Fowlicidin-1 and fowlicidin-2 are two newly identified chicken cathelicidins with potent antibacterial activities.. Unlike many other host defense peptides with antimicrobial activities t
Trang 1with potent antibacterial and
lipopolysaccharide-neutralizing activities
Yugendar R Bommineni1*, Huaien Dai2*, Yu-Xi Gong2, Jose L Soulages3, Samodha C Fernando1, Udaya DeSilva1, Om Prakash2and Guolong Zhang1
1 Department of Animal Science, Oklahoma State University, Stillwater, OK, USA
2 Department of Biochemistry, Kansas State University, Manhattan, KS, USA
3 Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK, USA
Cationic antimicrobial peptides comprise a large group
of small peptides with extremely diverse amino-acid
sequences but with conserved features in each family
[1,2] Acting as an important first line of defense, these
peptides are mostly produced by innate immune cells
such as phagocytes, mucosal epithelial cells, and skin
keratinocytes in vertebrates, capable of killing a broad range of bacteria, fungi, and viruses, including resist-ant strains [1,2] Because of nonspecific membrane-lytic activities, antimicrobial peptides have a low tendency
to develop resistance, a desirable feature as a new class
of antimicrobial agents [1,3,4]
Keywords
antibiotic resistance; antimicrobial peptide;
cathelicidin; host defense; structure–activity
relationship
Correspondence
O Prakash, Department of Biochemistry,
Kansas State University, Manhattan,
KS 66506, USA
Fax: +1785 532 7278
Tel: +1785 532 2345
E-mail: omp@ksu.edu
G Zhang, Department of Animal Science,
Oklahoma State University, Stillwater,
OK 74078, USA
Fax: +1405 744 7390
Tel: +1405 744 6619
E-mail: zguolon@okstate.edu
*These authors contributed equally to this
paper
(Received 13 October 2006, revised 7
November 2006, accepted 10 November
2006)
doi:10.1111/j.1742-4658.2006.05589.x
Cathelicidins are an important family of cationic host defense peptides in vertebrates with both antimicrobial and immunomodulatory activities Fowlicidin-1 and fowlicidin-2 are two newly identified chicken cathelicidins with potent antibacterial activities Here we report structural and func-tional characterization of the putatively mature form of the third chicken cathelicidin, fowlicidin-3, for exploration of its therapeutic potential NMR spectroscopy revealed that fowlicidin-3 comprises 27 amino-acid residues and adopts a predominantly a-helical structure extending from residue 9 to
25 with a slight kink induced by a glycine at position 17 It is highly potent against a broad range of Gram-negative and Gram-positive bacteria
in vitro, including antibiotic-resistant strains, with minimum inhibitory con-centrations in the range 1–2 lm It kills bacteria quickly, permeabilizing cytoplasmic membranes immediately on coming into contact with them Unlike many other host defense peptides with antimicrobial activities that are diminished by serum or salt, fowlicidin-3 retains bacteria-killing activit-ies in the presence of 50% serum or physiological concentrations of salt Furthermore, it is capable of suppressing lipopolysaccharide-induced expression of proinflammatory genes in mouse macrophage RAW264.7 cells, with nearly complete blockage at 10 lm Fowlicidin-3 appears to be
an excellent candidate for future development as a novel antimicrobial and antisepsis agent, particularly against antibiotic-resistant pathogens
Abbreviations
CCL, CC chemokine ligand; CFU, colony forming unit; EC50, 50% effective concentration; LPS, lipopolysaccharide; MCP-1, monocyte chemotactic protein-1; MIC, minimum inhibitory concentration; MIP-1a, monocyte inflammatory protein-1a; MDCK, Madin–Darby canine kidney cells; NOE, nuclear Overhauser effect.
Trang 2Besides having direct microbicidal activities,
anti-microbial peptides have increasingly been appreciated
to play a profound role in regulating host immune
responses to infections Many peptides have been
shown to be actively involved in binding and
neutral-ization of lipopolysaccharide (LPS), chemotaxis of
immune cells, regulation of dendritic cell
differenti-ation, induction of angiogenesis and
re-epithelializa-tion, and modulation of cytokine and chemokine gene
expression [5–7] To better reflect the pleiotropic effects
of antimicrobial peptides on various aspects of innate
and adaptive immunity, these peptides have been
pro-posed to be renamed as host defense peptides [6,7]
Both antimicrobial and immunomodulatory activities
of these peptides are being harnessed and manipulated
for therapeutic benefit It is possible to use these
pep-tides for antimicrobial therapy without provoking
detrimental proinflammatory responses [6–8]
Cathelicidins represent a major family of host
def-ense peptides that have been identified in fish, birds,
and mammals [9–11] All cathelicidins share a highly
conserved cathelin pro-sequence at the N-terminus,
with extremely variable C-terminal sequences having
antimicrobial and immune regulatory activities [9–11]
We recently identified three chicken cathelicidins,
fow-licidins-1–3 [12] On the basis of the conserved
ela-stase cleavage site present in the precursor sequences,
we predicted that mature forms of fowlicidins-1–3 are
likely to consist of 26, 31, and 27 amino-acid residues
in the C-terminal regions, respectively [12] We
fur-ther found that putatively mature fowlicidin-1 and
fowlicidin-2 are among the most efficacious
