Tài liệu Báo cáo khoa học: Antimicrobial effects of H4-(86–100), histogranin and related compounds – possible involvement of DNA gyrase ppt

Antimicrobial effects of H4-(86–100), histogranin and
related compounds – possible involvement of DNA gyrase
Simon Lemaire, Thuy-Tie
ˆ
n Trinh, Hoang-Thanh Le, Shun-Chii Tang, Maxwell Hincke,
Olivier Wellman-Labadie and Sophie Ziai
Department of Molecular and Cellular Medicine, University of Ottawa, Canada
Histones are highly conserved proteins that play a key
function in the packaging of DNA within eukaryotic
cells during their division [1]. Histone proteins and
fragments are also recognized to have some extra-
nuclear and extracellular functions [2]. Their anti-
microbial activity was first observed by Hirsch in 1958
[3], who reported that an arginine-rich preparation of
histones isolated from calf thymus (fraction B: a mix-
ture of histones H3 and H4) displayed potent bacterici-
dal activity mainly against Gram-negative bacteria, an
effect that was amplified by acid media, whereas
a lysine-rich preparation (fraction A: a mixture of
histones H1, H2A and H2B) was less effective. Since
then, a large body of evidence has indicated that
Keywords
antimicrobial peptide; DNA gyrase;
histogranin; histone H4 peptides; innate
immunity
Correspondence
S. Lemaire, Department of Cellular and
Molecular Medicine, Faculty of Medicine,
University of Ottawa, 451 Smyth Road,
Ottawa, ON, Canada K1H-8M5
Fax: +1 613 562 5434

Tel: +1 613 562 5800 ext. 8350
E-mail:
(Received 10 July 2008, revised 21 August
2008, accepted 26 August 2008)
doi:10.1111/j.1742-4658.2008.06659.x
Histone-derived antimicrobial peptides have been identified in various
organisms from plants to humans. The rat histone H4 mRNA variants,
H4-v.1 and rat histogranin (HNr) mRNAs, were recently reported to be
involved in the synthesis of H4-(86–100) and its related peptide HNr,
respectively. Herein, the two peptides were investigated for putative anti-
microbial activity and found to inhibit growth of Gram-negative
(Escherichia coli, Pseudomonas aeruginosa) and Gram-positive (Bacillus -
subtilis, Staphylococcus aureus) bacteria. Their inhibitory potencies in
E. coli (LD
50
: 3.48 and 4.34 lgÆmL
)1
) are comparable to that of the anti-
microbial peptide LL-37 (LD
50
: 4.10 lgÆmL
)1
). The antimicrobial activities
of H4-(86–100) and HNr depend upon the integrity of the molecules, as pre-
cursors [H4-(84–102), pro-HNr] and fragments [bovine histogranin (HNb)-
(1–13), HNb-(3–13), H4-(89–102) or OGP] are at least five times less potent
than the parent peptides. Among various HN-like compounds, cyclo-
(-Gly-pCl-Phe-Tyr-d-Arg) (compound 3) and N-5-guanidino pentanamide-
(2R)-yl-2-N-(p-hydroxyphenylacetyl)-4-(p-chlorobenzoyl)-phenylene diamine
(compound 8) display antimicrobial activities comparable to that of HNr.

Interestingly, the antimicrobial activities of H4-(86–100), HNr and com-
pound 3, like those of quinolone antibiotics acting as DNA gyrase poisons,
are potentiated by ATP (1 mm) and coumermycin A1 (a DNA gyrase-
linked ATPase inhibitor) and blocked by 2,4-dinitrophenol (DNP, an
uncoupler of oxidative phosphorylation) and fluoroacetic acid (a metabolic
poison). Finally, in vitro experiments indicate that H4-(86–100), HNr, com-
pound 3 and compound 8, but not HNb-(1–13) or HNb-(3–13), inhibit
DNA gyrase-mediated supercoiling of pBR322 DNA. These data indicate
that the naturally occurring H4-(86–100) and HNr display antimicrobial
effects that involve a modulation of ATP-dependent DNA gyrase.
Abbreviations
CFU, colony-forming unit; CFUv, virtual colony-forming unit; DNP, 2,4-dinitrophenol; HN, histogranin; HNb, bovine histogranin; HNr, rat
histogranin; NET, neutrophil extracellular trap; OGP, osteogenic growth peptide.
5286 FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS
histones H1, H2A and H2B are potent antimicrobial
agents [4–12]. Thus, antimicrobial histone H2A was
observed in skin exudates of rainbow trout [4], whereas
catfish skin [5] and salmon liver [6] extracts contained
antimicrobial histones H2B and H1, respectively. In
the toad stomach [7], histones H1, H2A and H2B were
observed in the cytoplasm of gastric gland cells along
with pepsinogen C, whereas the N-terminal his-
tone H2A fragment buforin-I formed a dense immuno-
reactive layer on the mucous surface of epithelial cells
[7]. The observation that pepsin C isozymes convert
histone H2A into buforin-I led the authors to propose
that histone-derived antimicrobial peptides may be
produced in the lumen of the stomach, where both
pepsinogen C and histone H2A are released, and there-
after histone H2A would be processed into buforin-I

