Antifungal activities of human beta-defensins HBD-1 to HBD-3 and their C-terminal analogs Phd1 to Phd3.

Abstract

The activities of defensins HBD-1, HBD-2, and HBD-3 and their C-terminal analogs Phd1, Phd2, and Phd3 against Candida albicans were investigated. Phd1 to Phd3 showed lower-level activities than HBD-1 to HBD-3, although metabolic inhibitors did not render Phd1 to Phd3 inactive. Their activities were also less salt sensitive than those of HBD-1 to HBD-3. Confocal microscope images indicated that the initial site of action was the fungal membrane.

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2009, p. 256–260
0066-4804/09/$08.00?0doi:10.1128/AAC.00470-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 53, No. 1
Antifungal Activities of Human Beta-Defensins HBD-1 to HBD-3 and
Their C-Terminal Analogs Phd1 to Phd3?
Viswanatha Krishnakumari, Nandini Rangaraj, and Ramakrishnan Nagaraj*
Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500 007, India
Received 9 April 2008/Returned for modification 6 July 2008/Accepted 14 September 2008
The activities of defensins HBD-1, HBD-2, and HBD-3 and their C-terminal analogs Phd1, Phd2, and Phd3
against Candida albicans were investigated. Phd1 to Phd3 showed lower-level activities than HBD-1 to HBD-3,
although metabolic inhibitors did not render Phd1 to Phd3 inactive. Their activities were also less salt sensitive
than those of HBD-1 to HBD-3. Confocal microscope images indicated that the initial site of action was the
fungal membrane.
Mammalian defensins comprising the alpha and beta fami-
lies are important components of the innate immune system (1,
8, 17, 18, 24, 25, 29, 30). HBD-1 and HBD-2 are active against
gram-negative bacteria. Their activities are attenuated by in-
creasing concentrations of NaCl (2, 9, 10). HBD-3 is active
against both gram-negative and gram-positive bacteria and is
not affected by NaCl (3, 11). The findings of extensive studies
have indicated that native disulfide bridges are not essential for
antibacterial activity and that segments of HBD-1 to HBD-3
shorter than the full-length defensins also exhibit antibacterial
activities (12–16, 21, 22, 26, 28, 35, 36). In recent years, there
has been considerable interest in the antifungal activities of
beta-defensins, as Candida albicans is responsible for causing
oral candidiasis, particularly in patients infected with human
immunodeficiency virus (5, 20). HBD-1 to HBD-3 have been
detected previously in salivary glands and salivary secretions
(4, 6, 7, 23, 27). The killing of C. albicans by HBD-2 and
HBD-3 is salt sensitive and energy dependent (33). We have
shown that single disulfide peptides spanning the C-terminal
segments of HBD-1 to HBD-3, i.e., Phd1 (ACPIFTKIQGTY
RGKAKCK), Phd2 (FCPRRYKQIGTGLPGTKCK), and
Phd3 (SCLPKEEQIGKSTRGRKCRRKK) (disulfide bridges
indicated by underlining), exhibit antibacterial activities (16).
In this report, we describe their activities against C. albicans
and compare the effects of salts and metabolic inhibitors on
these peptides with the effects on HBD-1 to HBD-3.
HBD-1, HBD-2, and HBD-3 were purchased from Peptides
International (Louisville, KY). Phd1, Phd2, and Phd3 were
synthesized as described earlier using 4-(hydroxymethyl)phe-
noxyacetamidomethyl resin and 9-fluorenylmethoxy carbonyl
chemistry (16). The formation of disulfide bonds was accom-
plished by air oxidation at a peptide concentration of 0.5 mg/ml
for 24 h at room temperature. Purified peptides were charac-
terized by matrix-assisted laser desorption ionization–time of
flight mass spectrometry on an ABI Voyager DE STR matrix-
assisted laser desorption ionization–time of flight mass spec-
trometer (PerSeptive Biosystems) using recrystallized ?-cyano-
4-hydroxycinnamic acid as a matrix (16). Peptide labeling with
carboxyfluorescein (CF) at a free amino group of the N-ter-
minal amino acid was carried out by treating 10 mg of resin-
bound peptide with 0.8 ml of dimethylformamide containing
CF and activating agents as described earlier (34). The depro-
tection of CF-labeled peptides (CF-Phd1 to CF-Phd3) from
the resin, purification, and characterization by mass spectrom-
etry were carried out as described earlier (16).
The activities of HBD-1 to HBD-3 and Phd1 to Phd3 in final
volumes of 50 ?l against C. albicans (ATCC 18804) in sterile
96-well plates were determined as described previously (33),
with slight modifications. Briefly, minimum fungicidal concen-
trations (MFC) of the peptides were determined by growing C.
albicans aerobically in yeast extract-peptone-dextrose (YEPD)
medium at 30°C. After 20 h, 0.5 ml from this suspension was
subcultured for 2 h in 20 ml of YEPD broth to obtain a
mid-log-phase culture. Cells were harvested by centrifugation,
washed with 10 mM phosphate buffer (PB), pH 7.4, and resus-
pended in the same buffer, and the concentration was adjusted
to 106cells/ml. Aliquots of diluted cells were incubated with
peptides in 50-?l volumes at 30°C for 2 h. Cell suspensions
were diluted and plated onto YEPD agar plates, and the plates
were incubated for 24 h at 30°C. Colonies were counted, and
the concentrations of the peptides at which no viable colonies
were formed were taken as the MFC. The averages of results
from three independent experiments done with duplicate sam-
ples were taken for the calculation of MFC. In order to deter-
mine ion specificity, various concentrations of NaCl, CaCl2,
and MgCl2were added to the incubation buffer. For the ex-
periments evaluating the energy requirements, mid-log-phase
cells (106/ml) in PB were preincubated with 5 mM sodium
azide or 50 ?M carbonyl cyanide m-chlorophenylhydrazone
(CCCP) (19) for 2 h at 30°C with shaking before being treated
with peptides.
Intracellular localization was analyzed by treating C. albi-
cans with CF-Phd1, CF-Phd2, and CF-Phd3 (at 50% of the
MFC) and propidium iodide (PI) for 15 min at 30°C. The cells
were examined with a Zeiss LSM 510 META confocal micro-
scope. Optical sectioning was done at 1 airy unit by using the
488- and 543-nm-wavelength laser lines with a 63? water lens
objective. Emission data were collected using 500- to 530-nm
* Corresponding author. Mailing address: Centre for Cellular and
Molecular Biology, Council of Scientific and Industrial Research, Up-
pal Road, Hyderabad 500 007, India. Phone: 91-40-27192589. Fax:
91-40-27160591. E-mail: nraj@ccmb.res.in.
?Published ahead of print on 22 September 2008.
256