cathelici-dins that have been reported, with fowlicidin-1 being
slightly more potent than fowlicidin-2 in killing
bac-teria [12]
To evaluate the potential of putatively mature
fow-licidin-3 as a model for the design of antimicrobial
agents, here we report structural and functional
char-acterization of fowlicidin-3, a third chicken
cathelici-din that is likely to have evolved from fowlicicathelici-din-1 by
gene duplication [12] Similar to fowlicidin-1,
puta-tively mature fowlicidin-3 peptide was found to be
largely a-helical with a kink in the central region and
a relatively flexible unstructured segment in the
N-ter-minal region Fowlicidin-3 is highly active against a
broad range of bacteria in vitro, including
antibiotic-resistant strains, but 4–6-fold less toxic to mammalian
host cells than fowlicidin-1 Moreover, fowlicidin-3 is
more potent than fowlicidin-1 in blocking
LPS-induced proinflammatory responses Collectively,
fow-licidin-3 represents an attractive antibacterial and
antisepsis drug candidate for further clinical
develop-ment
Results
Structural characterization of fowlicidin-3 Putatively mature fowlicidin-3 comprising 27 amino-acid residues was synthesized and purified to > 95% purity, and its mass was confirmed by MS to be 3095.1 Da, consistent with the calculated value (3094.8 Da) Putatively mature fowlicidin-1 comprising
26 amino acids was similarly synthesized and purified as
a reference peptide with an observed mass of 3141.6 Da and calculated mass of 3141.9 Da, as described [13]
CD spectroscopy was first performed to determine the secondary structure of fowlicidin-3 in the presence
of different concentrations of trifluoroethanol and sodium dodecyl sulfate (SDS) As shown in Fig 1A,
Fig 1 CD spectra of fowlicidin-3 in different concentrations of tri-fluoroethanol (TFE) (A) and SDS (B) with or without 0.15 M NaCl.
Trang 3fowlicidin-3 was largely unstructured in phosphate
buffer and began to transform into a typical a-helical
conformation after the addition of trifluoroethanol in
a dose-dependent manner Significant a-helical content
(86%) with virtually no b-sheet structure was observed
in fowlicidin-3 in 50–60% trifluoroethanol (Fig 1A)
Similarly, fowlicidin-3 exhibited 53% a-helical content
in the presence of 0.25% SDS, and the a-helical
con-tent remained largely unaltered in 0.5% or 2.0% SDS
micelles (Fig 1B) These results suggest that
fowlici-din-3 is likely to adopt a predominantly a-helical
con-formation when interacting with bacterial membranes
To further determine the tertiary structure of
fowlic-idin-3, 2D NMR spectroscopy was used Because the
a-helical content of fowlicidin-3 peaked in 50%
tri-fluoroethanol (Fig 1A) and NMR signals in
trifluoro-ethanol were much sharper and more intense than in
SDS micelles, fowlicidin-3 (4 mm) prepared in 50%
deuterated trifluoroethanol (trifluoroethanol-d3)⁄ 50%
water (v⁄ v) was selected for detailed NMR studies as
described [13] Complete proton resonance assignments
were obtained using spin system identification and
sequential assignments from NMR spectra recorded at
25C (Supplementary Figs S1 and S2) Consistent
with the CD results, the Ca-proton chemical shift
index, together with the presence of a number of
sequential dNN(i, i +1), nonsequential daN(i, i +3),
and dab(i, i +3) nuclear Overhauser effect (NOE)
peaks (Fig 2), clearly indicates an a-helical
conforma-tion for fowlicidin-3
A total of 205 NOE constraints, including 68
intra-residue, 86 sequential, and 51 medium-range
con-straints, were used to calculate the tertiary structure of
fowlicidin-3 (Table 1) From 100 calculated structures
that satisfied the experimental restraints, 20 structures
with the lowest total energy were selected for further
analysis A Ramachandran plot, produced by
pro-check-nmr [14], showed that 64.8% residues are in
the most favored region and 33.4% are in additional
allowed regions (Table 1) A superimposition of the 20
lowest-energy structures showed a considerable degree
of flexibility, with a pairwise rmsd of the backbone of 3.03 A˚ (Table 1) However, alignments along residues V9–A16 (Fig 3A) and N19–R25 (Fig 3B) of the 20 structures resulted in backbone rmsd values of
< 0.4 A˚ in both cases (Table 1), suggesting relative rigidity of these two a-helical segments
The energy-minimized average structure of fowlici-din-3 was further calculated, showing a predominantly a-helical structure extending from V9 to R25 with a relatively flexible N-terminal segment (Fig 3C) A clo-ser examination of the NMR structure revealed a kink within the long a-helix between residues 16 and 19, due to the presence of a glycine at position 17 This was indicated by the fact that the CaH chemical index showed no shift for G17 and I18 (Fig 2), consistent with the notion that glycine usually allows peptide backbone flexibility As evidenced by a lack of NOEs (Fig 2), such a kink indeed provides conformational flexibility between two short a-helical segments (com-pare Fig 3A,B), reminiscent of fowlicidin-1 [13] Superimposition of fowlicidin-1 on fowlicidin-3 indeed revealed substantial overlapping, except for the flexible N-sequences (Fig 3D) This is perhaps not surprising, given the fact that both peptides are likely to have evolved by duplication and share > 60% identity in
Fig 2 Schematic diagram of C aH chemical-shift index as well as
sequential and medium distance NOE connectivities for fowlicidin-3.
