Báo cáo khoa học: Identification, structure and differential expression of novel pleurocidins clustered on the genome of the winter flounder, Pseudopleuronectes americanus (Walbaum) ppt

Identification, structure and differential expression of novel
pleurocidins clustered on the genome of the winter flounder,
Pseudopleuronectes americanus
(Walbaum)
Susan E. Douglas, Aleksander Patrzykat, Jennifer Pytyck and Jeffrey W. Gallant
Institute for Marine Biosciences, Halifax, Nova Scotia, Canada
Antimicrobial peptides form one of the first lines of defense
against invading pathogens by killing the microorganisms
and/or mobilizing the host innate immune system. Although
over 800 antimicrobial peptides have been isolated from
many different species, especially insects, few have been
reported from marine fish. Sequence analysis of two genomic
clones (15.6 and 12.5 kb) from the winter flounder,
Pseudopleuronectes americanus (Walbaum) resulted in the
identification of multiple clustered genes for novel pleuro-
cidin-like antimicrobial peptides. Four genes and three
pseudogenes (Y) are encoded in these clusters, all of which
have similar intron/exon boundaries but specify putative
antimicrobial peptides differing in sequence. Pseudogenes
are easily detectable but have incorrect initiator codons
(ACG) and often contain a frameshift(s). Potential pro-
moters and binding sites for transcription factors implicated
in regulation of expression of immune-related genes
have been identified in upstream regions by comparative
genomics. Using reverse transcription-PCR assays, we have
shown for the first time that each gene is expressed in a tissue-
specific and developmental stage-specific manner. In addi-
tion, synthetic peptides based on the sequences of both genes
and pseudogenes have been produced and tested for anti-
microbial activity. These data can be used as a basis for
prediction of antimicrobial peptide candidates for both

human and nonhuman therapeutants from genomic
sequences and will aid in understanding the evolution and
transcriptional regulation of expression of these peptides.
Keywords: antimicrobial peptide; development; fish; gene
expression; promoter.
Antimicrobial peptides have been isolated from a wide
variety of plants, animals, fungi and bacteria, and play an
important role in defense against microbial infection. Many
of these small molecules are amphiphilic a-helices contain-
ing clusters of cationic amino acid residues that are well
separated in space from hydrophobic residues. These
characteristics play a role in how peptides insert into
biological membranes. Although the primary mode of
action of antimicrobial peptides has been described as
destruction of membranes, they may also exert their effects
by disrupting intracellular processes. In addition, some have
been reported to exert a variety of beneficial effects on host
cells such as mediating inflammation and modulating the
immune response (for a review, see [1]).
Of the over 800 antimicrobial peptide sequences depos-
ited in the described Antimicrobial Peptide Database
( sequences
have been reported from only 11 fish species. Although
relatively understudied, fish are proving to be a rich source
of antimicrobial peptides, possibly because of their more
pronounced reliance on innate immune functions in their
defense against pathogens than mammals [2,3]. Natural
antimicrobial peptides have been isolated from only a few
teleosts and include pleurocidin from winter flounder [4,5],
pardaxin from Red Sea Moses sole [6], misgurin from

loach [7], HFA-1 from hagfish [8], piscidins from hybrid
striped bass [9], moronecidin from hybrid striped bass [10],
hepcidin from bass [11] and winter flounder and Atlantic
salmon [12], chrysophsin from red sea bream [13], parasin
and hipposin, cleavage products of histone 2A from catfish
[14] and Atlantic halibut [15], respectively, two hydrophobic
proteins of 27 kDa and 31 kDa from mucous secretions of
carp [16] and a highly hydrophobic cationic peptide of
undetermined sequence from trout [17]. In addition, a
cationic steroidal antibiotic, squalamine, has been isolated
from the shark [18].
There is scant information on the structure and regulation
of expression of antimicrobial peptide genes, particularly in
fish. Most studies report the biochemical purification of
specific peptide sequences, and in some cases the subsequent
cloning and sequencing of the corresponding gene or
cDNA. Only in the case of the human defensins have genes
for antimicrobial peptides been conclusively demonstrated
to be clustered on the genome in vertebrates [19,20], and in
Correspondence to Institute for Marine Biosciences, 1411 Oxford
Street, Halifax, Nova Scotia, Canada, B3H 3Z1.
Fax: +1 902 426 9413, Tel.: +1 902 426 4991,
E-mail:
Abbreviations: AP-1, activator protein 1; ATF, activating transcription
factor-1; CAAT, CCAAT binding factor; C/EBP, CAAT/enhancer
binding protein; d.p.h., days posthatch; GATA, GATA motif; IFN,
interferon; IL, interleukin; NF-IL6, nuclear factor interleukin 6;
OCT1, octamer motif; Y, pseudogene; MIC, minimal inhibitory
concentration; RT, reverse transcription.
Note: Nucleotide sequence data are available in the GenBank database