and fixed to the mucous surface of epithelial cells to
prevent the entry of bacteria into the epithelial layer
and their invasion into the lower parts of the gastro-
intestinal tract [7]. Their finding of buforin-I in
porcine, bovine and human stomach lavage fluids sup-
ported the concept that such a defense mechanism also
exists in vertebrates. In chicken, antimicrobial
histones H2A and H2B were isolated from the liver
[8], and histones H1 and H2B were identified in the
reproductive system [9]. In humans, histone H1 and its
fragments derived from gastrointestinal epithelial cells
were active against Salmonella typhimurium [10], and
histones H2A and H2B were expressed on the surface
of and secreted from amnion epithelial cells [11]. A
recent report indicated that activated human neutroph-
ils contain and release chromatin and nuclear proteins
such as histone H2A that together form extracellular
fibers called neutrophil extracellular traps (NETs) for
the immobilization and killing of bacteria [12]. Thus,
the presence of antimicrobial proteins and peptides in
NETs was proposed to ensure a high concentration of
the antimicrobial compounds at sites of bacterial
killing. Interestingly, activated neutrophils, which are
known to play a preponderant role in immune defense
mechanisms, release factors such as ATP and acids
that may enhance the antimicrobial activities of NET
histones [13].
Histogranin (HN; Fig. 1), a slightly modified C-ter-
minal histone H4 peptide homologous to H4-(86–100),
was first isolated in our laboratory from bovine adre-

nal medulla [14]. The immunoreactive peptide was
detected in various rat tissues, including the pituitary,
adrenal glands, lungs, spleen, brain and plasma [15].
Our initial search to determine the structure of the HN
gene led to the discovery of the H4-v.1 mRNA variant
in a bovine adrenal medulla cDNA library [16]. Bovine
H4-v.1 was shown to be a polyadenylated mRNA
coding for unmodified histone H4. The presence of
H4-v.1 mRNA in various rat tissues and isolated
alveolar macrophages correlated well with the presence
of immunoreactive H4-(86–100), but not whole his-
tone H4, suggesting a role for H4-v.1 mRNA in the
synthesis of the unmodified C-terminal histone H4
peptide [17]. More recently, the mRNA coding for rat
HN (HNr), a slightly modified fragment of H4-(86–
100), was also identified [18]. Interestingly, the levels of
both H4-v.1 and HN mRNAs in isolated rat alveolar
macrophages were increased in the presence of lipo-
polysaccharide. As the production of histone-derived
antimicrobial peptides in insects [19], trout [4] and
humans [11] is enhanced by lipopolysaccharide, and as
such induction at the bacterial trap sites (NETs) on
activated human neutrophils [12] is accompanied by
the release of ATP [13,20], it became of interest to
verify whether the histone H4-derived peptides H4-
(86–100) and HN possess bactericidal activity and
whether such activity is modified by ATP.
Herein we report that H4-(86–100), HN and related
compounds structure-dependently inhibit growth of
bacteria in an ATP-dependent quinolone-like manner,

