Antimicrobial activity of human prion protein is mediated by its N-terminal region.
ABSTRACT Cellular prion-related protein (PrP(c)) is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP(c), and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypothesize that PrP(c) could exert antimicrobial activity.
Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking) liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-alpha in vitro.
The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.
- [show abstract] [hide abstract]
ABSTRACT: Antimicrobial peptides are ancient components of the innate immune system and have been isolated from organisms spanning the phylogenetic spectrum. Over an evolutionary time span, these peptides have retained potency, in the face of highly mutable target microorganisms. This fact suggests important coevolutionary influences in the host-pathogen relationship. Despite their diverse origins, the majority of antimicrobial peptides have common biophysical parameters that are likely essential for activity, including small size, cationicity, and amphipathicity. Although more than 900 different antimicrobial peptides have been characterized, most can be grouped as belonging to one of three structural classes: (1) linear, often of alpha-helical propensity; (2) cysteine stabilized, most commonly conforming to beta-sheet structure; and (3) those with one or more predominant amino acid residues, but variable in structure. Interestingly, these biophysical and structural features are retained in ribosomally as well as nonribosomally synthesized peptides. Therefore, it appears that a relatively limited set of physicochemical features is required for antimicrobial peptide efficacy against a broad spectrum of microbial pathogens. During the past several years, a number of themes have emerged within the field of antimicrobial peptide immunobiology. One developing area expands upon known microbicidal mechanisms of antimicrobial peptides to include targets beyond the plasma membrane. Examples include antimicrobial peptide activity involving structures such as extracellular polysaccharide and cell wall components, as well as the identification of an increasing number of intracellular targets. Additional areas of interest include an expanding recognition of antimicrobial peptide multifunctionality, and the identification of large antimicrobial proteins, and antimicrobial peptide or protein fragments derived thereof. The following discussion highlights such recent developments in antimicrobial peptide immunobiology, with an emphasis on the biophysical aspects of host-defense polypeptide action and mechanisms of microbial resistance.Biopolymers 02/2006; 84(5):435-58. · 2.88 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: An important class of cytolytic antimicrobial peptides (AMPs) assumes an amphipathic, alpha-helical conformation that permits efficient interaction with biological membranes. Host defence peptides of this type are widespread in nature, and numerous synthetic model AMPs have been derived from these or designed de novo based on their characteristics. In this review we provide an overview of the 'sequence template' approach which we have used to design potent artificial helical AMPs, to guide structure-activity relationship studies aimed at their optimization, and to help identify novel natural AMP sequences. Combining this approach with the rational use of natural and non-proteinogenic amino acid building blocks has allowed us to probe the individual effects on the peptides' activity of structural and physico-chemical parameters such as the size, propensity for helical structuring, amphipathic hydrophobicity, cationicity, and hydrophobic or polar sector characteristics. These studies furthermore provided useful insights into alternative modes of action for natural membrane-active helical peptides.Biochimica et Biophysica Acta 10/2006; 1758(9):1436-49. · 4.66 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Gene-encoded, ribosomally synthesised antimicrobial peptides (AMPs) are an ancient and pervasive component of the innate defence mechanisms used by multicellular organisms to control the natural flora and combat pathogens. Bacteria also produce such AMPs to maintain ecological niches free of rival strains. Several hundred different peptides have been characterised to date, and they show a marked degree of variability in both sequence and structure, having evolved to act against distinct microbial targets in different physiological contexts. Many of these peptides appear to function via a selective, but not receptor-mediated, permeabilisation of microbial membranes, while others interact with specific membrane associated or intracellular targets. This review presents a broad survey of different amp structural classes, emphasising both their molecular diversity and underlying similarities. The mode of action of these peptides and potential for biomedical and other application is also briefly discussed.Current Pharmaceutical Design 02/2002; 8(9):743-61. · 3.31 Impact Factor
Antimicrobial Activity of Human Prion Protein Is
Mediated by Its N-Terminal Region
Mukesh Pasupuleti1*, Markus Roupe2, Victoria Rydenga ˚rd1, Krystyna Surewicz3, Witold K. Surewicz3,
Anna Chalupka1, Martin Malmsten4, Ole E. So ¨rensen2, Artur Schmidtchen1*
1Division of Dermatology and Venereology, Department of Clinical Sciences, Lund University, Lund, Sweden, 2Division of Infection Medicine, Department of Clinical
Sciences, Lund University, Lund, Sweden, 3Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, United States of America,
4Department of Pharmacy, Uppsala University, Uppsala, Sweden
Background: Cellular prion-related protein (PrPc) is a cell-surface protein that is ubiquitously expressed in the human body.
The multifunctionality of PrPc, and presence of an exposed cationic and heparin-binding N-terminus, a feature
characterizing many antimicrobial peptides, made us hypotesize that PrPccould exert antimicrobial activity.
Methodology and Principal Findings: Intact recombinant PrP exerted antibacterial and antifungal effects at normal and
low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide
20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein.
Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa,
and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence
studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted
membrane-breaking effects similar to those seen after treatment with the ‘‘classical’’ human antimicrobial peptide LL-37. In
contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively
charged (bacteria-mimicking) liposomes. PrP furthermore showed an inducible expression during wounding of human skin
ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-a in vitro.
Conclusions: The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-
binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could
have a previously undisclosed role in host defense.