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band-pass and 565- to 615-nm band-pass filters for CF and PI,
respectively, in the multitrack mode. Z-sections were acquired
at 0.35-?m intervals and projected using the LSM-FCS soft-
ware version 3.2. The bright-field images were obtained simul-
taneously using the transmitted-light detector. The images
were assembled using Adobe Photoshop version 6.
Membrane permeabilization of C. albicans was determined
using the fluorescent dye Sytox green (Molecular Probes, Eu-
gene, OR) (31). Mid-log-phase C. albicans cells (107CFU per
ml) were washed and resuspended in PB containing 1 ?M
Sytox green. A greater number of organisms were used in this
experiment than in the antifungal assays in order to detect
changes in fluorescence. Aliquots of diluted cells were mixed
with the peptide concentrations specified in the figure legends
in 0.5-ml cuvettes held at 30°C. All measurements were carried
out on a FluoroLog model 3-22 fluorescence spectrophotom-
eter (Jobin Yvon) at an excitation wavelength of 488 nm (slit
width, 2 nm) and an emission wavelength of 540 nm (slit width,
5 nm).
The hemolytic activities of Phd1 to Phd3 were determined
using human erythrocytes as described earlier (32). Briefly,
erythrocytes were obtained by the centrifugation (800 ? g) of
heparinized blood and were washed three times with 5 mM
HEPES (pH 7.4) containing 150 mM NaCl. Aliquots contain-
ing 107red blood cells/ml were incubated in the presence of
different peptide concentrations in 0.5-ml tubes containing a
final volume of 100 ?l for 30 min at 37°C with gentle mixing.
The samples were centrifuged, and the absorbance of the su-
pernatants at 540 nm was measured. The level of erythrocyte
lysis occurring with 0.1% Triton X-100 was taken as the max-
imal level of lysis.
The antifungal activities of HBD-1 to HBD-3 and Phd1 to
Phd3 are summarized in Table 1. We observed that HBD-1,
obtained from Peptides International, showed substantial ac-
FIG. 1. Effect of metabolic inhibitors on the candidacidal activities
of HBD-1 to HBD-3 and Phd1 to Phd3 against C. albicans. Cells
pretreated with 50 ?M CCCP or 5 mM sodium azide for 2 h at 30°C
were further incubated with peptides at the MFC in PB for 2 h at 30°C.
Suitably diluted aliquots were plated onto YEPD agar plates, which
were incubated for 24 h. The colonies formed were counted, and the
percent killing of C. albicans cells was determined. Shaded bars and
dark bars represent peptide activities in the presence of 50 ?M CCCP
and 5 mM sodium azide, respectively. The values represent averages of
results from three independent experiments done with duplicate sam-
ples, and variations were 3%.
FIG. 2. Effect of salts on candidacidal activities of HBD-1 to
HBD-3 and Phd1 to Phd3 against C. albicans. Cells were incubated
with peptides at their MFC in PB containing different concentrations
of salts for 2 h at 30°C. Suitably diluted aliquots were incubated for
24 h on YEPD agar plates. The colonies formed were counted, and the
percent killing of C. albicans cells was determined. Panels: A, 25 mM
NaCl and 100 mM NaCl; B, 0.5 mM CaCl2and 5 mM CaCl2; and C,
0.5 mM MgCl2and 25 mM MgCl2. Light bars and dark bars represent
lower and higher concentrations of salts, respectively. The values rep-
resent averages of results from three independent experiments done
with duplicate samples.
TABLE 1. Antifungal activities of human beta-defensins HBD-1 to
HBD-3 and C-terminal analogs Phd1 to Phd3
Peptide
MFC (?M)afor
C. albicans
HBD-1........................................................................................... 7
Phd1...............................................................................................18
HBD-2........................................................................................... 8
Phd2...............................................................................................18
HBD-3........................................................................................... 2.5
Phd3...............................................................................................16
aThe values reported are averages of results from three different experiments