an effect that correlates with their in vitro inhibitory
effects on DNA gyrase.
Results
Antimicrobial effects of HNr and related peptides
and nonpeptides
H4-(86–100) and HNr (Fig. 1) were tested for their
bactericidal activity against Gram-negative and Gram-
positive bacteria (Table 1). The bactericidal activities
of HNr and H4-(86–100) were comparable, except for
Staphylococcus aureus, against which H4-(86–100) dis-
played higher bactericidal potency. The similarity of
their antimicrobial potencies can be explained by the
fact that their only structural difference is Thr4 in
HNr versus Ala89 in H4-(86–100) (Fig. 1A). Nonethe-
less, their antimicrobial potency was 1.19 to > 17.14
times higher in Gram-negative (Escherichia coli, Pseu-
domonas aeruginosa) than Gram-positive (S. aureus,
Bacillus subtilis) bacteria, with LD
50
values of 1.75–
4.34 lgÆmL
)1
and 5.16 to > 30 lgÆmL
)1
, respectively.
At the LD
90
, their potency differences were in the
same range; that is, they were 1.36 to > 6.25 times
more potent in Gram-negative than in Gram-positive

bacteria.
The bactericidal potencies of H4-(86–100), HNr and
related peptides and nonpeptides in E. coli were com-
pared with those of the cationic peptides protegrin and
LL-37 (Table 2). Among the various HN-related
S. Lemaire et al. Antimicrobial histone H4 peptides
FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS 5287
peptides tested, H4-(86–100) displayed the highest
potency, with an LD
50
comparable to that of LL-37
(3.48 versus 4.1 lgÆmL
)1
). Protegrin was somewhat
more potent, with an LD
50
of 0.70 lgÆmL
)1
. H4-(86–
100) and HNr had close LD
50
values (4.34 and
3.48 lgÆmL
)1
). All fragments of HN and H4-(86–100),
including osteogenic growth peptide (OGP) or H4-(89–
102) [21], were much less potent than the parent
peptides, stressing the importance of the integrity of
the molecule (15 amino acids) for bactericidal activity.
However, the addition of Gly-Gly in the C-terminal

portions of H4-(86–100) to provide H4-(86–102) was
A
BC
DE
Fig. 1. Structures of HN and related peptides and nonpeptides. (A) Amino acid sequences of naturally occurring HN-like peptides. (B) Theo-
retical amphipathic a-helical conformation of HNr ( (C) Structures of HN-like cyclic tetrapeptides.
(D) Structures of o-phenyldiamine derivatives (HN-like nonpeptides). (E) Structures of benzimidazole derivative (HN-like nonpeptide). Arrows
in (B) show protruding basic, phenyl and phenol groups in the C-terminal portion of HNr.
Antimicrobial histone H4 peptides S. Lemaire et al.
5288 FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS
better tolerated for antimicrobial activity (LD
50
:
4.94 lgÆmL
)1
) than the addition of two extra N-termi-
nal amino acids in H4-(84–100) and H4-(84–102)
(LD
50
: 26.25 and 55.25 lgÆmL
)1
, respectively). Arg in
position 6 of H4-(86–100) or HNr was also important
for their antimicrobial activity, as its replacement by
Gly in HNb is most likely responsible for its relatively
low potency (LD
50
: 88.3 lgÆmL
)1
). Interestingly, the

antimicrobial potency of HNb could be improved by
amidation of its C-terminal amino acid (LD
50
:
10.6 lgÆmL
)1
) or addition of Gly-amide (LD
50
:
15.3 lgÆmL
)1
).
HN-like cyclic tetrapeptides and nonpeptides were
recently designed to mimic the antinociceptive effects
of parent HN [22,23]. Among these compounds, com-
pounds 1–9 (Fig. 1C,D) contain basic, phenol and ⁄ or
phenyl groups that mimic key residues (Arg9, Tyr13
and Phe15) in HNr (Fig. 1B). As the opposite orienta-
tion of basic and hydrophobic amino acids in linear
amphipathic a-helical model peptides was shown to be
an important determinant for their antimicrobial activ-
ity [24], it was of interest to verify whether HN-like
compounds with similar structural arrangements
display antimicrobial activity. Table 2 indicates that
among the HN-like cyclic tetrapeptides, compound 3
[Fig. 1C: cyclo-(Gly-pCl-Phe-Tyr-d-Arg)] [22] is the
most potent E. coli-killing agent, with an LD
50
of
3.08 lgÆmL