Citation: Pasupuleti M, Roupe M, Rydenga ˚rd V, Surewicz K, Surewicz WK, et al. (2009) Antimicrobial Activity of Human Prion Protein Is Mediated by Its N-Terminal
Region. PLoS ONE 4(10): e7358. doi:10.1371/journal.pone.0007358
Editor: Neeraj Vij, Johns Hopkins School of Medicine, United States of America
Received July 10, 2009; Accepted September 14, 2009; Published October 7, 2009
Copyright: ? 2009 Pasupuleti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Swedish Research Council (projects 13471 and 621-2003-4022), the Welander-Finsen, Crafoord, So ¨derberg,
Schyberg, Alfred O¨sterlund, and Kock Foundations, and The Swedish Government Funds for Clinical Research (ALF). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (AS); email@example.com (MP)
provides a rapid and non-specific response against potentially invasive
pathogenic microorganisms. At present, over 900 different AMP
peptide sequences are known[1,2,3] (see also http://www.bbcm.univ.
by an amphipathic structure, composed of hydrophobic and cationic
amino acids spatially organized in sectors of the molecules. For
forming cysteine-linked antiparallel b-sheets, as well as cysteine-
constrained loop structures. AMPs may also, however, be found
among peptides not displaying such ordered structures as long as these
are characterized by an over-representation of certain amino
acids[1,4,5,6]. The interaction with bacterial membranes is a
prerequisite for AMP function. However, the modes of action of
AMPs on their target bacteria are complex, and can be divided into
membrane disruptive and non-membrane disruptive[1,3,7,8].
It has become increasingly clear, that AMPs belong to a
multifunctional group of molecules that interact with negatively
charged glycosaminoglycans (such as heparin), biomembranes,
and cell receptors. Apart from their antibacterial actions,
biological effects exerted by AMPs include growth stimulus and
angiogenesis, protease inhibition, anti-angiogenesis, and chemo-
taxis[9,10,11]. Conversely, cationic peptide motifs from proteins
not previously considered as AMPs have been shown to exert
antimicrobial activities. For example, the anaphylatoxin peptide
C3a and kininogen-derived peptides exert antimicrobial ef-
fects[12,13,14,15]. In conjunction with these findings, consensus
heparin-binding peptide sequences were shown to be antibacte-
rial and specifically interact with membranes.
Cellular prion-related protein (PrPc) is a cell-surface protein that
is ubiquitously expressed in the human body. Apart from a high
expression in the brain, the protein is also found throughout the
body in blood, skin, haematopoietic cells, gastric mucosa
, mammary glands and kidney. Although numerous
biological functions have been attributed to prions, their exact
PLoS ONE | www.plosone.org1October 2009 | Volume 4 | Issue 10 | e7358
physiological role is still not known. Hence, recent work has
suggested that PrPcis required for self-renewal of hematopoietic
cells and may be involved in T-cell activation. PrPcis
highly expressed in the neural system, and since this is the major
site of prion pathology, most interest has been focused on defining
the role of PrPcin neurons. However, PrPc2/2 mice are
relatively normal, only presenting subtle abnormalities in synaptic
transmission. Accumulated evidence suggests that PrPc may
function as a metal transporter, and binding of Cu2+also enables
PrPc to acquire a superoxide-dismutase like antioxidant activi-
ty[22,23]. Other ligands that have been reported to bind PrPc
include laminin, nucleic acids, as well as glycosaminoglycans such
as heparin. Considering the latter it was demonstrated that the
heparin-binding site was located in the N-terminus of PrPc,
involving amino acid residues 23–35. Interestingly, an increase in
prion protein expression has been demonstrated during bacterial
infection and inflammation, and hence it has been
hypothesized that PrPcmay act as a pattern recognition receptor
inducing the innate and adaptive immunity in a TLR independent
The multi-functionality of PrPc, and presence of an exposed
cationic and heparin-binding N-terminus made us raise the
question whether PrPccould exert antimicrobial activity. We here
show that the recombinant prion protein may indeed kill both
Gram-negative and Gram-positive bacteria and fungi, the activity
being mediated by the N-terminus of PrP. These findings, together
with a marked induction of PrP expression in skin wounds, suggest
that antimicrobial activity could be one potential role of the prion
Materials and Methods
Pep-screen peptides were from Sigma-Genosys, generated by a
peptide synthesis platform (PEPscreen, Custom Peptide Libraries,
Sigma Genosys) and yield was ,1–6 mg with an average crude
purity of 60–70%. Prior to biological testing, the peptides were
diluted in dH2O (5 mM stock) and stored at 220uC. This stock
solution was used for the subsequent experiments. The high
KKR20; KKRPKPGGWNTGGSRYPGQG, MAN28; MAN-
LGCWMLVLFVATWSDLGLCKKRPKP, GHH20; GHHPH-
GHHPHGHHPHGHHPH and AHH24; AHHAHAAHH AHA-
AHHAHAAHHAHA were synthesized by Biopeptide Co., San
Diego, USA, with the exception of LL-37, which was obtained
from Innovagen AB, Lund, Sweden. The purity (.95%) of these
peptides was confirmed by mass spectral analysis (MALDI-ToF
Voyager). The production of recombinant PrP and truncated
versions has been described previously . Human TGF-a was
from Peprotech (Rocky Hill, NJ). The polyclonal goat antibody
against prion protein (PrP27–30), used for imunnohistochemistry
and immunoblotting, was purchased from Chemicon.
Bacterial isolates Escherichia coli ATCC 25922, Pseudomonas
aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, Bacillus
subtilis ATCC 6633, Candida albicans ATCC 90028 and Candida
parapsilosis ATCC 90018 and were obtained from the Department
of Bacteriology, Lund University Hospital.
Viable count analysis
Escherichia coli ATCC 25922 was grown overnight in full-
strength (3% w/v) trypticase soy broth (TSB) (Becton-Dickinson,
Cockeysville, MD), whereas C. parapsilosis ATCC 90018 was grown
in YPD medium. The microbes were washed twice with 10 mM
Tris, pH 7.4, and diluted in 10 mM Tris, pH 7.4, 5 mM glucose
or in 10 mM MES pH 5.5, containing 5 mM glucose. Following
this, microbes (in 50 ml; 1226106cfu/ml) were incubated at 37uC
for 2 hours at the indicated concentrations with PrP and variants
thereof, or with peptides in appropriate buffers in presence/
absence of 0.15 M NaCl or 20% citrate plasma. In some
experiments, C. parapsilosis were incubated with peptides or recPrp
in 10 mM Tris, pH 7.4 mM glucose in the absence or presence of
50 mM Zn2+. For analyses in buffers mimicking human sweat
(40 mM NaCl, 10 mM KCl, 1 mM CaCl2, 1 mM MgCl2and
1 mM Na-dihydrogenphosphate, pH 5.5 or 6.5), C. parapsilosis
(1226107cfu/ml) was incubated with recPrP (1 mM) in sweat
buffer for 2 hours at 37uC as described previously. To
quantify the bactericidal activity, serial dilutions of the incubated
mixtures were plated on TH (for bacteria), or YPD agar (for fungi),
followed by incubation at 37uC overnight and the number of
colony-forming units was determined. 100% survival was defined
as total survival of bacteria in the same buffer and under the same
conditions as in the absence of peptide. Significance was
determined using the statistical software SigmaStat (SPSS Inc.,
Chicago, IL, USA).