The thickness of the bar reflects the strength of the NOE
connectiv-ities.
Table 1 Structural statistics of the 20 lowest-energy structures of fowlicidin-3.
NOE constraints
Energies (kcalÆmol)1)
Pairwise rmsds for residues 1–27 (A ˚ )
Rmsds to mean structure (A ˚ ) (residues 9–16)
Rmsds to mean structure (A ˚ ) (residues 19–25)
Percentage of residues in regions of /–w space
Trang 4amino-acid sequence in the putatively mature region
(Fig 3E) Despite structural similarities, it will be
interesting to study whether the two fowlicidins differ
in functional properties
Evaluation of antibacterial properties of
fowlicidin-3
Fowlicidin-1 was found to be among the most potent
cathelicidins in killing bacteria [12] To evaluate the
antibacterial spectrum and efficacy of fowlicidin-3, we
performed standard broth microdilution assays in
100% Muller-Hinton broth as recommended by the
Clinical and Laboratory Standards Institute [15] using
fowlicidin-1 as a reference peptide As shown in Table 2, fowlicidin-3 was active against a wide range
of Gram-negative and Gram-positive bacteria with minimum inhibitory concentrations (MICs) in the range 1–2 lm, often showing slightly higher potency than fowlicidin-1 Moreover, fowlicidin-3 exhibited
no diminished efficiency against antibiotic-resistant strains, including multidrug-resistant Salmonella
enteri-ca serovar Typhimurium DT104 and two methicillin-resistant Staphylococcus aureus strains tested
Most cationic host defense peptides, including cathe-licidins, are membrane-lytic agents, killing bacteria by physical interaction with and disruption of bacterial cell membranes, although increasing evidence suggests the presence of intracellular targets for certain peptides [1,16] To examine the mechanism of action and bac-terial killing kinetics of fowlicidin-3, Escherichia coli ML-35p, a strain that contains a plasmid giving consti-tutive expression of b-galactosidase in the cytosol, was incubated with different concentrations of peptides for
1 h in the presence of a chromogenic substrate, o-ni-trophenyl-b-d-galactopyranoside [17–19] It is conceiv-able that the amount of b-galactosidase released, as indicated by color change, is proportional to the degree of permeabilization of bacterial cytosolic mem-branes by fowlicidins As shown in Fig 4, membrane permeabilization began almost immediately upon the addition of 1 lm fowlicidin-3 or fowlicidin-1 to bac-teria, reaching a plateau at 30–40 min, entirely consistent with earlier colony counting assays with fowlicidin-1, in which bacteria were killed quickly with the maximum killing occurring 30 min after incubation
of the peptide with bacteria [12] Identical trends were
Fig 3 Tertiary structure of fowlicidin-3 in 50% trifluoroethanol (A)
Superimposition of the backbones of the 20 lowest-energy
struc-tures of fowlicidin-3 best-fitted to residues 9–16 (B)
Superimposi-tion of the backbones of the 20 lowest-energy structures of
fowlicidin-3 best-fitted to residues 19–25 (C) Ribbon diagram of the
minimized average structure of fowlicidin-3 (D) Superimposition of
the average structures of fowlicidin-3 with fowlicidin-1 The
struc-tures were generated by using MOLMOL (E) Sequence alignment of
fowlicidin-3 and fowlicidin-1 Dashes are created to maximize the
alignment, and the total amino-acid residue numbers are also
indi-cated Vertical bars connecting sequences denote identities, and
colons mean similarities The conserved glycine is boxed.
Table 2 MICs of fowlicidin-3 (Fowl-3) in comparison with fowlici-din-1 (Fowl-1) MICs were determined as the lowest peptide con-centration that gave no visible bacterial growth after overnight incubation in a standard broth microdilution assay using 100% Muller–Hinton broth The experiments were repeated at least twice for each bacterial strain with similar values MRSA, Methicillin-resistant Staph aureus.
