Cathelicidin-BF, a Snake Cathelicidin-Derived Antimicrobial Peptide, Could Be an Excellent Therapeutic Agent for Acne Vulgaris
Could Be an Excellent
Therapeutic Agent for Acne Vulgaris. PLoS ONE 6(7): e22120. doi:10.1371/journal.pone.0022120
Cathelicidin-BF, a Snake Cathelicidin-Derived Antimicrobial Peptide, Could Be an Excellent Therapeutic Agent for Acne Vulgaris
Yipeng Wang 0
Zhiye Zhang 0
Lingling Chen 0
Huijuan Guang 0
Zheng Li 0
Hailong Yang 0
Jianxu Li 0
Dewen You 0
Haining Yu 0
Ren Lai 0
Paul Proost, University of Leuven-Rega Institute, Belgium
0 1 Biotoxin Units of Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences , Kunming, Yunnan , China , 2 College of Life Sciences, Hebei Normal University , Shijiazhuang, Hebei , China , 3 School of Life Science and Biotechnology, Dalian University of Technology , Dalian, Liaoning, China, 4 Clinical Laboratory , The First Affiliated Hospital of Kunming Medical College , Kunming, Yunnan , China , 5 Graduate School of the Chinese Academy of Sciences , Beijing , China
Cathelicidins are a family of antimicrobial peptides acting as multifunctional effector molecules in innate immunity. Cathelicidin-BF has been purified from the snake venoms of Bungarus fasciatus and it is the first identified cathelicidin antimicrobial peptide in reptiles. In this study, cathelicidin-BF was found exerting strong antibacterial activities against Propionibacterium acnes. Its minimal inhibitory concentration against two strains of P. acnes was 4.7 mg/ml. Cathelicidin-BF also effectively killed other microorganisms including Staphylococcus epidermidis, which was possible pathogen for acne vulgaris. Cathelicidin-BF significantly inhibited pro-inflammatory factors secretion in human monocytic cells and P. acnesinduced O2.2 production of human HaCaT keratinocyte cells. Observed by scanning electron microscopy, the surfaces of the treated pathogens underwent obvious morphological changes compared with the untreated controls, suggesting that this antimicrobial peptide exerts its action by disrupting membranes of microorganisms. The efficacy of cathelicidin-BF gel topical administering was evaluated in experimental mice skin colonization model. In vivo anti-inflammatory effects of cathelicidin-BF were confirmed by relieving P. acnes-induced mice ear swelling and granulomatous inflammation. The antiinflammatory effects combined with potent antimicrobial activities and O2.2 production inhibition activities of cathelicidinBF indicate its potential as a novel therapeutic option for acne vulgaris.
. These authors contributed equally to this work.
Antimicrobial peptides play important roles in preventing
microorganism infections. Most of them are 1050 residues in
length. They can provide an effective and fast acting defense
against harmful microorganisms [1,2]. There are two major
vertebrate antimicrobial peptide families including cathelicidins
and defensins. Cathelicidins have been found in many
mammalians and birds. Recently, a few cathelicidin antimicrobial peptides
were identified from snake venoms [3,4]. They are the first report
of reptile cathelicidins.
Acne vulgaris is the most common skin disease. It often occured
in areas containing large skin oil glands, such as face, back, and
trunk . The pathogenesis of acne is currently attributed to
multiple factors such as hormonal factors, hyperkeratinization,
resident microbiota, sebum, nutrition, cytokines and toll-like
receptors . Propionibacterium acnes act on important roles in acne
pathogenesis although they belong to the resident microbiota. P.
acnes induce the expression of antimicrobial peptides and
proinflammatory cytokines/chemokines, which contribute to the
inflammatory responses of acne . Some P. acnes strains
may cause an opportunistic infection worsening acne lesions .
Antibiotics are employed as therapeutic agents for acne by
inhibiting inflammation or killing bacteria. However, antibiotic
resistance has been increasing in prevalence within the
dermatologic setting . Antimicrobial peptides have been considered as
new type of antimicrobial reagents because they have low potential
to induce drug resistance of microorganisms. The current work is
performed to evaluate the anti-P. acnes abilities of cathelicidin-BF
in vitro and in vivo.