Radial diffusion assay
Essentially as described earlier , bacteria were grown
overnight in 10 ml of full-strength (3% w/v) trypticase soy broth
(TSB) (Becton-Dickinson, Cockeysville, MD), whereas fungi were
grown in YPD medium and washed twice with 10 mM Tris,
pH 7.4. 46106colony forming units was added to 15 ml of the
underlay agarose gel, consisting of 0.03% (w/v) TSB, 1% (w/v)
low-electro endosmosis type (Low-EEO) agarose (Sigma, St Louise
MO) and a final concentration of 0.02% (v/v) Tween 20 (Sigma).
Three different underlay gels were used, each based on different
compositions (10 mM Tris, pH 7.4, 10 mM MES, pH 5.5, and
10 mM Tris, pH 7.4, 50 mM Zn2+). The underlay was poured into
a Ø 144 mm petri dish. After agarose solidification, 4 mm-
diameter wells were punched and 6 ml of peptide was added to
each well. Plates were incubated at 37uC for 3 hours to allow
diffusion of the peptides. The underlay gel was then covered with
15 ml of molten overlay (6% TSB and 1% Low-EEO agarose in
dH2O). Antimicrobial activity of a peptide was visualized as a clear
zone around each well after 18–24 hours of incubation at 37uC.
The impermeant probe FITC (Sigma-Aldrich, St. Louis, USA)
was used for monitoring of bacterial membrane permeabilization.
S. aureus ATCC 29213 were grown to mid-logarithmic phase in
TSB medium. Bacteria were washed and resuspended in buffer
(10 mM Tris, pH 7.4, 0.15 M NaCl, 5 mM glucose) to yield a
suspension of 16107CFU/ml. 100 ml of the bacterial suspension
was incubated with 30 mM of the respective peptides at 30uC for
30 min. Microorganisms were then immobilized on poly (L-
lysine)-coated glass slides by incubation for 45 min at 30uC,
followed by addition onto the slides of 200 ml of FITC (6 mg/ml) in
buffer and a final incubation for 30 min at 30uC. The slides were
washed and bacteria fixed by incubation, first on ice for 15 min,
then in room temperature for 45 min in 4% paraformaldehyde.
The glass slides were subsequently mounted on slides using
Prolong Gold antifade reagent mounting medium (Invitrogen,
Eugene, USA). Bacteria were visualized using a Nikon Eclipse
TE300 (Nikon, Melville, USA) inverted fluorescence microscope
equipped with a Hamamatsu C4742-95 cooled CCD camera
(Hamamatsu, Bridgewater, USA) and a Plan Apochromat 6100
objective (Olympus, Orangeburg, USA). Differential interference
Antimicrobial Prion Protein
PLoS ONE | www.plosone.org2 October 2009 | Volume 4 | Issue 10 | e7358
contrast (Nomarski) imaging was used for visualization of the
EDTA-blood was centrifuged at 800 g for 10 min, whereafter
plasma and buffy coat were removed. The erythrocytes were
washed three times and resuspended in PBS, pH 7.4 to get a 5%
suspension. The cells were then incubated with end-over-end
rotation for 60 min at 37uC in the presence of peptides (60 mM).
2% Triton X-100 (Sigma-Aldrich) served as positive control. The
samples were then centrifuged at 800 g for 10 min and the
supernatant was transferred to a 96 well microtiter plate. The
absorbance of hemoglobin release was measured at l 540 nm and
is in the plot expressed as % of TritonX-100 induced hemolysis.
Lactate dehydrogenase (LDH) assay
HaCaT keratinocytes were grown to confluency in 96 well
plates (3000 cells/well) in serum-free keratinocyte medium (SFM)
supplemented with bovine pituitary extract and recombinant EGF
(BPE-rEGF) (Invitrogen, Eugene, USA). The medium was then
removed, and 100 ml of the peptides investigated (at 60 mM,
diluted in SFM/BPE-rEGF or in keratinocyte-SFM supplemented
with 20% human serum) were added. The LDH-based TOX-7 kit
(Sigma-Aldrich, St. Louis, USA) was used for quantification of
LDH release from the cells. Results represent mean values from
triplicate measurements, and are given as fractional LDH release
compared to the positive control consisting of 1% Triton X-100
(yielding 100% LDH release).
LPS binding ability of the peptides were examined by slot-blot
assay. Peptides (2 and 5 mg) were bound to nitrocellulose
membrane (Hybond-C, GE Healthcare BioSciences, UK), pre-
soaked in PBS, by vacuum. Membranes were then blocked by 2
wt% BSA in PBS, pH 7.4, for 1 h at RT and subsequently
incubated with125I-labelled LPS (40 mg/mL; 0.136106cpm/mg)
or125I-labelled heparin (Sigma) for 1 h at RT in 10 mM Tris,
pH 7.4, 0.15 M NaCl, or 10 mM MES, pH 5.5, 0.15 M NaCl.
After LPS binding, membranes were washed 3 times, 10 min each
time in the above buffers and visualized for radioactivity on Bas
2000 radioimaging system (Fuji, Japan).