Gram-negative
Gram-positive
Trang 5also observed with 0.5 and 2 lm fowlicidin
concentra-tions (data not shown) These results imply that, as
with most other host defense peptides, physical
mem-brane disruption appears to be a major mechanism of
killing bacteria for fowlicidin-3 and fowlicidin-1
Physiological concentrations of salt prove to be
inhibitory to the antibacterial activities of many
anti-microbial peptides, such as human cathelicidin LL-37
[18] and a-defensin and b-defensin [20,21] However,
the presence of 100 mm NaCl had little impact on
membrane permeabilization, with only a minimal delay
in killing kinetics for fowlicidin-3 (Fig 4), consistent
with our direct colony counting assay (data not
shown) Indeed, the presence of physiological
concen-trations of NaCl did not affect the structure of
fowlici-din-3 in membrane mimetic environments (Fig 1A,B)
These data suggest that, similar to fowlicidin-1 and
fowlicidin-2 [12], fowlicidin-3 kills bacteria in a
salt-independent manner, in contrast with many other
pep-tides, the activities of which are severely suppressed in
the presence of salt [18,20,21]
Serum has been found to be another important
inhibitory factor in bactericidal activities of many host
defense peptides, probably because of the presence of
certain salts, bivalent cations, and peptide-binding
pro-teins To examine the effect of serum on antibacterial
efficacy of fowlicidin-3, a radial diffusion assay [22] was performed with E coli O157:H7 ATCC 700728 and Staph aureus ATCC 25923 and peptides diluted with and without 50% human or chicken serum The results revealed that both fowlicidin-3 and fowlicidin-1 retained > 80% activity against Gram-negative E coli O157:H7 in either serum (Fig 5) The same trend was also true with Gram-positive Staph aureus (data not shown) These results imply in vivo therapeutic poten-tial for fowlcidin-3 and fowlcidin-1 for systemic appli-cations
Evaluation of the toxicity of fowlicidin-3
to mammalian cells
As compared with b-sheet defensins, a considerably higher degree of toxicity to mammalian cells occurs with a-helical cathelicidins, limiting their potential as antimicrobial agents To study the toxicity of fowlici-din-3, Madin-Darby canine kidney (MDCK) epithelial cells were first incubated with different concentrations
0 10 20 30 40 50 60
0.0
0.5
1.0
1.5
No Peptide
No Peptide + Salt
Fowl-3
Fowl-1
Fowl-3 + Salt
Fowl-1+ Salt
Time (min)
Fig 4 Peameabilization of bacterial cytoplasmic membrane by
fow-licidins E coli ML-35p was diluted to (2.5–5) · 10 7
CFUÆmL)1and incubated with 1 l M fowlicidin-3 or fowlcidin-1 in 10 m M sodium
phosphate, pH 7.4, in the presence and absence of 100 m M NaCl
at 37 C A chromogenic substrate for b-galactosidase,
o-nitrophe-nyl-b- D -galactopyranoside, was also added to a final concentration
of 1.5 m M The absorbance at 405 nm was monitored every 2 min
for the production of p-nitrophenol for up to 1 h Data shown are
representative of two independent experiments with highly similar
results.
Fowl-3
Fowl-1
Chicken Serum
Human Serum
No Serum
0 20 40 60 80 100
Fowlicidin-3 Fowlicidin-1
A
B
Fig 5 Effect of serum on the antibacterial activity of fowlicidins by radial diffusion assay Fowlicidin-3 or fowlcidin-1, 1 lg diluted in 0.01% acetic acid with and without 50% human or chicken serum, was added to the wells of the underlay gel containing E coli O157:H7 ATCC 700728 (4 · 10 5 CFUÆmL)1) After overnight incuba-tion, bacterial clearance zones were recorded, and antibacterial activities (%) in the presence of serum were calculated relative to the activities without serum In (B), open bars represent no serum controls, and striped and solid bars are 50% human and chicken serum, respectively Data shown are mean ± (SEM) from two inde-pendent experiments.
Trang 6of fowlicidins in the presence or absence of 10% fetal
bovine serum, and then a cell viability assay was
per-formed as described [12] As compared with fowlicidin-1
with a 50% effective concentration (EC50) of 2 lm,
fowlicidin-3 killed 50% MDCK cells at 12 lm
(Fig 6A) Moreover, the presence of 10% serum further
reduced the toxicity of fowlicidin-3 by twofold
(Fig 6A)
To test the hemolytic activity of fowlicidin-3 further,
freshly isolated human erythrocytes were incubated
with fowlicidins with and without 10% fetal bovine
serum, and erythrocyte lysis was measured according
to the release of hemoglobin [12] In the absence
of serum, 50% hemolysis occurred at 9 lm for
fowlici-din-3, whereas fowlicidin-1 was considerably more
toxic with an EC50of 1.5 lm (Fig 6B) Serum
sub-stantially reduced hemolysis of both peptides, with
EC50 values of 80 lm for fowlicidin-3 and 13 lm for
fowlicidin-1 in 10% fetal bovine serum Taking the
results together, fowlicidin-3 is slightly more potent
than fowlicidin-1 in killing many bacterial strains
tes-ted, but is 4–6-fold less toxic to mammalian cells than
fowlicidin-1, indicating higher therapeutic potential for fowlicidin-3
Inhibition of LPS-induced proinflammatory gene expression by fowlicidin-3
Because fowlicidin-1 and fowlicidin-2 were found to be able to bind LPS directly and suppressed LPS-induced cytokine gene expression [12], we sought to determine whether fowlicidin-3 has a similar LPS-neutralizing activity Mouse macrophage RAW264.7 cells were sti-mulated for 4 h with 100 ngÆmL)1LPS in the presence and absence of different concentrations of fowlicidins, followed by real-time RT-PCR analysis of the expres-sions of three common proinflammatory genes, inclu-ding interleukin-1b, CC chemokine ligand 2 (CCL2)⁄ monocyte chemotactic protein-1 (MCP-1), and CCL3⁄ monocyte inflammatory protein-1a (MIP-1a)
As shown in Fig 7, fowlicidin-3 dose-dependently inhibited the expression of interleukin-1b or CCL3⁄ MIP-1a genes, with a concentration of 10 lm reducing
0
20
40
60
80
100
A
B
Fowl-3 Fowl-3 + FBS Fowl-1 Fowl-1 + FBS
Peptide (µM)
0
20
40
60
80
100
Fowl-3 + FBS Fowl-3
Fowl-1 + FBS Fowl-1
Peptide (µM)
Fig 6 Toxicity of fowlicidins to MDCK cells (A) and human
erythro-cytes (B) in the presence and absence of 10% fetal bovine serum
(FBS) EC 50 is indicated as dotted lines in both panels Data shown
are mean ± SEM from two to four independent experiments.