Antimicrobial activities of cathelicidin-BF
As listed in Table 1, cathelicidin-BF showed strong
antimicrobial abilities against several microorganisms, which are related to
acne vulgaris. The MICs value of cathelicidin-BF, LL-37 and
clindamycin against two P. acnes strains are 4.7 mg/ml (1.3 mM),
9.4 mg/ml (2.2 mM), and 2.3 mg/ml (5.2 mM), respectively.
Cathelicidin-BF showed strong antimicrobial functions (MIC of
1.22.3 mg/ml, 0.330.65 mM) against two strains of S. epidermidis
while LL-37 has no activity against them (no amtimicrobial
activity was seen when the concentration of LL-37 was up to
200 mg/ml, 46.8 mM). Clindamycin showed only antimicrobial
ability (MIC of 1.2 mg/ml, 2.6 mM) against one (S.
epidermidis09B2490) of the two S. epidermidis strains. The MIC values of
cathelicidin-BF and LL-37 against S. aureus ATCC2592 are
4.7 mg/ml (1.3 mM for cathelicidin-BF, 1.1 mM for LL-37) while
that of clindamycin is 1.2 mg/ml (2.6 mM).
Bacteria killing kinetics
Using clindamycin as a positive control, antibacterial properties
of cathelicidin-BF against P. acnes ATCC6919 were tested by the
colony counting assay. As illustrated in Fig. 1, cathelicidin-BF
could rapidly exert its antibacterial activities. It just took less than
160 minutes to kill all the P. acnes at the concentration of one time
of MIC. The antibacterial activity was proved to be lethal for P.
acnes. P. acnes were not capable of resuming growth on agar plates
after a 6-h treatment with concentrations above the corresponding
MICs. In contrast, the antibiotics, clindamycin could not clean the
bacteria at the concentration of one time of MIC. Besides, P. acnes
treated by one time MIC of clindamycin was capable of resuming
growth after 80 min of the treatment (Fig. 1).
The Effects on membrane morphology
The morphology difference of untreated, clindamycin-treated
and cathelicidin-BF-treated P. acnes was studied by SEM as
illustrated in Fig. 2. There are clear morphology differences
among these P. acnes. The outer membranes of untreated P. acnes
were long, spindle-shaped, and smooth (Figs. 2A). But once treated
with cathelicidin-BF, the intracellular inclusions were found
effluxed extracellularly (the representatives are indicated by
arrows), indicating that the breaks might be formed in the plasma
membranes of P. acnes. In addition, the cell swell was observed
obviously in the cathelicidin-treated P. acnes (Fig. 2B).
Clindamycin-treated P. acnes had no significant morphology difference from
the untreated bacterium (Fig. 2C), suggesting that it does not act
on membranes. In fact, clindamycin kills bacteria by inhibiting
Cathelicidin-BF inhibits P. acnes-induced O2.2 production
Previous work by Grange et al has indicated that P. acnes
significantly induces O2.2 production, which affects IL-8 levels
. The effects of cathelicidin-BF on P. acnes-induced O2.2
production was tested in this work. Both live and heat-killed P.
acnes significantly induced O2.2 production as the results from
S. epidermidis 09A3726 2.3 (0.65 mM)
MIC: minimal peptide concentration required for total inhibition of cell growth
in liquid medium. These concentrations represent mean values of three
independent experiments performed in duplicates.
BF: canthelicidin-BF; CL: clindamycin.
Grange et al . As illustrated in Fig. 3, cathelicidin-BF with 16
or 0.16 MIC could significantly inhibited P. acnes-induced O2.2
production. Different from cathelicidin-BF, only 16 MIC
clindamycin significantly inhibited P. acnes-induced O2.2
production but 0.16 MIC clindamycin had little effect.
Cytokine production inhibition by cathelicidin-BF
Several pro-inflammatory cytokines including TNF-a, IL-1b,
IL-8, and MCP-1 were induced by heat-killed P. acnes as listed in
Fig. 4. Both cathelicidin-BF and clindamycin could significantly
inhibit cytokines secretion induced by P. acnes in a dose-dependent
manner. For example, TNF-a, one of the most important
proinflammatory cytokines was induced to a concentration of 400 pg/
ml by the heat-killed P. acnes (Fig. 4A). 0.5, 1, and 2 times of MIC
of cathelicidin-BF could inhibit 32.5, 40.3, and 43.3% of the
induced TNF-a secretion, respectively, while the inhibition rate of
clindamycin was 14.7, 20.7, and 27%, respectively. To account for
any reduction in pro-inflammatory cytokines resulting from
cytotoxic effects of cathelicidin-BF, the cytotoxicity induced by
these extracts was determined by MTT assays in THP-1 cells.
cathelicidin-BF had little cytotoxic effects with only 0.3, 0.7, and
1.4% cell growth inhibition at concentration of 0.5, 1, and 2 times
of MIC, respectively. In addition, after an 18-h incubation, only
cathelicidin-BF (no heat-killed P. acnes) did not increase the
secretions of either TNF-a, IL-8, IL-1b, or MCP-1 by THP-1 cells.