under the accession numbers AY282498 and AY282499.
(Received 5 June 2003, revised 9 July 2003, accepted 17 July 2003)
Eur. J. Biochem. 270, 3720–3730 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03758.x
no case has the differential expression of different members
of a vertebrate antimicrobial peptide gene family been
reported.
Expression of pleurocidin peptide in skin and intestine
has been demonstrated using immunohistochemical tech-
niques [21] and we have recently localized expression of one
pleurocidin gene (WF2) to circulating eosinophilic granule
cells of winter flounder gill [22]. In a previous study, we
reported the existence of multiple genes encoding pleuro-
cidin-like peptides and demonstrated generalized pleuroci-
din transcripts early in the development of winter flounder
larvae [5]. However, we could not discriminate between the
different pleurocidin genes; expression of the multiple genes
encoding pleurocidins or, in fact any antimicrobial peptide
in any organism, during different stages of development and
in different tissues has never been reported. In order to
investigate this and also to determine whether other
previously unreported pleurocidin genes may be present
on the winter flounder genome, we have sequenced two
genomic fragments that gave positive hybridization signals
with a pleurocidin probe. Furthermore, we have used the
power of comparative genomics to identify potential
regulatory sequences that may be involved in transcriptional
control and to shed light on the role of these peptides in host
immunity.
In most cases, identification of antimicrobial peptides
has involved laborious, time-consuming biochemical puri-

fication followed by antimicrobial activity assays. Using a
genomics approach, we have successfully identified addi-
tional variants of this antimicrobial peptide family,
predicted their sequences and determined the activity of
synthetic peptides corresponding to these sequences against
a variety of pathogens. With the wealth of genomic data
now available from a wide variety of organisms, this
genomic screening approach should be of value in future
studies aimed at uncovering multiple genes encoding
families of novel antimicrobial peptides and elucidating
their roles in vivo.
Materials and methods
Fish rearing and sampling
All animal procedures were approved by the Dalhousie
University Committee for Laboratory Animals and the
National Research Council, Halifax Local Animal Care
Committee. Winter flounder larvae were reared as described
[23]. All fish were killed with an overdose of tricaine
methanesulfonate (MS 222, 0.1 gÆL
)1
,ArgentChemical
Laboratories, Inc., Redmond, WA, USA) prior to sampling.
Tissues were removed into RNALater (Ambion, Austin,
TX, USA) and kept at )80 °C until used. Samples of larvae
at different stages (hatch, 5, 9, 15, 20, 25, 30 and 36 days
posthatch; d.p.h.) and juveniles were rinsed in RNALater
(Ambion), transferred into 1.5 mL Eppendorf tubes
containing 0.5–1.25 mL RNALater, and kept at )80 °C
until used.
Sequencing and data analysis

A winter flounder genomic k-GEM11 library was screened
by standard procedures using a pooled radioactively labeled
probe comprised of PCR-amplified bands corresponding to
pleurocidins WF1, WF2, WF3 and WF4 [5]. Positively
hybridizing clones were picked and replated until 100%
purity was achieved and mapped using BamHI, SstI, XhoI
and EcoRI. Clones were completely sequenced from both
strands using an ABI377 automated sequencer and the
AmpliTaqFS Dye Terminator Cycle Sequencing Ready
Reaction kit (Perkin Elmer). Sequence data were analyzed
using Sequencher (Gene Codes, Inc., Ann Arbor, MI, USA)
and DNA Strider (Marck, 1992). Signal peptide cleavage
sites were predicted using the SignalP Web Prediction Server
( with the neural
networks trained on sequences from eukaryotes. Transcrip-
tion factor binding sites were identified using WWW Signal
Scan ( with the
TRANSFAC [24] and TFD databases.
Expression of pleurocidin genes by reverse
transcription-PCR (RT-PCR)
Total RNAs were isolated from esophagus, pyloric stom-
ach, cardiac stomach, pyloric caeca, liver, spleen, intestine,
rectum, gill, brain, muscle and skin (20–50 mg tissue) of an
adult winter flounder, and from pooled samples of 20 whole
larvae (hatch and 9 d.p.h.), 10 whole larvae (15, 20, 25 30
and 36 d.p.h.) and five whole juveniles using the RNA Wiz
kit (Ambion, Austin, TX, USA) according to the manu-
facturer’s recommendations. The isolated RNA was treated
with DNA Free Kit (Ambion) as directed by the manufac-
turer, except that a 1-h incubation was performed rather