Liposome preparation and leakage assay
The liposomes investigated were either zwitterionic (DOPC/
cholesterol 60/40 mol/mol or DOPC without cholesterol) or
anionic (DOPE/DOPG 75/25 mol/mol). DOPG (1,2-Dioleoyl-
sn-Glycero-3-Phosphoglycerol, monosodium salt), DOPC (1,2-
dioleoyl-sn-Glycero-3-phoshocholine), and DOPE (1,2-dioleoyl-
sn-Glycero-3-phoshoetanolamine) were all from Avanti Polar
Lipids (Alabaster, USA) and of .99% purity, while cholesterol
(.99% purity), was from Sigma-Aldrich (St. Louis, USA). Due to
the long, symmetric and unsaturated acyl chains of these
phospholipids, several methodological advantages are reached.
In particular, membrane cohesion is good, which facilitates very
stable, unilamellar, and largely defect-free liposomes (observed
from cryo-TEM) and well defined supported lipid bilayers
(observed by ellipsometry and AFM), allowing detailed values on
leakage and adsorption density to be obtained. The lipid mixtures
were dissolved in chloroform, after which solvent was removed by
evaporation under vacuum overnight. Subsequently, 10 mM Tris
buffer, pH 7.4, was added together with 0.1 M carboxyfluorescein
(CF) (Sigma, St. Louis, USA). After hydration, the lipid mixture
was subjected to eight freeze-thaw cycles consisting of freezing in
liquid nitrogen and heating to 60uC. Unilamellar liposomes of
about Ø140 nm were generated by multiple extrusions through
polycarbonate filters (pore size 100 nm) mounted in a LipoFast
miniextruder (Avestin, Ottawa, Canada) at 22uC. Untrapped CF
was removed by two subsequent gel filtrations (Sephadex G-50,
GE Healthcare, Uppsala, Sweden) at 22uC, with Tris buffer as
eluent. CF release from the liposomes was determined by
monitoring the emitted fluorescence at 520 nm from a liposome
dispersion (10 mM lipid in 10 mM Tris, pH 7.4). An absolute
leakage scale was obtained by disrupting the liposomes at the end
of each experiment through addition of 0.8 mM Triton X-100
(Sigma-Aldrich, St. Louis, USA). A SPEX-fluorolog 1650 0.22-m
double spectrometer (SPEX Industries, Edison, USA) was used for
the liposome leakage assay, and also for monitoring W absorption
spectra of GKH17-WWW in Tris buffer in the absence and
presence of liposomes under conditions described above. Mea-
surements were performed in triplicate at 37uC.
The CD spectra of the peptides in solution were measured on a
Jasco J-810 Spectropolarimeter (Jasco, U.K.). The measurements
were performed at 37uC in a 10 mm quartz cuvet under stirring
and the peptide concentration was 10 mM. The effect on peptide
secondary structure of liposomes at a lipid concentration of
100 mM was monitored in the range 200–250 nm. The only
peptide conformations observed under the conditions investigated
were a-helix and random coil. The fraction of the peptide in a -
helical conformation, Xa, was calculated from
where A is the recorded CD signal at 225 nm, and Aaand Acare
the CD signal at 225 nm for a reference peptide in 100% a-helix
and 100% random coil conformation, respectively. 100% a -helix
and 100% random coil references were obtained from 0.133 mM
(monomer concentration) poly-L-lysine in 0.1 M NaOH and
0.1 M HCl, respectively[29,30]. For determination of effects of
lipopolysaccharide on peptide structure, the peptide secondary
structure was monitored at a peptide concentration of 10 mM,
both in Tris buffer and in the presence of E. coli lipopolysaccharide
(0.02 wt%) (Escherichia coli 0111:B4, highly purified, less than 1%
protein/RNA, Sigma, UK). To account for instrumental differ-
ences between measurements the background value (detected at
250 nm, where no peptide signal is present) was subtracted.
Signals from the bulk solution were also corrected for.
Primary human keratinocytes were obtained from Cascade
Biologics (Portland, OR) and cultured in serum-free medium
(KGM2-Bullet kit) from Cambrex (Walkersville, MD). 24 hours
after complete confluence was reached cells were stimulated with
TGF-a (50 ng/mL) for 48 hours or left non-stimulated for control
Human skin wounds
Non-wounded human skin was obtained by taking punch
biopsies from three donors, while skin wound samples were
retrieved by making new punch biopsies from the edges of the
initial biopsies. A small fraction of these samples were fixed in
formalin for immunohistochemistry or prepared for miccroarray
analysis as previously described. The material was obtained
under protocols approved by the Ethics Committee at Lund
University, Lund, Sweden.
Antimicrobial Prion Protein
PLoS ONE | www.plosone.org3October 2009 | Volume 4 | Issue 10 | e7358
Model of ex vivo injured human skin
Surplus, normal, skin was obtained as previously described from
three donors following surgery according to protocols approved by
the Ethics Committee at Lund University. In brief the skin was cut
into slices of 1610 mm and incubated in culture for 4 days (ex vivo
injured skin). The skin samples were cultured in serum-free
keratinocyte medium (KGM2-Bullet kit) from Cambrex (Walkers-
ville, MD) supplemented with transferrin, hEGF (0.15 ng/mL),
0.5 mg/mL hydrocortisone, gentamicin, amphotericin B, and
epinephrine but without insulin (all supplied by Cambrex).