)1
. With the exception of compound
8 [Fig. 1D: N-5-guanidinopentanamide-(2R)-yl-2-N-
(p-hydroxyphenylacetyl)-4-(p-chlorobenzoyl)-phenylene-
diamine] [23], the HN-like nonpeptides were much less
potent, with LD
50
values varying between 117 and
> 300 lgÆmL
)1
. However, the relatively high bacterici-
dal potency of compound 8 (LD
50
: 13.9 lgÆmL
)1
)
makes this structure attractive for further consi-
deration.
Potentiation of the antimicrobial activity of HN
and related compounds by ATP
Inflammation caused by infection or wounds triggers
the stimulation of neutrophils, which form traps
(NETs) for the attachment, capture and destruction of
invading microorganisms. The NETs contain nuclear
materials that include DNA and bactericidal proteins
and histone-derived peptides such as buforins and pos-
sibly HN. The inflammation is also accompanied by
the release of large amounts of ATP from stimulated
cells [13,25]. A large body of evidence indicates that
cationic antimicrobial peptides create channels through

the bacterial membrane through which they enter and
allow the entry and release of ions and other cell
constituents [26]. In most instances, the pores are
Table 1. Bactericidal activity of HNr and H4-(86–100) in Gram-nega-
tive and Gram-positive bacteria. Structures of HNr and H4-(86–100)
are illustrated in Fig. 1.
Compound
LD
50
± SEM
(lgÆmL
)1
)
LD
90
± SEM
(lgÆmL
)1
)
Escherichia coli (Gram-negative)
HNr 4.34 ± 0.39 10.33 ± 0.78
H4-(86–100) 3.48 ± 0.15 6.68 ± 0.73
Pseudomonas aeruginosa (Gram-negative)
HNr 3.50 ± 0.86 8.70 ± 1.32
H4-(86–100) 1.75 ± 0.03 4.80 ± 0.41
Bacillus subtilis (Gram-positive)
HNr 5.16 ± 0.84 14.06 ± 0.84
H4-(86–100) 5.16 ± 0.61 17.10 ± 2.43
Staphylococcus aureus (Gram-positive)
HNr > 30 > 30

H4-(86–100) 5.83 ± 0.03 14.66 ± 0.24
Table 2. Relative bactericidal activity of HN and related compounds
in E. coli. Structures of HN and related compounds are illustrated in
Fig. 1. Significance is expressed as P £ 0.05 as compared to HNr
*
or H4-(86–100)
**
.
Compound
LD
50
± SEM
(lgÆmL
)1
)
LD
90
± SEM
(lgÆmL
)1
)
1. HN-like peptides
HNr 4.34 ± 0.39 10.3 ± 0.78
Pro-HNr 22.50 ± 4.47
*
59.7 ± 2.7
*
HNb 88.3 ± 5.36
*
155 ± 17

*
HNb-(1–13) > 300
*
HNb-(3–13) > 300
*
HNb-(7–15) 16.25 ± 0.21
*
>30
*
HNb-(8–15) 150 ± 20
*
> 150
*
HNb-amide 10.6 ± 1.7
*
21.2 ± 2.8
HNb-Gly-amide 15.3 ± 2.8
*
22.5 ± 4.5
H4-(86–100) 3.48 ± 0.15
*
6.68 ± 0.73
*
H4-(86–102) 4.94 ± 0.94 17.7 ± 5.8
H4-(84–100) 26.25 ± 5.45
**
76.0 ± 17.2
**
H4-(84–102) 55.25 ± 1.77
**

144 ± 6
**
OGP or H4-(89–102) 14.40 ± 2.77
**
67.2 ± 15.2
**
H4-(92–100) > 150
**
2. HN-like cyclic tetrapeptides
Compound 1 11.30 ± 2.05
*
21.00 ± 6.83
Compound 2 30.25 ± 5.07
*
59.25 ± 4.71
*
Compound 3 3.08 ± 0.42
*
4.66 ± 0.23
*
3. HN-like nonpeptides
Compound 4 > 300
*
> 300
*
Compound 5 119 ± 19
*
220 ± 22
*
Compound 6 > 300

*
> 300
*
Compound 7 117 ± 24
*
264 ± 18
*
Compound 8 13.90 ± 0.30
*
19.63 ± 2.25
Compound 9 180 ± 34
*
> 300
*
4. Reference peptides
LL-37 4.10 ± 0.44 6.93 ± 0.07
*
Protegrin 0.70 ± 0.10
*
1.00 ± 0.12
*
S. Lemaire et al. Antimicrobial histone H4 peptides
FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS 5289
short-lived and do not induce cell killing per se, but
they allow the entry of the cationic peptides into the
cells and their interaction with cytoplasmic polyanions,
nuclear proteins and DNA to induce bacteriostatic
and ⁄ or bactericidal activities [26,27]. We then pre-
sumed that the pores could also allow the entry of
ATP for potentiation of peptide activities. For