than the specified 30 min.
First-strand cDNA was synthesized from 2 lgtotal
RNA using the RetroScript kit (Ambion) and aliquots of
the reaction products were subjected to PCR using rTaq
polymerase (Amersham Biosciences) and gene-specific
primers (Table 1). As Y2andY3 showed significant
sequence degeneration (see Results), it was assumed that
they are no longer functional and they were therefore not
assayed. The amplification conditions were: 2 min at 94 °C;
32 cycles of 30 s at 94 °C, 30 s at 52 °C, 90 s at 72 °C; and
2 min at 72 °C. Amplification of b-actin mRNA was
performed to confirm the steady-state level of expression of
a housekeeping gene. Amplification products were resolved
on a 2% PCR Plus agarose gel (EM Sciences, Gibbstown,
NJ, USA) with a 100-bp ladder as a marker (Amersham
Biosciences). Controls were performed using single primers
to eliminate single primer artifacts and without template to
eliminate amplification products arising from contami-
nating genomic DNA.
Synthesis of peptides
The sequences of selected peptides used for testing and their
physical properties are provided in Table 2. All anti-
microbial peptides used in this study were synthesized by
N-(9-fluorenyl) methoxy carbonyl (Fmoc) chemistry at the
Nucleic Acid Protein Service unit at the University of
British Columbia. Peptide purity was confirmed by HPLC
and MS analysis in each case. Due to the high cost of
peptide synthesis, only a subset of peptides was made. Two
peptides, WFY and WFZ, based on alternative splice
products were predicted from pseudogene 1 (which con-

Ó FEBS 2003 Pleurocidin differential gene expression (Eur. J. Biochem. 270) 3721
tained no frameshifts) but no peptides were synthesized
based on pseudogenes 2 or 3 (which exhibited significant
sequence degeneration). WF-YT was not synthesized as it
did not exhibit the typical GEC signature upstream of the
mature peptide and the position of the carboxy-terminal
residue was ambiguous. A previously synthesized peptide,
NRC-01, which differs from the predicted amino acid
sequence of WF1-like peptide by only one residue (a Glu vs.
an Asp at position 7 of the mature peptide) was used to
estimate the activity of WF1-like pleurocidin.
Antimicrobial assays
All strains used in this study are listed in Table 3. As
inhibition of microbial growth in humans is of interest,
nonfish bacterial strains as well as Candida albicans were
assayed and grown at 37 °C in Mueller-Hinton Broth
(MHB; Difco Laboratories, Detroit, MI, USA), while the
bacteria pathogenic to fish were maintained at 16 °Cin
tryptic soy broth (Difco, 5 gÆL
)1
NaCl). All strains were
stored at )70 °C until they were thawed for use and
subcultured daily. Two field isolates of the salmonid
pathogen Aeromonas salmonicida are from the Institute
for Marine Biosciences strain collection. The following
strains, P. aeruginosa K799 (parent of Z61), P. aeruginosa
Z61 (antibiotic supersusceptible), Salmonella typhimurium
14028s (parent of MS7953s), Salmonella typhimurium
MS7953s (defensin supersusceptible), as well as Staphylo-
coccus epidermidis (human clinical isolates), methicillin-

resistant Staphylococcus aureus (isolated by A. Chow,
University of British Columbia, Canada) and C. albicans
were provided by R. E. W. Hancock, University of British
Columbia, Canada. Antibiotic-supersusceptible strains were
included with the specific intent of determining if these
mutations also result in increased susceptibility to peptides,
which may imply a specific mode of action.
Escherichia coli strain CGSC 4908 (his-67, thyA43, pyr-
37), auxotrophic for
L
-histidine, thymidine and uridine [25]
was obtained from the E.coliGenetic Stock Centre (Yale
University, New Haven, CT, USA). MHB supplemented
with 5 mgÆL
)1
thymidine, 10 mgÆL
)1
uridine and 20 mgÆL
)1
L
-histidine (Sigma Chemical Co.), was used to grow E.coli
CGSC 4908 unless otherwise specified.
The activities of the antimicrobial peptides were deter-
mined as minimal inhibitory concentrations (MICs) using
the microtitre broth dilution method of Amsterdam [26], as
modified by Wu and Hancock [27]. Serial dilutions of the
peptide were made in water in 96-well polypropylene
microtiter plates (Costar, Corning Incorporated, Corning,
NY, USA). Bacteria or C. albicans were grown overnight to
Table 1. Nucleotide sequences of oligonucleotides used for assay of pleurocidin-like gene expression in different tissues and at different stages of

development of winter flounder.
Gene sequence Primer Amino acid Nucleotide sequence (5¢fi3¢)
WF1-like RTWF1
KGRWLER AAGGGCAGGTGGTTGGAAAGG
RTWF1/3¢ YQEGEE
a
CCCTCCCCCTCCTGGTA
WF2 RTWF2 KAAHVG AAGGCTGCTCACGTTGGC
PL2 3¢ untranslated CTGAAGGCTCCTTCAAGGCG
WFYT RTWFYT GFLFHG GGGATTTCTTTTTCATGG
RTWFYT/3¢ SFDDNP
a
GGGTTGTCATCGAATGAG
WFX RTWFX RSTEDI CGTTCTACAGAGGACATC
RTWFX/3¢ DDDDSP
a
GGGGCTGTCATCATCATC
WFY (Y1) RTWF5.1/5¢ IVMFEP CATCGTCATGTTTGAACC
RTWF5.1/3¢ GYLNAA
a
GGCCGCATTGAGATAACC
WFZ (Y1) RTWF5.1/5¢ IVMFEP CATCGTCATGTTTGAACC
RTWF5.1a/3¢ PFIKPR
a
CCTGGGTTTAATAAATGG
Actin ActF(WF) AALVVD TCGCTGCCCTCGTTGTTGAC
ActR(WF) VLLTEAP
a
GGAGCCTCGGTCAGCAGGA
a