In vivo mice ear colonization inhibition of P. acnes and
anti-inflammation by cathelicidin-BF
Intradermally injected P. acnes induced severe inflammation in the
ears of Kunming mice as illustrated in Fig. 5A. One day after the
injection, the ear thickness was about two time of the control. Both
cathelicindin-BF and clindamycin 0.2% gels could inhibit the
inflammation induced by P. acnes. After one day treatment, 0.2%
cathelicindin-BF (425 times of MIC) and clindamycin (950 times of
MIC) gel could inhibit 42% and 47% of the ear swelling, respectively.
The numbers of P. acnes colonized within the ears were
illustrated in Fig. 5B. The number of P. acnes colonized within
the ear treated by the vehicle is about 1700. The number of P.
acnes in the cathelicindin-BF and clindamycin group is about 470
and 430, respectively. Furthermore, data from MTT assays
showed that at concentration of 1, 10, and 30 times of MIC,
cathelicindin-BF inhibited 0.2, 1.1, and 1.9% cell growth,
respectively, indicating almost no cytotoxicity on human HaCaT
keratinocyte cells. These data suggest that dermal application of
cathelicindin-BF can effectively relieve P. acnes-induced
inflammation without detrimental effects on skin cells.
Over the past years, natural antimicrobial peptides (AMPs)
have attracted considerable interests as a new type of
antimicrobial agents for several reasons including their relative
selectivity towards targets (microbial membranes), their rapid
mechanism of action and, above all, the low frequency in
selecting resistant strains . Cathelicidin-BF is an
antimicrobial peptide identified from the snake venoms of B.
fasciatus. Our previous work has indicated that cathelicidin-BF
exerted strong and rapid antimicrobial activities against many
microorganisms including Gram-negative, Gram-positive
bacteria and fungi, especially some clinically isolated
drugresistance microorganisms. Besides, cathelicidin-BF has no
hemolytic and cytotoxic activity on human cells . However,
its effect toward P. acnes has not been studied.
Several antimicrobial peptides including epinecidin- and
granulysin-derived peptides, and frog skin peptides have been
found to exert anti-P. acnes functions . Recently,
sebocytes are found to express functional cathelicidin
antimicrobial peptides with activity to kill P. acnes . Considering
anti-inflammatory activities of some antimicrobial peptides, they
are suggested to be potent agents for acne vulgaris treatment
. The current work indicated that cathelicidin-BF contained
potential antimicrobial activity against P. acnes in vitro (Table 1,
Fig. 1). Its MIC against two P. acnes strains is 4.7 mg/ml
(1.3 mM), which is comparable to the anti-P. acnes potential
antibiotics of clindamycin (2.3 mg/ml, 5.2 mM) (Table 1). SEM
study indicated that cathelicidin-BF acted on the membrane of
P. acnes (Fig. 2).
Previous work by Grange et al has indicated that the whole P.
acnes bacteria or the extract of its surface proteins had the same
effects to induce O2.2 production in keratinocytes. In addition,
their results also indicted that the toxicity of reactive oxygen
species on P. acnes-stimulated keratinocytes is mainly caused by the
O2.2 overproduction . In this study, cathelicidin-BF was found
to obviously inhibit O2.2 production induced by P. acnes in the
HaCaT keratinocyte cells (Fig. 3). By O2.2 production inhibition,
cathelicidin-BF may inhibit inflammation because O2.2 can exert
a positive effect on IL-8 production .
Some factors of P. acnes, such as heat shock protein HSP60, can
stimulate the production of pro-inflammatory cytokines IL-1b and
TNF-a . In turn, these released cytokines lead to the
inflammatory reactions . The anti-inflammatory function of
cathelicidin-BF was evaluated by measuring its effects on
proinflammatory cytokine secretion. It could significantly inhibit P.
acnes-induced secretion of several pro-inflammatory factors
including TNF-a, IL-8, IL-1b, and MCP-1 In vitro (Fig. 4). In vivo
anti-inflammatory effect of cathelicidin-BF was confirmed by
relieving P. acnes-induced ear swelling and granulomatous
inflammation (Fig. 5). The anti-inflammatory effects combined
with potent antimicrobial activities and O2.2 production inhibition
activities of cathelicidin-BF indicate its pontential as a novel
therapeutic option for acne vulgaris.