instance, it is known that the quinolone antibiotics act
on intracellular DNA gyrase, and their inhibitory
effect on the enzyme is potentiated by ATP [28].
Therefore, we verified whether the presence of ATP
could affect the antimicrobial activities of H4-(86–
100), HNr, compound 3 and the potent quinolone
antibiotic ciprofloxacin (Fig. 2). ATP (10
)3
m) alone
did not affect E. coli growth, but it significantly poten-
tiated the antimicrobial activities of HN and related
compounds as well as that of ciprofloxacin. Thus, their
LD
50
values were reduced by a factor varying between
1.29 (HNr) and 2.43 (compound 3), whereas that of
ciprofloxacin was reduced by a factor of 2.8. Similar
reductions in LD
90
values were observed in the pres-
ence of ATP. On the other hand, the bactericidal
potencies of LL-37 (Fig. 2E) and protegrin (Fig. 2F)
were not affected by ATP. Therefore, the antimicrobial
activity of these two latter peptides may involve a
different mechanism.
The concept of a possible role for ATP in the mech-
anism of action of H4-(86–100) and ciprofloxacin was
strengthened by the observations that their antimicro-
bial effects were blocked by 2,4-dinitrophenol (DNP),
an uncoupler of oxidative phosphorylation (Fig. 3A,B)

and fluoroacetic acid, a potent metabolic poison
(Fig. 3C,D), whereas coumermycin A1, a blocker of
Gyr-B-linked ATPase, enhanced their antimicrobial
activities (Fig. 3E,F).
Time dependence of the bactericidal activity of
H4-(86–100) and ciprofloxacin
In order to evaluate the rapidity of action of his-
tone H4 peptides and compare it with that of the
quinolone antibiotic ciprofloxacin, the LD
50
values of
Fig. 2. ATP potentiation of the bactericidal
activity of H4-(86–100) (A), HNr (B), com-
pound 3 (C) and ciprofloxacin (D) against
E. coli. LL-37 (E) and protegrin (F) were
used as negative controls. LD
50
and LD
90
values are expressed in lgÆmL
)1
.*P £ 0.05
as compared to control.
Antimicrobial histone H4 peptides S. Lemaire et al.
5290 FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS
H4-(86–100) and ciprofloxacin were measured after dif-
ferent periods (15 min, 30 min and 2 h) of incubation
in phosphate buffer at 37 °C with E. coli (Fig. 4). The
bactericidal effects of H4-(86–100) and ciprofloxacin
were completed within 30 min, as incubation of the

cells for a longer period (2 h) in the presence of
increasing concentrations of the compounds did not
significantly affect their LD
50
values. However, a
shorter duration (15 min) of incubation of the cells in
the presence of H4-(86–100) and ciprofloxacin resulted
in a significant decrease in their bactericidal potencies
as assessed by higher LD
50
values (3.55-fold and 1.73-
fold higher, respectively; Fig. 4).
Radial diffusion assay
The antimicrobial activities of H4-(86–100) and some
related compounds were also assessed by the radial dif-
fusion assay (Fig. 5). In this test, H4-(86–100) was
equipotent with HNr and compound 3, and about 4.2
and > 8.5 times as potent as compound 8 and OGP,
respectively. H4-(86–100) was 390 times less potent
than ciprofloxacin (Fig. 5), as compared with being
324 times less potent than ciprofloxacin as measured
by the microwell turbidimetric assay (Fig. 4). The
control peptides protegrin and LL-37 had potencies
slightly lower than (1.8 and 1.1 times, respectively) but
still comparable to those of H4-(86–100), HNr and
compound 3 (Fig. 5). Their lower potencies in this
assay may be due to their lower ability to diffuse out
of the paper disk and come into contact with bacteria.
In vitro blockade of DNA gyrase-induced
supercoiling activity