Primer based on complement.
Table 2. Properties of predicted peptides. To estimate the net charge K and R were assumed to have a value of + 1, H of + 1/2, D and E of )1,
and C-terminal amidation was counted as an additional +1.
Label Residues Amino acid sequence M
r
Charge
WF1-like
a
24 GKGRWLERIGKAGGIIIGGALDHL-NH
2
2487 +3.5
NRC-01
b
24 GKGRWLDRIGKAGGIIIGGALDHL-NH
2
2473 +3.5
WF2 25
GWGSFFKKAAHVGKHVGKAALTHYL-NH
2
2711 +6.5
WFX 21
RSTEDIIKSISGGGFLNAMNA-NH
2
2180 +2.0
WFY (Y1) 19
FFRLLFHGVHHGGGYLNAA-NH
2
2112 +3.5
WFZ (Y1) 19
FFRLLFHGVHHVGKIKPRA-NH

2
2260 +6.5
a
WF1-like contains an N-terminal insertion RRKKKGSKRKGSKGKGSK.
b
NRC-01, which differs from WF1-like by a single Glu-Asp
substitution, was tested instead of WF1-like.
3722 S. E. Douglas et al. (Eur. J. Biochem. 270) Ó FEBS 2003
mid-logarithmic phase as described above, and diluted to
give a final inoculum size of 10
6
c.f.u.ÆmL
)1
. A suspension
of bacteria or yeast was added to each well of a 96-well plate
and incubated overnight at the appropriate temperature.
Inhibition was defined as growth lesser or equal to one-half
of the growth observed in control wells where no peptide
was added. However, in all cases except P. aeruginosa,
where growth inhibition was indeed gradual, complete
inhibition (no growth) was achieved at the lowest inhibitory
concentration. Growth was assessed visually. Three repli-
cates of each MIC determination were performed. MHB
supplemented with 200 m
M
NaCl was used to test salt
resistance.
Survival of bacteria and C. albicans upon exposure to
selected peptides applied at their MICs and 10 times their
MICs was measured using standard methodology. The test

organisms were grown in MHB and exposed to the peptides.
At specified time intervals, equal aliquots were removed
from the cultures, plated on MHB plates, and the resulting
colonies were counted. Percentage survival was plotted
against time on a logarithmic scale. Two replicates of each
experiment were performed.
Results
Genomic structure
Four lambda clones giving positive hybridization signals
with the pleurocidin probe were isolated from the winter
flounder genomic library. Two clones differing markedly
in restriction endonuclease cleavage pattern were selected
for sequencing. Complete sequencing of these two clones
(k1.1 and k5.1) revealed inserts of 15.6 and 12.5 Kb,
respectively. k1.1 contained three genes encoding pleuro-
cidin-like antimicrobial peptides (referred to as WFYT,
WFX and WF1-like) and one pseudogene (Y3). k5.1
contained one gene encoding pleurocidin (WF2) and two
pseudogenes (Y1andY2). Sequence analysis demonstra-
ted that all pleurocidin genes contained four exons and
three introns. Interestingly, the pseudogenes all had a
similar genomic structure but were missing ATG start
codons and correct splicing sites, or contained frameshifts
(asterisks, Fig. 1). Intron and exon sizes varied quite
markedly (Table 4). Pronounced degeneration of the
sequences upstream of the pseudogenes made it impossible
to predict the locations of the first introns and exons of
Y1, Y2andY3.
An alignment of the peptides predicted from the
pleurocidin genes and pseudogenes is shown in Fig. 1. All

of the genes, and even most of the pseudogenes, encode
highly conserved signal sequences and anionic propieces.
The exception is Y2, which encodes a very long carboxy-
terminal extension. The mature peptides, predicted by
SignalP to start after the motif GEC, GES or GEG and
to terminate at the glycine adjacent to the acidic propiece
(by comparison with the published amino acid sequence of
pleurocidin [4]), are somewhat variable in sequence, net
positive charge and length (Table 2).
Computer searches for promoters revealed common
promoter motifs with very high scores (0.98–1.00) in the
upstream regions of all four genes (Fig. 2A). However,
only aberrant motifs with much lower scores (0.88–0.93)
could be detected in the upstream regions of the
pseudogenes. All of the high-scoring promoters exhibited
significant sequence identity with one another (Fig. 2B)
but no similarity to the motifs upstream of pseudogenes.
Table 3. Strains used to test antimicrobial activity of pleurocidins and
the corresponding MICs.
MIC in lgÆmL
)1
NRC-01 WF2 WFX
WFY
(Y1)
WFZ
(Y1)
Aeromonas salmonicida
A449 field isolate 64 2 >64 >64 >64
97–4 field isolate 64 2 >64 >64 32
Salmonella typhimurium