Materials and Methods
Two cathelicidins (snake cathelicidin-BF,
KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF, and human cathelicidin, LL-37,
synthesized by GL Biochem (Shanghai) Ltd. (Shanghai, China) and
analyzed by HPLC and mass spectrometry to confirm their purity
higher than 98%.
Figure 4. Effects of cathelicidin-BF and clindamycin on cytokine production by P. acnes-stimulated monocytic cells. Human monocytic
THP-1 cells (16106 cells/ml) were incubated with heat-killed (incubated at 80uC for 30 min to kill the bacteria) P. acnes (wet weight 100 mg/ml) alone
or in combination with different concentrations (0.01, 0.05, and 0.1 mg/ml) of tested sample for 18 h. These represent mean values of three
independent experiments. The values for cathelicidin-BF and clindamycin were significant different from the value for the HPC group (*P,0.05 and
**p,0.01). NC: Negative control; HPC: heat-killed P. acnes; CA: cathelicidin-BF; CL: clindamycin.
Microorganism strains and growth conditions
Propionibacterium acnes (ATCC6919 and ATCC 11827),
Staphylococcus epidermidis (09A3726 and 09B2490) and Staphylococcus aureus
(ATCC 2592) were obtained from Kunming Medical College. P.
acnes were cultured in brain heart infusion (BHI) broth
(HKM,Guangzhou, China) with 1% glucose at 37uC for 3 days
to exponential-phase and for 5 days to stationary phase. The
bacteria were cultured in an anaerobic atmosphere using MGC
Anaeropack systems (Mitsubishi Gas Chemical Co., Inc, Japan); S.
epidermidis and S. aureus were grown in LB (Luria-Bertani) broth as
our previous report .
MIC (minimal inhibitory concentration) of antimicrobial
peptides against microorganisms was determined using broth
dilution determination as our previous methods . Peptides
were prepared as a stock solution in H2O at a series of
concentration. 890 ml special broth (BHI broth for P. acnes, LB
broth for S epidermidis and S. aureus), 100 ml bacterial suspension
(108 CFU/ml) and 10 ml test peptides were put together in the test
tube and shaken at 37uC for 24 h. A tube with corresponding
volume of H2O was used as control. The MIC was defined as the
lowest concentration of test peptides inhibiting microorganisms
Bacteria killing kinetics
The bacterial effect of cathelicidin-BF against P. acnes
(ATCC6919) was tested using clindamycin as positive control. P.
acnes was grown to log phase in BHI broth and centrifuged at
5000 rpm for 5 min. The collected bacterium pellet was washed
twice by BHI broth and diluted to 16106 CFU/ml with BHI
broth. Cathelicidin-BF or clindamycin with one time of MIC was
added into the BHI broth containing P. acnes and cultured at 37uC
with shaking. The colony counting was performed at different
times as described by Mygind et al .
Scanning electron microscopy (SEM)
SEM was performed to study the possible mechanisms of action
of cathelicidin-BF on bacteria according to the methods described
by Lu et al  with minor modification. Propionibacterium acnes
ATCC6919 was cultured in BHI liquid medium to
exponentialphase. After washing with 0.15 M sodium chloride solution for
two times, the bacteria were resuspended and incubated with
cathelicidin-BF (16 MIC) at 37uC for 30 min. The pellets after
centrifuging at 1000 rpm for 10 min were fixed with 2.5%
buffered glutaraldehyde at 4uC for 2 h. The bacteria were then
postfixed in 1% buffered osmium tetroxide for 2 h, dehydrated in
a graded series of ethanol, frozen in liquid nitrogen cooled
tertbutyl alcohol and vacuum dried overnight. After mounting
onto aluminum stubs and vacuum sputter-coating with gold, the
samples were analyzed with a Hitachi S-3000N SEM under
standard operating conditions.