done with duplicate samples, and variations were 3%.
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tivity. Phd1 to Phd3 showed activities against C. albicans, but
with lower potencies than those of HBD-1 to HBD-3.
The data shown in Fig. 1 compare the candidacidal activities
of the peptides at the MFC in the presence of CCCP and
sodium azide. HBD-3 and analogs Phd1 to Phd3 were active in
the presence of sodium azide and CCCP, whereas HBD-1 and
HBD-2 were inactive. The results indicate that HBD-3 and
Phd1 to Phd3 kill C. albicans by energy-independent mecha-
nisms, unlike HBD-1 and HBD-2.
The effects of different concentrations of salts on the candi-
dacidal activities of HBD-1 to HBD-3 and Phd1 to Phd3 at
their MFC are indicated in Fig. 2. The data in Fig. 2A show
that Phd1 and Phd2 exhibited activities at 25 mM NaCl, unlike
HBD-1 and HBD-2, which were inactive. HBD-3 and Phd3
showed comparable activities at 25 mM NaCl. At 100 mM
NaCl, all peptides at their MFC showed very little activity
whereas 50% killing was observed at double their MFC (data
not shown). As summarized in Fig. 2B and C, Phd1 and Phd2
showed considerably greater activities than HBD-1 and HBD-2
in the presence of 0.5 mM Ca2?or Mg2?whereas HBD-3 and
Phd3 showed comparable activities. At a 5 mM CaCl2concen-
tration, Phd1 and Phd2 were inactive, like HBD-1 and HBD-2,
while HBD-3 exhibited greater activity than Phd3. Unlike the
full-length peptides HBD-1 to HBD-3, Phd1 and Phd2 were
active at 0.5 mM MgCl2while Phd3 was active even at 25 mM
MgCl2.
The cellular localization of Phd1 to Phd3 in C. albicans was
investigated using CF-Phd1 to CF-Phd3 and confocal micros-
copy analysis as presented in Fig. 3. The cells exhibited intense
fluorescence at the locations indicated in the fluorescence im-
ages and the corresponding bright-field images. The data in-
dicate that the peptides were localized on the membrane. A
diffuse intracellular staining pattern was also observed, which
indicates the translocation of the peptides into the cells. These
cells showed intense PI staining, indicating membrane damage
(Fig. 3B, C, and D). Control cells showed negative staining for
PI (Fig. 3A).
Membrane damage was also assessed by an increase in flu-
orescence due to the influx of Sytox green, a high-affinity nu-
cleic acid stain that does not cross the membranes of live cells.
However, it penetrates cells with damaged plasma membranes
and binds to nucleic acids, resulting in the enhancement of its
FIG. 3. Confocal microscope images showing the localization of CF-Phd1, CF-Phd2, and CF-Phd3 incubated with C. albicans. Cells were
treated with 50% MFC of peptide and 4 ?g/ml of PI. Arrows in the panels show membranes in the fluorescence images (CF-peptide) and also in
the corresponding bright-field (BF) images. Panels: A, control cells without peptide; B, CF-Phd1; C, CF-Phd2; and D, CF-Phd3. The bar represents
5 ?m.
258KRISHNAKUMARI ET AL.ANTIMICROB. AGENTS CHEMOTHER.

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fluorescence intensity (31). The data shown in Fig. 4 indicate
that Phd1 and Phd2 caused greater membrane permeabiliza-
tion than the parent peptides HBD-1 and HBD-2. However,
HBD-3 showed more fluorescence enhancement than Phd3.
Although the data shown in Fig. 4 correspond to changes in
fluorescence at one concentration, the increase in fluorescence
was concentration dependent. HBD-3 and Phd3 caused
greater membrane permeabilization than HBD-1 and HBD-2
and Phd1 and Phd2, respectively.
Phd1 to Phd3 showed no hemolytic activities at concentra-
tions of up to 75 ?M, which exceeds the MFC by three- to
fourfold. At 100 ?M, 15% lysis was observed.
Although Phd1 to Phd3 were less active than HBD-1 to
HBD-3, their activities were not lost in the presence of meta-
bolic inhibitors. Also, the activities were less salt sensitive than
those of the parent peptides HBD-1 and HBD-2. Hence, Phd1
to Phd3 and possibly the C-terminal regions of other defensins
may be attractive candidates for development as therapeutic
agents as well as for analysis to understand the mechanism of
action.
Funding from CSIR Network project NWP-05 is gratefully acknowl-
edged.
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