MS7953s supersusceptible 16 2 >64 64 16
14028s parent of
MS7953s
>64 16 >64 >64 >64
Pseudomonas aeruginosa
K799 parent of Z61 >64 8 >64 >64 32
Z61 supersusceptible 32 4 >64 64 8
Escherichia coli
CGSC4908 triple
auxotroph
32 2 >64 64 32
UB1005 parent of DC2 32 4 >64 >64 32
DC2 outer membrane
mutant
32 2 >64 >64 32
Staphylococcus epidermidis
Clinical isolate >64 8 >64 >64 32
Staphylococcus aureus
Methicillin resistant
clinical isolate
>64 8 >64 >64 64
Candida albicans
Clinical isolate 64 8 >64 >64 >64
Fig. 1. Predicted amino acid sequences of peptide precursors encoded by pleurocidin genes and pseudogenes. Single letter amino acid code is used and
deletions are indicated by dashes (–). Insertions in WF1-like and Y2 peptides relative to the other peptides are indicated by arrows. Positions of
frameshifts in the ÔMature peptideÕ region of Y2andY3 are indicated by asterisks (*).
Ó FEBS 2003 Pleurocidin differential gene expression (Eur. J. Biochem. 270) 3723
Apart from a 5-nucleotide deletion that was present in
the promoters from WF1 and WFX relative to WF2
and WFYT, the sequences differed at only five locations

and four of these were in a stretch of eight positions
at the 5¢ end. Classical TATA and CAAT boxes could
be found in the upstream regions of all four genes
but, of the pseudogenes, only Y3hadaTATAbox.
No CAAT boxes could be detected in any of the
pseudogenes.
Stage-specific gene expression of pleurocidin genes
The results of RT-PCR expression assays of each pleuro-
cidin gene during development are shown in Fig. 3.
Expression of WF1-like pleurocidin could not be detected
at any stage of development. Expression of WFX was just
discernable at 20 d.p.h. whereas expression of WFYT and
WF2 was readily detectable in premetamorphic larvae and
juveniles.
Tissue-specific gene expression of pleurocidin genes
The results of RT-PCR expression assays of each pleuro-
cidin gene in different tissues of adult fish are shown in
Fig. 4. Expression of WFX appears to be confined to the
Table 4. Sizes (in bp) of introns (I) and exons (E) of pleurocidin genes
and pseudogenes (Y) encoded on k1.1 and k5.1 clones. ND, Not deter-
mined.
Name E1 I1 E2 I2 E3 I3 E4
WF1-like 25 120 155 539 31 95 112
WFX 25 119 101 453 19 97 85
WFYT 25 115 101 386 19 97 88
WF2 25 101 101 523 31 109 76
WFY (Y1) ND ND 101 2120 19 113 76
WFZ (Y1) ND ND 101 544 41 1656 87
Y2 ND ND 93 368 8 108 160
Y3 ND ND 102 524 30 97 67

Fig. 2. Locations of transcription factor binding sites of pleurocidin genes and pseudogenes (A) and an alignment of predicted promoters (B). (A)
Promoters are indicated by solid boxes with the corresponding score above. The first two exons (hatched boxes) and the first intron (stippled box) of
each gene are shown. Asterisks above and below the lines represent GAAA motifs on the coding and noncoding strands, respectively. AP-1,
activator protein 1; ATF, activating transcription factor-1; CAAT, CCAAT binding factor; GATA, GATA motif; a-IFN, a-interferon; NF-IL6,
nuclear factor-interleukin 6; OCT1, octamer motif. (B) Upper case letters indicate nucleotides present in the pleurocidin transcripts and lower case
letters indicate nucleotides present in the 5¢ nontranscribed portions of the genes. The predicted promoter is underlined.
3724 S. E. Douglas et al. (Eur. J. Biochem. 270) Ó FEBS 2003
skin whereas that of the other three genes is more
widespread, particularly in the gill, skin and gut tissues. In
addition, WFYT transcripts were detected in spleen and
WF2 transcripts were found in liver.
Pseudogene expression
No expression could be detected from Y1 either at different
stages of development (Fig. 3) or in different tissues (Fig. 4),
Fig. 3. RT-PCR of expression of pleurocidin genes and pseudogenes throughout larval development. Larvae at 0, 5, 9, 15, 20, 25, 30 and 36 d.p.h. and
juveniles (J) were analyzed. Controls using single primers (5¢,3¢) and no template (NT) are also shown. Exons and introns are represented as in
Fig. 2A. No reading frames could be identified in regions represented by solid lines. Portions of the clones flanking the pleurocidin gene clusters are
not represented. Markers (M) are a 100-bp ladder (100–400 bp shown).
Fig. 4. RT-PCR assays of expression of pleurocidin genes and pseudogenes in different tissues. Esophagus (E), pyloric stomach (PS), cardiac stomach
(CS), pyloric caeca (PC), liver (L), spleen (Sp), intestine (I), rectum (R), gill (G), brain (B), muscle (Mu) and skin (Sk) were analysed. Markers (M)
and representations of genes and pseudogenes are the same as in Fig. 3.
Ó FEBS 2003 Pleurocidin differential gene expression (Eur. J. Biochem. 270) 3725
consistent with the lack of a strong promoter motif, TATA
or CAAT boxes (Fig. 2A). Primers based on alternatively
spliced transcripts that would give rise to either WFY or
WFZ peptides (Table 1), were both negative for expression.
Although expression of Y2andY3 was not tested, it is
assumed that their aberrant genomic structure and lack of
promoters would preclude transcription.
Antimicrobial activity of synthetic peptides