0 5 10 15 20 25 30
Control Fowlicidin-3 Fowlicidin-1
LPS - + - - - + + + - - + + Peptide
-0 1000 2000 3000 4000 5000 6000 7000 8000
Fig 7 Inhibition of LPS-induced expression of interleukin-1b and CCL3 ⁄ MIP-1a in RAW264.7 cells Cells were pretreated for 30 min with and without fowlicidin-3 (0.5, 2.5, and 10 l M ) or fowlicidin-1 (2.5 and 10 l M ) in duplicate, followed by stimulation for another 4 h with 100 ngÆmL)1LPS Total RNA was then isolated and subjected
to real-time RT-PCR analysis Data shown are mean ± SEM from two independent experiments.
Trang 7expression of both genes by > 95% A similar
block-age of CCL2⁄ MCP-1 expression was also observed
(data not shown) Because treating cells with
fowlici-din-3 alone had no effect on gene expression (Fig 7),
such LPS-neutralizing activity was specific It is
note-worthy that, compared with fowlicidin-1, fowlicidin-3
is more potent in inhibiting LPS-induced gene
expres-sion (Fig 7), suggesting that fowlicidin-3 may be more
effective in antisepsis therapy
Discussion
Our previous analyses of genomic sequences have
revealed that the genes for 1 and
fowlicidin-3 are almost identical in the first three exons and first
three introns [12] The fourth exon, which primarily
encodes biologically active, mature sequences, also
shares > 60% identity between the two peptides
(Fig 3E) Therefore, these two fowlicidins were most
likely to be duplicated from each other during
evolu-tion The putatively mature fowlicidin-3 peptide
con-sists of 27 amino acid residues with a charge of +6
and no anionic residues, whereas fowlicidin-1 is
com-posed of 26 amino acids with a net charge of +8
Evo-lution of two highly similar antimicrobial peptides
with potent antibacterial activities may represent an
enforcement of innate host defense It is also plausible
that fowlicidin-1 and fowlicidin-3 may have some
non-overlapping biological functions yet to be discovered
Because of a similarity in primary sequence, it is not
surprising that the two fowlicidins adopt a similar
a-helical conformation in membrane-mimicking
envi-ronments (Fig 3D) Moreover, both peptides contain
a kink near the central helical region due to the
pres-ence of a conserved glycine residue (Fig 3E)
Interest-ingly, such a glycine-induced hinge is not unique to
fowlicidins, but appears to be a common feature for
many a-helical cationic host defense peptides
[13,23,24] The presence of a hinge structure has been
shown to be beneficial in enhancing molecular
flexibil-ity while reducing the toxicflexibil-ity of otherwise rigid
pep-tides to mammalian cells [23,24]
Amphipathicity is another hallmark of most
a-heli-cal cationic peptides [23,24] However, unlike typia-heli-cal
a-helical peptides, the long helices of fowlicidin-1 and
fowlicidin-3 are much less amphipathic, with no
obvi-ous segregation of hydrophobic residues from
hydro-philic residues (Fig 8) Furthermore, the a-helical
region is highly hydrophobic (Fig 8) in that
fowlici-din-3 is composed of only one cationic (K22) and three
polar uncharged residues (N12, T13 and N19), whereas
fowlicidin-1 consists of only two cationic (R11 and
R21) and two polar uncharged residues (T12 and N18)
(Fig 3E) Instead, positively charged residues are mostly concentrated at both tails (Figs 3E and 8)
A series of antibacterial tests revealed that, similar
to fowlicidin-1, fowlicidin-3 possesses potent, broad-spectrum, and fast-acting bactericidal activities with similar efficiency against both antibiotic-susceptible and antibiotic-resistant bacterial strains Killing of bac-teria by fowlicidins starts immediately on contact with bacteria, in sharp contrast with human cathelicidin LL-37, which takes up to 20–30 min before permeabili-zation of bacterial inner membranes occurs [17,18] More significantly, bacterial killing activity is largely unaffected by salt or serum, making fowlicidins attractive therapeutic candidates for potential in vivo systemic applications
In spite of similarities in structural and antibacterial properties, fowlicidin-3 is much less toxic to mamma-lian cells than fowlicidin-1 Because the cytotoxicity (EC50) of fowlicidin-3 is at least 10–40-fold (in the presence of serum) higher than MICs against all bac-terial strains tested, a therapeutic window clearly exists for fowlicidin-3, particularly for systemic applications
Fig 8 Surface accessibilities of fowlicidins (A) Front view of the solvent-accessible surface of fowlicidin-3 (B) Back view of the vent-accessible surface of fowlicidin-3 (C) Front view of the sol-vent-accessible surface of fowlicidin-1 (D) Back view of the solvent-accessible surface of fowlicidin-1 Positively charged resi-dues are in blue, polar uncharged resiresi-dues are in pink, and hydro-phobic residues are in yellow The N-terminus is on the top The figures were generated using PYMOL (http://pymol.sourceforge.net).
