOOTNOTES |
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* Corresponding author. Mailing address: ACTA, Department of Oral Biochemistry, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Phone: 3120-4448674. Fax: 3120-4448685. E-mail: EJ.Helmerhorst.obc.acta{at}med.vu.nl.
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Antimicrobial Agents and Chemotherapy, March 1999, p. 702-704, Vol. 43, No. 3
0066-4804/99/$04.00+0
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