Minimal inhibitiory concentrations of the five tested pep-
tides against a range of bacteria and C. albicans are shown
in Table 3. While WFX and WFY pleurocidins appeared to
be inactive in our hands, NRC-01 (similar to WF1-like) and
WFZ pleurocidins possessed moderate antimicrobial acti-
vity. WF2, an amidated version of the original pleurocidin,
showed activity similar to that described in previous studies
[4]. All peptides with detectable activity against P. aerugi-
nosa Z61 (NRC-01, WF2, WFY and WFZ) retained that
activity in the presence of 200 m
M
NaCl (Table 5). The
ability of NRC-01 pleurocidin to kill A. salmonicida A449,
Salmonella typhimurium MS7953s and C. albicans at its
MIC and 10 times MIC is shown in Fig. 5. NRC-01
pleurocidin added at 10 times MIC showed strong bacte-
ricidal and modest fungicidal activity against the pathogens
tested. NRC-01 pleurocidin added at its MIC was less active
although it still showed bactericidal activity against
Salmonella typhimurium and A. salmonicida.
Discussion
Antimicrobial peptides such as cecropins [28], apidaecins
[29], dermaseptins [30] and defensins [31] are known to be
encoded by multigene families. Southern blot analysis of
apidaecin [29] and more recently, pleurocidin [5] and
hepcidin [12], indicate that antimicrobial peptide genes
may exist in clusters on the genome. However, with the
exception of human defensins [32], definitive proof of a
clustered gene arrangement has never been demonstrated.
In fact, genomic sequencing of the defensin cluster has

recently been used to uncover additional previously
unknown members of this antimicrobial peptide family [19].
Our data definitively demonstrate that pleurocidin genes
occur at at least two distinct loci on the winter flounder
genome. Furthermore, we predict that there must be at least
one other locus encoding the previously described WF1,
WF1a, WF3 and WF4 pleurocidins [5]. These may be
located on the other two lambda clones we isolated that had
slightly different restriction patterns from the two that were
sequenced.
Given the high degree of sequence conservation among
the pleurocidin genes and their flanking regions (Figs 1
and 2), it is possible that recombination between these
conserved modules has allowed the generation of variants
with diverse sequences, and presumably antimicrobial
properties. Some of these changes may be very subtle or
even vary between winter flounder from different loca-
tions; the WF2 pleurocidin we describe differs from the
gene sequence determined by Cole et al.[21]atseven
Table 5. Minimum inhibitory concentrations of peptides active against
P. aeruginosa Z61 in the presence or absence of 200 m
M
NaCl.
MIC in lgÆmL
)1
against P. aeruginosa Z61
No salt 200 m
M
NaCl
WF1-like 32 32

WF2 4 8
WFY (Y1) 64 64
WFZ (Y1) 8 16
Fig. 5. Ability of NRC-01 to kill A. salmonicida (A), Salmonella
typhimurium (B) and C. albicans (C). Peptide was added at its MIC (h),
10 times MIC (n) and a no-peptide control was included in each group
(r).
3726 S. E. Douglas et al. (Eur. J. Biochem. 270) Ó FEBS 2003
positions in the upstream region and four positions in the
first intron, including a large deletion of 17 nucleotides.
This amount of sequence divergence between different
individuals of the same species indicates that the pleuro-
cidin genes, like defensin genes, are evolving very rapidly.
In addition, the identification of pseudogenes with a
relatively low amount of degeneration indicates a fairly
recent evolutionary event; the sequences were highly
similar to those found in active genes and easily detect-
able. The ability to generate multiple genes encoding
diverse antimicrobial peptides may be one way that fish
are able to capitalize on this component of innate
immunity, both in killing microbes and in modulating
host immunity.
Three pseudogenes were present in the cluster containing
the four functional pleurocidin genes. In mammalian
genomes, pseudogenes are nearly as abundant as genes
[33], possibly as a result of the genomic processes involved in
generating multigene families, such as those concerned with
immunity. The general assumption that pseudogenes rep-
resent dysfunctional relics has recently been contradicted by
the discovery of a pseudogene that regulates expression of