Trang 8More desirably, fowlicidin-3 is highly potent in
block-ing LPS-induced proinflammatory gene expression
Collectively, fowlicidin-3 appears to have promising
therapeutic potential for further development as a
novel antimicrobial and antisepsis agent
It is interesting to note that the higher toxicity
asso-ciated with fowlicidin-1 is probably due to limited
flexi-bility of the a-helix, which is a result of the physical
hindrance caused by the side chain of a nearby tyrosine
[13] Although fowlicidin-3 is devoid of aromatic
resi-dues adjacent to the conserved glycine (Fig 3E), it will
be important to examine the impact of further
enhan-cing its flexibility on the functional properties In fact,
the flexibility of the hinge region has often been found
to be positively correlated with a decrease in the
toxic-ity of many a-helical peptides [23,24] Because
amphi-pathicity, hydrophobicity, and helicity are among the
most important factors that influence the antibacterial
and toxicity of a-helical cationic peptides [23,24],
rational changes of these structural and
physicochemi-cal parameters are likely to further improve the
thera-peutic potential of fowlicidin-3
Experimental procedures
Peptide synthesis
Putatively mature fowlicidin-1 (RVKRVWPLVIRTVIA
GYNLYRAIKKK) and fowlicidin-3 (KRFWPLVPVAIN
TVAAGIN LYKAIRRK) were chemically synthesized
using the standard solid-phase method of SynPep (Dublin,
CA, USA) and Bio-Synthesis (Lewisville, TX, USA),
respectively Both peptides were purified to > 95% purity
by RP-HPLC The mass and purity of each peptide were
further confirmed by MS using the Voyager DE-PRO
instrument (Applied Biosystems, Foster City, CA, USA)
housed in the Recombinant DNA⁄ Protein Core Facility
of Oklahoma State University Lyophilized peptides were
reconstituted in 0.01% acetic acid, and concentrations were
measured by UV absorbance at 280 nm in the presence of
6 m guanidine hydrochloride [25], based on the absorption
coefficients for aromatic tryptophan and tyrosine residues
present in both peptides
CD spectroscopy and secondary-structure
determination
The secondary structure of fowlicidin-3 was determined on
a Jasco-715 spectropolarimeter (JASCO, Tokyo, Japan)
using a 0.1-cm path length cell over the 180–260 nm range
as described [13] The CD spectra were acquired at 25C
every 1 nm with a 2-s averaging time per point and a 1-nm
band pass Fowlicidin-3 (10 lm) was measured in 50 mm
potassium phosphate buffer, pH 7.4, with or without differ-ent concdiffer-entrations of trifluoroethanol (0%, 10%, 20%, 40%, 50% and 60%) or SDS micelles (0.25%, 0.5% and 2.0%) CD analyses were also performed in 50% trifluoro-ethanol and 2.0% SDS in phosphate buffer with addition
of 150 mm NaCl Mean residue ellipticity (MRE) was expressed as [h]MRE (degreesÆcm2Ædmol)1) The contents of the secondary-structural elements, including regular and distorted a-helix, regular and distorted b-sheet, turns, and unordered structures, were analyzed using the program selcon3 [26]
NMR spectroscopy and tertiary structure calculations
The NMR experiments were performed with 500-MHz Varian UNITY plus NMR spectrometer (Varian, Palo Alto, CA, USA) as previously described [13] Because NMR signals in 50% trifluoroethanol-d3⁄ 50% water mix-ture were much sharper and intense than in SDS micelles, fowlicidin-3 (4 mm) prepared in trifluoroethanol⁄ water (1 : 1, v⁄ v) was selected for detailed NMR studies The data sets were acquired at different temperatures ranging from 10 to 35C The 2D 1H-1H TOCSY spectra with an isotropic mixing time of 100 ms at a B1 field strength of
8 kHz and 2D1H-1H NOESY spectra with mixing times of
100, 200, 300, 400 and 500 ms were collected The trifluoro-ethanol peak (3.88 p.p.m at 25C) was considered as the reference for chemical shift assignments A mixing time of
300 ms was initially used for distance constraint measure-ments, and the assigned NOE peaks were then checked with the spectra obtained with a 100-ms mixing time For molecular modeling calculations, only NOE peaks present
in the NOESY spectra obtained with a mixing time of
100 ms were used to rule out the peaks due to spin diffu-sion The intensities of the cross-peaks in NOESY spectra were classified as strong, medium, and weak, corresponding
to distance restraints of 1.8–2.8, 1.8–4.0, and 1.8–5.0 A˚, respectively The distance restraints were then used to cal-culate structures using the program cns (version 1.1) [27], using a simulated annealing protocol for torsion angle dynamics From all 100 calculated structures accepted, 20 structures with the lowest total energy were selected and an-alyzed with molmol [28] and procheck-nmr [14] The atomic co-ordinates and structure factors of putatively mature fowlicidin-3 have been deposited under accession code 2HFR in the Protein Data Bank, Research Collabora-tory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA (http://www.rcsb.org/)
Bacterial culture and antibacterial testing
Gram-negative bacteria (E coli ATCC 25922, S enterica serovar Typhimurium ATCC 14028, S enterica serovar
Trang 9Typhimurium DT104 ATCC 700408, and Klebsiella
pneu-moniaeATCC 13883), and Gram-positive bacteria (Listeria