RNA isolation and microarray analysis
Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA)
according to the recommendations of the supplier and resus-
pended in 0.1 mmol/L EDTA. The concentration was deter-
mined by spectrophotometric measurement. For gene expression
analysis, total RNA was biotinylated and hybridized to Human
Genome U133 Plus 2.0 GeneChipsH (Affymetrix, Santa Clara,
CA) according to the instructions by the manufacturer. The
microarray fluorescence signals were normalized using the
GeneChip Operation Software (GCOS ver. 1.4, Affymetrix). All
probe set lists were annotated with locus link identifications (IDs)
provided by the NetAffx database and converged into gene/EST
(expressed sequence tag) lists by exclusion of redundant probe sets
with identical locus ID. Genes were defined as expressed in cell
populations if all replicates were assigned a present call by the
GCOS software (Affymetrix). Genes of potential interest for
wound healing were therefore only genes with at least three
present calls in either the in vivo control condition or in the in vivo
wound condition. The microarray data present in this study has
been sent in the MAIME (Minimum Information About a
Microarray Experiment) compliant MAGE-TAB format to the
ArrayExpress database (www.ebi.ac.uk/arrayexpress) where it
soon will be made publicly available under an accession
The wound specimens (described earlier) that were fixed in 10%
formalin, were dehydrated and embedded in paraffin. Sections of
5 mm thickness were placed on poly-lysine coated glass slides,
deparaffinized in xylene and rehydrated in graded alcohols. The
slides were then treated with Dako antigen retrieval solution
(Dako) for 40 min at 97uC. The slides were incubated for 24 hours
at room temperature in a 1:1000 dilution of polyclonal antibodies
(adcam, England) The antibodies were diluted in TBS with 1%
BSA, 5% serum from the same species as the secondary antibody,
0.05% Tween 20 (Sigma). After three 20 min washes in TBS with
0.05% Tween 20 the slides were incubated with alkaline
phosphatase conjugated secondary anti IgG (Dako) diluted
1:1000 in the same buffer as the first antibody and incubated for
another 24 hours followed by three 20 min washes. Color was
developed with Vulcan Fast Red chromogen (Biocare Medical,
Concord, CA) and the slides were counterstained with Harris
Hematoxylin (EM Science, Gibbstown, NJ).
SDS-PAGE and immunoblotting
SDS-PAGE and immunoblotting were performed according to
the instructions from the manufacturer (BioRad, Hercules, CA).
After transfer of proteins from the polyacrylamide gels, the PVDF-
membrane was fixed for 30 min in TBS with 0.05% glutaralde-
hyde (Sigma) and blocked with 3% skimmed milk. For
visualization of the poly(peptides), polyvinylidene difluoride
(PVDF) membranes were incubated overnight with primary
antibodies. The following day, the membranes were incubated
for 2 hours with HRP-conjugated secondary antibodies (Dako,
Glostrup, Denmark) and visualized by SuperSignal West Pico
Chemiluminescent Substrate (Thermo Scientific, Rockford, IL).
The PVDF membrane was stripped for 20 minutes in 0.2 mmol/L
glycine (pH 2.5), 1% SDS, washed twice with TBS with 0.05%
Tween-20 and finally blocked with 3% skimmed milk before
incubating overnight with a different antibody.
Values are reported as means6standard deviation of the means.
To determine significance, analysis of variance with ANOVA
(SigmaStat, SPSS Inc., Chicago, USA), followed by post hoc testing
using the Holm-Sidak method, or Student’s t-test, were used as
indicated in the figure legends, where ‘‘n’’ denotes number of
independent experiments. Significance was accepted at p,0.05.
This study was conducted according to the principles expressed
in the Declaration of Helsinki. The study was approved by the
Institutional Review Board of Lund University hospital. Written
informed consent for the collection of samples and subsequent
analysis was obtained.
To investigate whether human prion protein could exert
antimicrobial effects, we tested the activity of protein against the
Gram-negative E. coli and the fungus C. parapsiliosis. The protein
was shown to be antimicrobial against these microbes at normal
(pH 7.4) as well as low pH (pH 5.5). An increase in antimicrobial
activity, particularly against C. parapsilosis, was noted at low pH
(Fig. 1A, for positive controls, see Figure S1). In order to explore
which part of PrP that could harbour the antimicrobial activity,
the full-length PrP protein (containing amino acids 23–231) was
compared with two truncated forms, either lacking the C-terminal
part (PrP23–144), or with a N-terminal truncated form (PrP90–231)
(Fig. 1B). Since the latter form, lacking the first 90 amino acids of
PrP significantly lost its antimicrobial potential, the results
indicated that the major antimicrobial activity was dependant on
an intact N-terminal region of PrP. In analogy with the
antimicrobial activity, slot-binding experiments with iodinated
LPS showed that intact PrP bound LPS at normal as well as low
pH, contrasting to the result with the N-terminally truncated form
(Fig. 2). Notably, PrP bound LPS similarly to human LL-37.
Experiments using iodinated heparin paralleled the findings with
LPS, and confirmed that the N-terminal region of PrP mediates
heparin binding (Fig. 2). Furthermore, heparin completely blocked
the interaction with LPS as well as antimicrobial activity,
confirming that heparin interacting sequences of PrP mediate
LPS binding, and thus, antimicrobial activity (not shown).
In order to further explore the structure-function relationship of
epitopes of PrP, overlapping peptide sequences comprising
20mers, as well as shorter variants (Fig. 3A, see also Figure S2
for illustration of sequence and peptides) of PrP were synthesized
and screened against the Gram-negative E. coli, Gram-positive S.
aureus and the fungus Candida parapsilosis using radial diffusion
assays under low salt conditions. The experiments demonstrated
that the antimicrobial activity was mainly found in two regions
comprising the N-terminal peptide containing the previously
identified heparin-binding motif of PrP; KKRPK (peptides no:
3 and 4, and 16 in the list in Fig. 3A). It has been reported that the
signal sequence of PrPc, normally cleaved of by a signal peptidase,
may be retained, leading to the secretion of unprocessed protein
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forms. Notably, the KKRPK motif, containing a part of the
signal sequence (peptide no: 3), was also antimicrobial. We have
previously shown that antimicrobial activities of histidine-rich
regions, such as those found in kininogen and histidine-rich
glycoprotein, rely on the presence of Zn2+or low pH [34,35].
Considering that stretches of PrP are histidine-rich, and given the
Figure 2. Interactions of PrP with LPS and heparin. 2 and 5 mg of recPrP and the truncated PrP90–231were applied onto nitrocellulose
membranes, followed by incubation with iodinated (125I) heparin or LPS in either Tris pH 7.4 or MES pH 5.5 (all at 10 mM, with 0.15 M NaCl).
Radioactivity of bound heparin or LPS was visualized using a phosphorimager system. LL-37 is included for comparison.