the functional gene from which it arose via an RNA-
mediated mechanism [34]. It will be of interest to probe
whether a similar process may be involved in the regulation
of expression of pleurocidin (and possibly other antimicro-
bial peptide) genes.
Little is known about the promoter or regulatory
sequences involved in pleurocidin gene expression. In a
recent study comparing human and mouse genomes, it was
apparent that conserved noncoding genomic sequence (5¢
upstream and intron sequence) is often enriched in signals
involved in transcriptional regulation compared to coding
sequence [35]. Furthermore, co-occurring pairs of transcrip-
tion factors could be identified using this approach. By using
a similar comparative genomics approach, we have been
able to identify putative biologically active regulatory
elements upstream of pleurocidin genes and within the first
intron, including one with a high prediction score for a
eukaryotic promoter, and also to predict which transcrip-
tion factors are likely to interact.
Transcription factors usually work in complexes to
regulate transcription [36] and their binding sites are often
clustered into modular units known as cis-regulatory
modules. Recent analysis of clusters of transcription factor
binding sites in the Drosophila genome indicated that
multiple transcription factors are required to modulate
eukaryotic gene expression [37]. While the consensus
sequences of some transcription factor binding sites are
known, their short length and low sequence complexity
often results in a high number of false positives in computer
searches due to random occurrences.

Binding sites for various transcription factors known to
be involved in host defense [activator protein 1 (AP-1),
a-interferon (a-IFN), nuclear factor interleukin 6 (NF-IL6),
octamermotif(OCT1)]aswellasGAAAmotifs(commonly
found upstream of interferon-induced genes) were identified
in the upstream regions of both genes and pseudogenes or
within the first intron. Those present in functional genes
conformed better to the consensus sequences than those
upstream of pseudogenes, as the latter appear to have
undergone substantial sequence drift. As seen from Fig. 2A,
many of these consensus binding sites are at similar
positions.
GAAA motifs were abundant upstream of genes (6–10
motifs) but rare upstream of pseudogenes (1–4 motifs).
Binding sites for activator protein-1 (AP-1), a family of
transcription factors consisting of homodimers and hetero-
dimers of Jun, Fos or activating transcription factor 1
(ATF) were also located upstream of three of the four
pleurocidin genes (for review of AP-1 function and regula-
tion see [38]). Cis-regulatory elements containing AP-1 sites
are regulated by multiple sets of bZIP transcription factor
dimers with different binding and transactivation properties
[39]. ATF motifs were found upstream of only one gene
(WF2) but in the identical position upstream of all three
pseudogenes. AP-1 factors are involved in cell differenti-
ation and survival and are also produced after viral
transformation of cells. The presence of GAAA and AP-1
motifs indicates that pleurocidins may be induced by viral
infection and play a role in clearing these pathogens
although at this time we do not have antiviral activity data

to support this hypothesis.
GATA motifs (WGATAR), which together with nuclear
factor (NF)-jB motifs are necessary for tissue-specific
expression of immunity genes in larval fat body (the insect
equivalent of the liver) and hemocytes of Drosophila [40]
and in erythroid-specific gene expression in mammals [41],
were found within and/or upstream of WF2 and WFYT.
They may be responsible for the transcripts in liver (WF2)
and spleen (WFYT). The more widespread pleurocidin
expression in gill, skin and gut tissues is not surprising, given
their proximity to the external environment (gill and skin) or
bacteria in the gut (intestine, pyloric caecae, stomach and
rectum). Specific cDNAs for WF1a, WF2 and WF3 have
been cloned and sequenced from intestine [5] and immuno-
gold electron microscopy revealed WF2-like pleurocidin
transcripts in mucus-producing cells of skin and goblet cells
of small intestine in winter flounder [21]. In addition, Paneth
cells within the human intestine have been shown to express
human defensin-5 [42]. It is possible that pleurocidin
transcripts also originate from immune cells circulating
through these tissues. In support of this, we have recently
localized WF2 pleurocidin (both transcripts and peptide) to
circulating eosinophilic granule cells in the gill of winter
flounder [22]. Human neutrophils contain defensins [31] and
bovine neutrophils have been shown to store the anti-
microbial peptide bactenecin in granules [43]. In situ hybridi-
zation experiments designed to elucidate which cell types are
responsible for production of the various pleurocidins are
underway in our lab.
A cluster of NF-IL6 and/or a-IFN transcription factor