monocytogenes ATCC 19115, Staph aureus ATCC 25923,
Staph aureus ATCC BAA-39, and Staph aureus ATCC
43300) were purchased from either ATCC (Manassas, VA,
USA) or MicroBiologics (St Cloud, MN, USA) and tested
individually against fowlicidin-1 and fowlicidin-3 The
MICs were determined by a standard broth microdilution
assay as recommended by the Clinical and Laboratory
Standards Institute [15] Briefly, overnight bacterial culture
was subcultured in fresh trypticase soy broth with shaking
at 250 r.p.m at 37C for 3 h to reach the mid-exponential
phase of growth Bacteria were then washed twice in
10 mm sodium phosphate buffer, pH 7.4, and diluted to
5· 105CFUÆmL)1in Muller-Hinton broth (BBL,
Cockeys-ville, MD, USA) After 90 lL bacteria had been dispensed
into 96-well cell culture plates, 10 lL peptides in serial
two-fold dilutions were added in duplicate The MIC value of
each peptide was determined as the lowest peptide
concen-tration that gave no visible bacterial growth after overnight
incubation at 37C
Assay of cytoplasmic membrane
permeabilization
E coliML-35p was kindly provided by R Gallo (UCSD, La
Jolla, CA, USA) and used as described [17–19] Briefly,
mid-exponential phase bacteria were washed twice in 10 mm
sodium phosphate buffer, pH 7.4, diluted to 0.03 A600
[equiv-alent to (2.5–5)· 107 CFUÆmL)1) in the same phosphate
buffer containing 1% trypticase soy broth with and without
100 mm NaCl After 80 lL bacteria had been dispensed into
each well of a 96-well tissue culture plate, different
concen-trations of fowlicidins and 1.5 mm
o-nitrophenyl-b-d-gal-actopyranoside (Sigma, St Louis, MO, USA) were added to
a total volume of 100 lL per well The production of
p-nitro-phenol was monitored spectrophotometrically at 37C and
405 nm every 2 min for up to 1 h with periodic shaking
Serum effect on the antibacterial activity
of fowlicidin-3
The radial diffusion assay [22] was used to study the effect
of serum on the antibacterial activity of fowlicidins Briefly,
after solidification of the underlay gel containing
4· 105CFUÆmL)1 Staph aureus ATCC 25923 or E coli
O157:H7 ATCC 700728, small wells ( 3 mm in diameter)
were punched Then 1 lg fowlicidin-1 or fowlicidin-3 was
diluted to a total of volume of 4 lL in 0.01% acetic acid
with or without 50% chicken or human serum and added
separately to the wells After 3 h of diffusion at 37C, the
nutrient-rich overlay gel was poured and incubated at
37C overnight The diameters of the bacterial clearance
zones were measured
Cytotoxicity assay
The toxicity of fowlicidin-3 toward mammalian epithelial cells was evaluated by using MDCK cells (ATCC) and an Alamar Blue dye (Biosource, Camarillo, CA, USA) as des-cribed [12] Briefly, MDCK cells were seeded in 96-well plates with 1.5· 105 cells⁄ well and allowed to grow over-night in Dulbecco’s modified Eagle medium (DMEM), con-taining 10% fetal bovine serum to 80–90% confluence After cells had been washed with serum-free DMEM, 90 lL fresh DMEM with or without 10% fetal bovine serum was added
to each well, followed by the addition of 10 lL serially dilu-ted peptides in duplicate After 18 h of incubation at 37C under 5% CO2, 10 lL Alamar Blue dye was added to each well and incubated for another 6 h The fluorescence was read with excitation at 545 nm and emission at 590 nm Per-centage cell death (%) was calculated as [1) (Fpeptide)
Fbackground)⁄ (Facetic acid) Fbackground)]· 100, where Fpeptideis the fluorescence of cells exposed to different concentrations
of peptides, Facetic acidis the fluorescence of cells exposed to 0.01% acetic acid only, and Fbackground is the background fluorescence of 10% AlamarBlue dye in cell culture medium without cells Cytotoxicity (EC50) was defined as the peptide concentration that caused 50% cell death
Hemolysis assay
Freshly collected chicken and human blood were used for evaluating hemolytic activity as described [12,13] The pro-tocols for collection of human and chicken blood were approved by the Institutional Review Board and Institu-tional Animal Care and Use Committee of Oklahoma State University, respectively Briefly, EDTA-anticoagulated blood was washed twice in NaCl⁄ Piand diluted to 0.5% in NaCl⁄ Piwith or without 10% fetal bovine serum Erythro-cytes (90 lL aliquots) were then dispensed into a 96-well plate, followed by the addition of 10 lL serially diluted fow-licidins in 0.01% acetic acid in duplicate After incubation for 2 h at 37C, supernatants were colleted by centrifuga-tion and transferred to a fresh 96-well plate to measure the absorbance of released hemoglobin at 405 nm Controls for 0% and 100% hemolysis were erythrocytes exposed to
10 lL 0.01% acetic acid and 1% Triton X-100, respectively Percentage hemolysis (%) was calculated as [(A405, peptide)
A405, 0.01% acetic acid)⁄ (A405, 1% Triton X-100) A405, 0.01% acetic acid)]· 100 EC50was determined as the peptide concentra-tion that lysed 50% erythrocytes
Real-time PCR analysis of the effect of fowlicidins on LPS-induced proinflammatory gene expression
Mouse macrophage RAW 264.7 cells were used to study the modulation of LPS-induced cytokine⁄ chemokine gene
Trang 10expression by fowlicidin-3 in comparison with fowlicidin-1.