Figure 1. Antimicrobial effects of recPrP and variants. (A) In viable count assays, Escherichia coli and Candida parapsilosis were subjected to
increasing doses of PrP in 10 mM Tris pH 7.4 (left panel) or 10 mM MES pH 5.5 (both containing 5 mM glucose) and the number of cfu was
determined. The values represent mean of triplicate samples, and a representative experiment (of three) is shown. The difference in activity against C.
parapsilosis at 1 uM is statistically significant (pooling of three experiments yielded P,0.001, n=9, t-test) (B) Comparison of antibacterial effects of
PrP with truncated variants. E. coli was incubated with 1 mM of the full-length PrP protein, or with variants PrP23–144or PrP90–231. LL-37 is shown for
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Figure 3. Activities of peptide sequences of PrP. (A) Antimicrobial activity of selected peptides (at 100 mM in RDA) against the indicated
microbes. For determination of antimicrobial activities, E. coli ATCC 25922, S. aureus ATCC 29213 isolates (46106cfu) or C. parapsilosis ATCC 90018
(16105cfu) was inoculated in 0.1% TSB agarose gel. Each 4 mm-diameter well was loaded with 6 ml of peptide. The zones of clearance correspond to
the inhibitory effect of each peptide after incubation at 37 uC for 18–24 h (mean values are presented, n=3). (B) Antimicrobial activity (at 100 mM in
RDA) against a panel of microbes of the selected highly pure peptides LVL20 and KKR20, and comparison with LL-37. (C) In viable count assays, the
indicated microbes were subjected to the N-terminal PrP peptide MAN28 (at 30 mM) in 10 mM Tris pH 7.4 containing 5 mM glucose. Identical buffers
without peptide were used as controls. (D) In viable count assays, E. coli bacteria were subjected to the indicated peptides in 10 mM Tris pH 7.4
containing 0.15 M NaCl in absence or presence of 20% human citrate-plasma. Identical buffers without peptide were used as controls. (E)
Permeabilizing effects of peptides on E. coli. E. coli was incubated with the indicated peptides and permeabilization was assessed using the
impermeant probe FITC. (F) Effects of recPrP (23–231) in presence of buffers mimicking the salt content and pH of human sweat. In viable count
assays, Candida parapsilosis were subjected to PrP (1 mM) in sweat buffer at pH 5.5 or 6.5 and the number of cfu was determined.
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slight increase of PrP-mediated antimicrobial activity against C.
parapsilosis at low pH (Fig. 1A), we next investigated the activity of
the 20mers in RDA at pH 5.5. Whereas the previously described
H-rich peptides GHHPHGHHPHGHHPHGHHPH (GHH20),
derived from histidine-rich glycoprotein  and AHHAHAAH-
HAHAAHHAHAAHHAHA (AHH24) both showed en-
hanced activity at low pH, the PrP-derived 20mers yielded no
additional clearance zones at low pH (Figure S3A, B). In viable
count assays, Zn2+at 50 mM did not enhance the activity of PrP
(Figure S4A), nor were additional antimicrobial 20mer peptides
detected using RDA in the presence of Zn2+(Figure S4B,C).
Although valuable for the initial evaluation, the PEP peptides
were only used for initial screening purposes and for selection of
putative active peptides. Still, inspection of the mass-spectrometry
data showed that contaminants consisted of smaller peptides,
being truncated version of the major peptide of relatively minor
effect on the observed antimicrobial activity. Based on the
data obtained from the initial screening, highly pure (.95%)
peptides were synthesized and further analysed; the previously
described LVLFVATWSDLGLCKKRPKP (LVL20), KKRPKP-
GGWNTGGSRYPGQG (Fig. 3A), but also a longer form extending
from the N-terminus and encompassing the KKRPKP sequence;
MANLGCWMLVLFVATWSDLGLCKKRPKP (MAN28). Antimi-
crobial assays confirmed the high activity of these peptides, when
compared to the benchmark peptide LL-37 (Figure 3B, C). First, the
activity of the peptides LVL20 and KKR20 was analysed using RDA.
These peptides all exerted antimicrobial activities against the above
microbes, as well as P. aeruginosa and B. subtilis (Fig. 3B). It was noted
that the activities were comparable to those demonstrated for the
shown that the peptides derived from the unprocessed N-termini of
mouse and bovine prion proteins, comprising hydrophobic sequences
followed by the charged domain KKRPKP, cause membrane
perturbation and lysis of phospholipid vesicles When tested in
highly hydrophobic peptides may aggregate/and or associate, or
antimicrobial activity. Hence, the peptide was tested in viable
AMPs are dependent of the microenvironment. For example, various
chemokines, defensins, as well as LL-37 are partly, or completely,
antagonized by high salt conditions or the presence of plasma proteins
in vitro [41,42]. The results showed that MAN28 retained some
antimicrobial activity in 0.15 M NaCl, however, its activity was lost in
the presence of plasma proteins (Fig. 3D). The former LVL20 and
KKR20 were inactive in the presence of salt and plasma. The
membrane permeabilizing effects of MAN28, LVL20, and KKR20
are illustrated in Figure 3E, where an increased uptake of the
impermeant dye propidium iodide is noted after subjection of E. coli to
the peptides. Similarly to the above peptides, PrP was also inactive in
the presence of salt and plasma (not shown). It should be noted,
salt and pH environment of human skin (Fig. 3F).
AMPs that kill bacteria may also exhibit hemolytic and
membrane permeabilising activities against eukaryotic cells. It
was noted that particularly MAN28, but also to lesser extent
LVL20, both containing the hydrophobic signal sequence,
displayed hemolytic effects which exceeded those observed for
LL-37 (Fig. 4A). Furthermore, the peptide MAN28 significantly
permeabilized HaCat cells (a keratinocyte cell line), whereas LL-37
as well as LVL20 where less active in this respect (Fig. 4B). In
liposome models, the peptides containing the KKRPKP sequence
caused CF release thus indicating a direct effect on lipid
membranes (Fig. 4C). In contrast to the classical helical peptides
LL-37, circular dichroism showed that there was no helix
induction upon incubation of the peptides with (negatively
charged) DOPG/DOPG liposomes (Fig. 4D).