Measurement of O2.2 production
Measurement of O2.2 production was carried out following the
protocol described by Grange et al . The human HaCaT
keratinocyte cells (16105 cells/ml, obtained from Cell Bank of
Kunming Institute of Zoology, Chinese Academy of Sciences)
were cultured in 96-well plates with Dulbeccos modified Eagles
medium (DMEM, Gibco) containing 10% fetal calf serum and
penicillin (100 u/ml)streptomycin (100 mg/ml) at 37uC in a
humidified 5% CO2 atmosphere. The cell line was routinely tested
to assess the absence of Mycoplasma infection. The monolayer cells
were washed three times with PBS (1.5 mM KH2PO4, 2.7 mM
Na2HPO4.7H2O, 0.15 M NaCl, pH 7.4), and cultured with
serum-free and antibiotic-free DMEM medium, incubating with
heat-killed P. acnes (16106) in the presence of test samples. After
co-culturing for 18 h, the cells were washed with PBS and
incubated with 100 ml per wells of 5 mM dihydroethidium solution
(DHE, a fluorescent superoxide anion indicator) for 30 min. The
level of intracellular O2.2 was assessed by spectrofluorimetry
(excitation/emission maxima: 480/610 nm) on a
spectrofluorimeter (FlexStation 3, Molecular Devices, USA).
Measurement of cytokine production in human
Human monocytic THP-1 cells (16106 cells/ml, Shanghai
Caoyan Biotechnology Co. Ltd, China) were cultured in 24-well
plates containing serum-free medium (RPMI 1640 medium, Gibco
Life Technologies). They were added with heat-killed (incubated
at 80uC for 30 min to kill the bacteria) P. acnes (wet weight 100 mg/
ml) alone or in combination with different concentrations (0.01,
0.05, and 0.1 mg/ml) of tested samples for an 18-h incubation.
Cell-free supernatants were collected, and concentrations of
MCP1, TNFa, IL-1b, and IL-8 were measured using corresponding
enzyme immunoassay kits (Adlitteram Diagnostic Laboratories,
In vitro cytotoxicity of cathelicidin-BF on human skin and
THP-1 and HaCaT cells were cultured in 96-well plates as
described above. Cell viability was evaluated by conventional
(MTT) reduction assays. After a 24-h treatment by the test
samples, 0.1 ml of MTT (5 mg/ml) was added to each well. The
supernatant was removed after 2 h of incubation, and 100 ml
acidic isopropanol was mixed with the precipitate. The absorbance
at 540 nm of the resulting solution was measured. The
experiments were performed in triplicate.
In vivo mice ear colonization of P. acnes
P. acnes (ATCC6919) was grown to the exponential-phase in
BHI broth. The bacterium was centrifuged at1000 rpm for
10 min. The bacterium pellet was washed twice with 0.15 M
sodium chloride solution, and re-suspended in 0.15 M sodium
chloride solution (56108 CFU/ml). P. acnes (16107 CFU per
20 ml) was intradermally injected into left ears of Kunming mice
(2062 g). Right ears received the same volume of 0.15 M sodium
chloride solution. Placebo gel, cathelicidin-BF or clindamycin
0.2% gel (Polyethylene Glycol (PEG) 400: PEG 4000, 1:1) were
administered (nine mice per group) on the skin surfaces of ears and
the sites were covered with OpSite dressings and occlusively sealed
with adhesive tape. The increase of ear thickness after 24 h
bacterial injection was measured using a micro caliper. The
increase in ear thickness of the left ear was calculated as
percentage of the right ear.
To determine P. acnes number in the ear, the left ear was cut off
after 24 h bacterial injection and wiped to remove gel. The ear
was homogenized in 0.15 M sodium chloride solution (1 ml per
ear) withith a hand tissue grinder. CFUs of P. acnes in the ear were
enumerated by plating serial dilutions of the homogenate on BHI
plates. The plates were anaerobically incubated at 37uC for
72 hours and the bacterial numbers were counted. All the
experimental protocols to use animals were approved by the
Animal Care and Use Committee at Kunming Institute of
Zoology, Chinese Academy of Sciences. The approval ID for this
study was syxk2009-0026.
Data were analyzed by X2 and by t test or repeated measure
analysis of variance (ANOVA) comparison of means.
Conceived and designed the experiments: YPW ZYZ LLC HJG ZL HLY
JXL DWY. Performed the experiments: YPW ZYZ LLC HJG ZL HLY
JXL DWY. Analyzed the data: HNY RL. Contributed reagents/materials/
analysis tools: HNY RL. Wrote the paper: HNY RL.
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