Cells were seeded in 12-well tissue culture plates with
5· 105 cells⁄ well in DMEM containing 10% fetal bovine
serum After overnight incubation, cells were pretreated for
30 min with 0.5, 2.5, and 10 lm fowlicidins in duplicate,
followed by stimulation for 4 h with 100 ngÆmL)1 LPS
from E coli O114:B4 (Sigma) Total RNA was then
isola-ted from cells using TRIzol (Invitrogen, Carlsbad, CA,
USA) according to the manufacturer’s instructions
Quanti-tative real-time RT-PCR was used to analyze the expression
of three common proinflammatory genes, namely
inter-leukin-1b, CCL2⁄ MCP-1, and CCL3 ⁄ MIP-1a, using
exon-spanning primers as described [12]
The first-strand cDNA from 1.5 lg each RNA sample
was synthesized in a reaction volume of 20 lL at 42C
for 30 min using a QuantiTect Reverse Transcription
Kit (Qiagen, Valencia, CA, USA), which included
removal of genomic DNA contamination before cDNA
synthesis Real-time PCR was performed using 0.2 lg of
the first-strand cDNA, gene-specific primers, SYBR
Premix Ex Taq (Takara Bio, Shiga, Japan), and
MyiQ Real-Time PCR Detection System (Bio-Rad,
Hercules, CA, USA) in a total volume of 10 lL PCR
cycling conditions were as follows: 95C for 30 s,
fol-lowed by 40 cycles of 95C for 15 s, 55 C for 30 s, and
72C for 30 s The comparative DDCT method was used
to quantify the gene expression levels, where b-actin was
used as an internal control for normalization [12]
Relat-ive fold changes in gene expression were calculated using
the formula 2–DDCt Melting curve analysis (55–95C)
was performed and confirmed amplification of a single
product in each case
Acknowledgements
This work was supported by grants from the National
Science Foundation (grants MCB0236039 and
EPS0236913), NIH (S10-RR022392), Oklahoma
Cen-ter for the Advancement of Science and Technology
(grant HR03-146), and Oklahoma Agricultural
Experi-ment Station (Project H-2507) We thank Robert Gallo
from the University of California, San Diego, CA,
USA for kindly providing E coli ML-35p for use in
the inner membrane permeabilization assays, and Steve
Hartson of Oklahoma State University for helping
with MS We are grateful to Haobo Jiang and Ulrich
Melcher for critical reading of the manuscript The
constructive comments from anonymous reviewers are
also appreciated
References
1 Zasloff M (2002) Antimicrobial peptides of multicellular
organisms Nature 415, 389–395
2 Brogden KA, Ackermann M, McCray PB Jr & Tack
BF (2003) Antimicrobial peptides in animals and their role in host defences Int J Antimicrob Agents 22, 465– 478
3 Hancock RE & Patrzykat A (2002) Clinical develop-ment of cationic antimicrobial peptides: from natural to novel antibiotics Curr Drug Targets Infect Disord 2, 79–83
4 Zanetti M, Gennaro R, Skerlavaj B, Tomasinsig L & Circo R (2002) Cathelicidin peptides as candidates for a novel class of antimicrobials Curr Pharm Des 8, 779–793
5 Yang D, Biragyn A, Hoover DM, Lubkowski J & Oppenheim JJ (2004) Multiple roles of antimicrobial defensins, cathelicidins, and eosinophil-derived neuro-toxin in host defense Annu Rev Immunol 22, 181–215
6 McPhee JB & Hancock RE (2005) Function and thera-peutic potential of host defence peptides J Pept Sci 11, 677–687
7 Bowdish DM, Davidson DJ & Hancock RE (2005) A re-evaluation of the role of host defence peptides in mammalian immunity Curr Protein Pept Sci 6, 35–51
8 Finlay BB & Hancock RE (2004) Can innate immunity
be enhanced to treat microbial infections? Nat Rev Microbiol 2, 497–504
9 Zanetti M (2004) Cathelicidins, multifunctional peptides
of the innate immunity J Leukoc Biol 75, 39–48
10 Zaiou M & Gallo RL (2002) Cathelicidins, essential gene-encoded mammalian antibiotics J Mol Med 80, 549–561
11 Lehrer RI & Ganz T (2002) Cathelicidins: a family of endogenous antimicrobial peptides Curr Opin Hematol
9, 18–22
12 Xiao Y, Cai Y, Bommineni YR, Fernando SC, Prakash
O, Gilliland SE & Zhang G (2006) Identification and functional characterization of three chicken cathelicidins with potent antimicrobial activity J Biol Chem 281, 2858–2867
13 Xiao Y, Dai H, Bommineni YR, Soulages JL, Gong
YX, Prakash O & Zhang G (2006) Structure-activity relationships of fowlicidin-1, a cathelicidin antimicrobial peptide in chicken FEBS J 273, 2581–2593
14 Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R & Thornton JM (1996) AQUA and PRO-CHECK-NMR: programs for checking the quality of protein structures solved by NMR J Biomol NMR 8, 477–486
15 National Committee for Clinical Laboratory Standards (2003) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 6th edn Approved Standard M7-A6 National Committee for Clinical Laboratory Standards, Wayne, PA
16 Brogden KA (2005) Antimicrobial peptides: pore form-ers or metabolic inhibitors in bacteria? Nat Rev Micro-biol 3, 238–250