Next, western blot was performed on extracts of keratinocyte
cultures stimulated with TGF-a. A stronger signal for PrP was
noted demonstrating that increased amounts of PrP are produced
by hyperproliferative keratinocytes (Fig. 5A). Immunohistochem-
istry with antibodies against the N-terminal part of PrP showed a
prominent staining of PrP that was particularly apparent 4 days
after wounding as compared to the low amounts found in normal
non-wounded epidermis (Fig 5B). Furthermore, hybridization
values for PrP were investigated in a microarray experiment
performed on in vivo skin wounds. In wounded skin of all three
donors, high levels of Prp expression were detected, when
compared to unwounded skin (not shown). Additional microarray
experiment using skin wounded ex vivo (n=3) yielded similar results
(p-value=0.005) (Figure 5C). These data clearly demonstrate an
inducible expression of PrP during inflammatory/hyperprolifera-
tive conditions in human skin ex vivo and in vivo.
The key findings in this study are the identification of an
antimicrobial activity of PrP together with the characterization of
epitopes mediating this effect, as well as mechanistic data showing
that the antimicrobial activity is mediated by bacterial membrane
permeabilisation. This new property of PrP is in line with several
observations indicating that heparin-binding proteins, attributed
various roles in biology, such as complement C3, kinino-
gen[12,15], heparin-binding protein, heparin-binding epider-
mal growth factor and other growth factors, b2-glycopro-
holoproteins or after fragmentation, also exert antimicrobial
activities in vitro, and in several cases, in vivo[12,15,36] .
Mature PrPccontains a folded globular C-terminal domain and
an N-terminal region which is largely unstructured. From a
structural perspective, several lines of evidence suggest that the
antimicrobial activity relies on the sequence KKRPK in the
unstructured N-terminal domain. However, since this domain also
contains stretches of histidines, as well as other sequence motifs,
mediating interactions with Zn2+and Cu2+, it cannot be
completely excluded that additional antimicrobial effects of PrP
may be mediated by this region, particularly at low pH and
perhaps in concert with contributions from additional helical
motifs in the globular part. Having said this, investigations
addressing this, e.g. testing effects of the overlapping 20mer
peptides at low pH as well as in presence of Zn2+, did not detect
any additional antimicrobial regions. However, it should be noted
that the 20mers obviously show limitations with respect to both
length and structure, and may not reflect the possible contribution
of the H-rich region to the observed antimicrobial activity of intact
PrP. With respect to the N-terminal signal peptides containing the
KKRPKP sequence, MAN28 (and its shorter variant LVL20) it is
particularly interesting that related peptides of mouse and bovine
origin permeabilize liposomes, in agreement with the antibacterial
activity reported here on the human peptide sequence. The
findings that the N-terminal peptide of mature PrP (KKR20), as
well as recombinantly produced PrP is antimicrobial, indicate
however, that the signal peptide is not required for antimicrobial
activity of the protein. Considering the N-terminal peptide
KKR20, the peptide likely resembles other linear peptides having
a low helical content. For example, AMPs derived from growth
factors display a low helical content in buffer and in presence of
may, either as
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membranes, reflecting their low content of features typical of
‘‘classical’’ helical peptides, such as regularly interspersed
hydrophobic residues. Furthermore, studies utilizing ellipso-
metry, CD, fluorescence spectroscopy, and z-potential measure-
ments on a kininogen-derived antimicrobial peptide, HKH20
HKH20 peptide display primarily random coil conformation in
buffer and at lipid bilayers, the interactions dominated by
electrostatics, as evidenced by strongly reduced adsorption and
membrane rupture at high ionic strength Both HKH20
and GKR22 (GKRKKKGKGLGKKRDPCLRKYK) , a
peptide derived from heparin-binding growth factor, retain
antibacterial activity in physiological buffers as well as in plasma.
In contrast to these observations, the prion protein-derived
KKR20 lost its antibacterial activity at high salt concentrations
(0.15 M NaCl), illustrating that cationicity alone is not a sufficient
parameter for determining antimicrobial activity of a given
peptide, and exemplifying that amphipathicity, as well as
hydrophobicity, enabling bacterial membrane interactions, are
necessary for activity of many AMPs, especially at physiological
salt concentrations, where initial electrostatic interactions with
anionic cell wall components are diminished by the ionic
A number of mechanisms by which AMPs induce membrane
defects have been observed. For some peptides, e.g., melittin,
alamethicin, magainin 2 and gramicidin A [7,48,49,50], as well as
the N-terminal PrP-derived signal peptides of mouse and bovine
origin , transmembrane structures have been reported,
forming transient pores. For disordered and highly charged
peptides membrane disruption is obtained by other mechanisms,
e.g., induction of a negative curvature strain, membrane thinning,
or local packing defects associated with peptide localization
primarily in thephospholipid
gion[7,17,47,51,52,53]. Although the data reported here demon-
strate that peptide-induced bacterial membrane damage correlates
to membrane defect formation in a model lipid membrane system,
additional studies are warranted in order to study the exact mode
of action of human PrP and related peptides.
As previously mentioned, many antimicrobial peptides, includ-
ing LL-37, C3a , and kininogen derived peptides [12,15]
are released during proteolysis. Whether N-terminal fragments of
the prion protein are generated in vivo remains to be investigated.
However, the observation that cleavage of the N-terminal part of
PrP occur in response to oxidative stress and reactive oxygen
species (ROS), releasing low molecular weight fragments of about
6 kDa , point at the interesting possibility that antimicrobial
fragments of the prion proteins may be released during
inflammation. Furthermore, although evidence suggest that
extracellular PrP may be released from cells , research is
needed whether this shed PrP may reach sufficiently high
antimicrobial levels in vivo. It should also be noted, that PrP, as
well as the studied peptides (with the exception of the signal
Figure 4. Activities on eukaryotic cells and liposomes. (A) Hemolytic effects of MAN28, LVL20, and KKR20 were investigated. The cells were
incubated with different concentrations of the peptides, 2% Triton X-100 (Sigma-Aldrich) served as positive control. The absorbance of hemoglobin
release was measured at l 540 nm and is expressed as % of Triton X-100 induced hemolysis (note the scale of the y-axis). Effects of LL-37 are shown
for comparison. (B) HaCaT keratinocytes were subjected to the indicated peptides prion-derived peptides as well as LL-37. Cell permeabilizing effects
were measured by the LDH based TOX-7 kit. LDH release from the cells was monitored at l 490 nm and was plotted as % of total LDH release. (C)
Effects of the indicated peptides on liposome leakage. The membrane permeabilizing effect was recorded by measuring fluorescence resulting from
the release of carboxyfluorescein from negatively charged DOPE/DOPG liposomes. Values represents mean of triplicate samples. (D) Helical content
of the indicated peptides in the presence or absence of negatively charged liposomes (DOPE/DOPG). Only LL-37 showed a marked helix induction
upon addition of the liposomes.