binding sites within 150 nucleotides of the promoter co-
occur in three out of the four pleurocidin genes (WFX, WF2
and WFYT). NF-IL6 is a member of the CCAAT/
Enhancer Binding Protein (C/EBP) class of leucine-zipper
transcription factors that binds to an IL-1-responsive
element in the IL-6 gene. It is induced by IL-1, IL-6, and
lipopolysaccharide during the acute phase response; it has
been termed the Ômaster regulator of the immune responseÕ
[44] and is involved in activating transcription of the
inflammatory cytokines IL-6 and IL-8 [45]. As antimicrobial
peptides can be induced in response to infection [46], the
detection of NF-IL6 sites is not surprising. One of the AP-1
Ó FEBS 2003 Pleurocidin differential gene expression (Eur. J. Biochem. 270) 3727
sites was also found clustered with an NF-IL6 site upstream
of WF2. Similar association of these two sites has been
reported in the enhancer region of one of the winter
flounder antifreeze protein genes, where a sequence element
containing an AP-1 site binds two transcription factors, one
of which is a C/EBP family member, C/EBPa [47]. Both
transcription factors are proposed to interact to direct liver-
specific gene expression in winter flounder. Possibly the
expression of WF2 in the liver is regulated in a similar
manner.
NFjB-like motifs have been found upstream of antimi-
crobial peptide genes in mammals, amphibia and Drosophila
[48], often adjacent to binding sites for NF-IL6 with which it
interacts to effect transcription of immune-relevant genes
[49]. The lack of NF-jB motifs upstream of the pleurocidin
genes described in this study may mean that inducible
expression of pleurocidin occurs via some other transcrip-

tional pathway involving NF-IL6 either alone or with
another unidentified factor, or that additional pleurocidin
genes possessing NFjB binding sites in their upstream
sequences may exist elsewhere in the genome.
Octamer motifs (ATGCAAAT) are present in the
noncoding sequences of all of the pleurocidins. These motifs
are often found in enhancers, where they promote tran-
scription of immune-relevant genes. In fish, such sequences
have been found in the salmon transferrin promoter [50],
trout MX genes [51] and catfish [52]. The catfish IgH
enhancer contains 11 octamer motifs, of which nine are
functional [53]. OCT1 sites have been detected in the human
IL-2 promoter [54]. The occurrence of between one and four
OCT1 sites in pleurocidin genes indicates that they may be
involved in promoting transcription of antimicrobial pep-
tide genes as well as other immune-relevant genes in fish.
Expression of WFX, WFYT and WF2 was detectable in
premetamorphic larvae and juveniles, but no expression of
WF1-like pleurocidin or any of the pseudogenes could be
detected at any stage of development. Interestingly, the
cluster of NF-IL6 and a-IFN transcription factor binding
sites found upstream of WFX, WF2 and WFYT are absent
from WF1-like pleurocidin and the pseudogenes, indicating
that these motifs may be necessary for expression in the
larval stages. Both WFYT and WF2 genes contain GATA
motifs, which have been shown to be essential for larval
expression of insect genes including those for two anti-
microbial peptides, cecropin and drosocin [40]. Our earlier
studies showed the expression of pleurocidin at 5 d.p.h. [5],
but the primers used could not discriminate between the

different variants described here. It is likely that another, as
yet undescribed member of the pleurocidin gene family, is
expressed earlier in development.
Although the focus of our study was not to identify
peptides with highly active antimicrobial properties, but
rather to demonstrate the power of genomics in identifying
novel peptide-encoding sequences, the modest activities we
detected against the microbes in our panel are encouraging.
The peptides may be active against other species of bacteria
more commonly encountered in fish. In addition, as many
antimicrobial peptides are known to synergize with each
other and with other components of the innate immune
system such as lysozyme [55], the in vivo effects may be more
powerful than shown in the in vitro assays performed in our
study. The ability of antimicrobial peptides to exert positive
modulatory effects on the innate immune system [56] may
result in further beneficial effects to the host. The peptides
are both inhibitory and cidal, although both activities vary
from pathogen to pathogen, with C. albicans counts being
reduced only by one log order, even at 10 times the MIC.
Also, the peptides are able to exert their effects in the
presence of 200 m
M
NaCl, a characteristic that is promising
for application to cystic fibrosis treatment. These results
encourage us to scout the genome of winter flounder for
more peptides with unique characteristics that could be of
value in combating bacterial infections.
Interestingly, the WFZ peptide exhibited some activity
even though the gene is no longer functional. There is no

detectable promoter, few transcriptional control sequences,
no initiator methionine, and frameshift mutations are
present. Nonetheless, the predicted peptide encoded by this
pseudogene is highly positively charged (Table 2). This
underscores the advantage of scanning genomic informa-
tion for possible antimicrobial peptide sequences: even if the
genes are no longer functional, the peptides they once
encoded may be of interest.
In conclusion, we have identified two clusters containing
several pleurocidin-like antimicrobial peptide genes and
pseudogenes and determined the antimicrobial activities of
synthetic peptides predicted from these sequences. We have
performed a comprehensive survey of the differential
expression of these genes and pseudogenes and shown for
the first time that the different antimicrobial peptide genes
are expressed in different tissues and at different times
during development. Using comparative genomics, we have
been able to identify sequence motifs that potentially bind
transcription factors involved in the regulation of expression
of immunity-related genes. This has provided us with a
window into the complex regulatory processes that appear
to govern the transcription of different members of this
multigene family. Future studies aimed at dissecting the
promoter and critical cis-regulatory sequences will further
enhance our understanding of the regulation of this
important component of innate immunity.
Acknowledgements
We thank Dr Vanya Ewart for critically reviewing this manuscript prior
to submission. This is NRCC publication 42402.
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