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peptide, MAN28) lost their antimicrobial activity at high salt
strength, casting doubt of the potential antimicrobial role of PrP in
vivo. Nevertheless, there is now convincing evidence that AMPs,
such as similarly salt-sensitive defensins and chemokines[57,58]
contribute to enhanced bacterial killing in vivo, likely reflecting the
necessity of their compartmentalization, presence of ionic
microenvironments (as illustrated by retained activity of PrP in
sweat buffers), or synergism between AMPs.
As mentioned above, prion protein is not only confined to the
nervous system, but instead ubiquitously found in many other cells
and tissues, and the physiological role for this protein are still
enigmatic. In a previous study, it was reported that human
keratinocytes express PrPcin vitro and during inflammatory skin
disease . Although that previous work was focusing on prion
infectivity routes, our current findings on increased expression of PrP
during wounding, together with the observation of its antimicrobial
activity, clearly indicate that PrPs could have a previously undisclosed
role in host defense. In this context, experiments with PrP deficient
animals in infection models should be valuable in order to further
delineate a possible role of PrP in innate defense.
pH. In viable count assays, Candida parapsilosis were subjected to
increasing doses of the peptides AHH24; AHHAHAAHH AH-
AAHHAHAAHHAHA (left panel) and GHH20; GHHPHGH-
HPHGHHPHGHHPH (right panel) in 10 mM Tris pH 7.4 or in
10 mM MES pH 5.5 and the number of cfu was determined.
Found at: doi:10.1371/journal.pone.0007358.s001 (0.40 MB TIF)
Antimicrobial effects of histidine-rich peptides at low
The peptides used in the study are indicated. In addition to the
Sequence of PrP and overlapping 20 mer peptides.
overlapping peptides, regions of specific interest, eg. high charge,
and content of helical structures were selected.
Found at: doi:10.1371/journal.pone.0007358.s002 (0.35 MB TIF)
low pH. (A) Antimicrobial activity of selected peptides (at 100 uM
in RDA) against C. parapsilosis ATCC 90018 (16105 cfu). The
fungi were inoculated in a 0.1% TSB agarose gel containing
10 mM Tris, pH 7.4 or 10 mM MES, pH 5.5. Each 4 mm-
diameter well was loaded with 6 ul of peptide. The zones of
clearance correspond to the inhibitory effect of each peptide after
incubation at 37uC for 18–24 h (mean values are presented, n=3).
(B) In a similar setup as above, the control peptides AHH24;
AHHAHAAHHAHAAHHAHAAHHAHA and GHH20; GHH-
PHGHHPHGHHPHGHHPH were tested at the indicated doses.
The activity of these control peptides was enhanced at low pH.
Found at: doi:10.1371/journal.pone.0007358.s003 (0.97 MB TIF)
Activities of peptide sequences of PrP at normal and
absence and presence of Zn2+. (A) In viable count assays, Candida
parapsilosis was subjected to PrP at 1 mM in 10 mM Tris pH 7.4 in
absence and presence of 50 uM Zn2+, and the number of cfu was
determined (n=3). There was no significant difference in PrP activity
in absence and presence of Zn2+. The peptides AHH24;
AHHAHAAHH AHAAHHAHAAHHAHA (center panel) and
GHH20; GHHPHGHHPHGHHPHGHHPH (right panel) showed
no antimicrobial activity in 10 mM Tris, but showed a dose-
dependent killing of C. parapsilosis in presence of 50 uM Zn2+. (B)
Antimicrobial activity of PrP-derived peptides (at 200 uM in RDA)
against C. parapsilosis ATCC 90018 (16105 cfu). The fungi were
inoculated in a 0.1% TSB agarose gel containing 10 mM Tris,
pH 7.4 with or without 50 uM Zn2+. Each 4 mm-diameter well was
loaded with 6 ul of peptide. The zones of clearance correspond to the
Activities of PrP and derived peptide sequences in
Figure 5. Expression and microarray hybridization levels of PrP in wounded skin ex vivo and in vivo. (A) PrP expression in keratinocyte
cultures. Cell extracts from keratinocytes grown in absence or presence of TGF-a were subjected to SDS-PAGE, blotted and probed with antibodies to
PrP 27–30. recPrP23–144(0.5 mg) was used as positive control. (B) Expression of PrP in wounds in vivo. Normal skin biopsies and biopsies of wound
edges (at day 4) were immunostained for PrPc. Hematoxylin was used for counterstaining. (C) Hybridization levels of PRPN in ex vivo wounded and
non-wounded skin after four days in culture are presented.
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inhibitory effect of each peptide after incubation at 37uC for 18–24 h
(mean values are presented, n=3). (C) In a similar setup as in B, the
control peptides AHH24 and GHH20 were tested at 200 uM in
RDA. The activity of these control peptides was significantly
enhanced at low pH (n=3, p,0.05).
Found at: doi:10.1371/journal.pone.0007358.s004 (1.25 MB TIF)
We thank Ms. Mina Davoudi, Ms. Lise-Britt Wahlberg for valuable
support and input.
Conceived and designed the experiments: MP MM AS. Performed the
experiments: MP MKR VR KS AC MM OES. Analyzed the data: MP
MKR VR MM OES AS. Contributed reagents/materials/analysis tools:
KS WKS. Wrote the paper: MP MM AS.
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