The observation that the antimicrobial effects of H4-
(86–100), HNr and the compound 3, like those of the
quinolone class of antibiotics acting on DNA gyrase,
are potentiated by ATP and modulated by chemical
agents that affect cell levels of ATP led us to verify the
effects of HN and related compounds on the in vitro
supercoiling activity of DNA gyrase (Fig. 6). Both
HNr (Fig. 6A) and H4-(86–100) (data not shown)
inhibited supercoiling of pBR322 DNA in a dose-
dependent manner, total inhibition being observed
with 5–7 lgÆ20 lL
)1
assay. Inhibition of DNA gyrase
supercoiling activity was also observed with the potent
antimicrobial compounds 3 and 8, but not bovine
histogranin (HNb)-(1–13) or HNb-(3–13) (Fig. 6B),
two inactive HN fragments, in the antimicrobial assay
Fig. 3. Effects of the uncoupler of
oxidative oxidation DNP (A, B), the
aconitase inhibitor fluoroacetate (C, D) and
the inhibitor of DNA gyrase-linked ATPase,
coumermycin A1 (E, F) on the bactericidal
activity of H4-(86–100) and ciprofloxacin
against E. coli.LD
50
is expressed in
lgÆmL
)1
.*P £ 0.05 as compared with
control.

S. Lemaire et al. Antimicrobial histone H4 peptides
FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS 5291
(Table 2). The relative inability of OGP [or H4-(89–
102)] to inhibit DNA gyrase was also in accordance
with its low antimicrobial activity (Fig. 6B and
Table 2).
Comparison of the DNA gyrase inhibitory activity
of HN with those of ciprofloxacin, LL-37 and amp-
icillin (an antibiotic acting as an inhibitor of cell wall
synthesis) indicated that the inhibitory effect of the
potent quinolone ciprofloxacin was mimicked by HNr
[and H4-(86–100)] but not LL-37 or ampicillin, which
served as negative controls (Fig. 6C). Histones are
known to associate with chromatin DNA and regulate
its replication. If the inhibitory effect of the C-terminal
histone H4 peptides HNr and H4-(86–100) on DNA
gyrase activity was due to a direct interaction of the
peptides with pBR322 DNA (either relaxed or super-
coiled DNA), such an effect would result in shifts
in gel migration of peptide-bound DNA. Figure 6D
indicates that H4-(86–100) and HNr, as well as cipro-
floxacin, LL-37 and ampicillin, do not change the
electrophoretic mobility of standard supercoiled
pBR322 DNA (SS sample from Topogen). The migra-
tion of the small amount of relaxed DNA contained
within this sample was also not affected by any one of
these agents (Fig. 6D).
Discussion
Structurally, HNr and its related peptide H4-(86–100)
belong to one major group of antimicrobial peptides:

linear amphipathic peptides without cysteine. Most of
these short antimicrobial peptides have random struc-
tures in water [27]. In this regard, H4-(86–102) dis-
solved in water displayed the characteristics of a fully
random conformation [29]. However, antimicrobial
peptides are known to form a structure, namely the
a-helical structure, when they bind to a membrane or
another hydrophobic cellular organelle [30,31]. His-
tone H4 contains two a-helical regions. One region
spans amino acids 55–67 [32]. A second region, that
spans amino acids 70–90, has been demonstrated to
adopt the a-helical conformation when histone H4 is
associated with the nucleosome but not when purified
histone H4 polymerizes in solution [29]. Thus, the pos-
sibility that extracellular HNr and its related peptide
H4-(86–100) form a-helical structures pertains to their
ability to associate with plasma membranes and other
hydrophobic cellular organelles. The theoretical helical
wheel conformation of HNr shows the basic and
hydrophobic or neutral amino acids on opposite sur-
faces of the molecule (Fig. 1B). Such an arrangement
of basic and hydrophobic groups was reported to be a
good prediction factor for the antimicrobial activity of
model [24] and naturally occurring cationic peptides
[33]. Some members of this group, such as buforin-II
[33,34], which act rapidly without any lag time, are
believed to kill bacteria by penetrating their membrane
and binding to the DNA without inducing cell lysis. In
the present study, HNr and H4-(86–100), like the
quinolone antibiotic ciprofloxacin, displayed rapid

(within 30 min; Fig. 4) inhibition of bacterial cell
growth at concentrations (nanomolar) that are compa-
rable to those observed with the potent antimicrobial
peptides protegrin and LL-37 (Table 2). The bacterici-
dal activity of the peptides was assessed by both turbi-
dimetric and radial diffusion (Fig. 5) procedures in
Gram-negative and Gram-positive bacteria (Table 1).
The enhancement of the antimicrobial potencies of
H4-(86–100), HN and compound 3 by ATP led us to
believe that the peptides might be acting at an intra-
cellular level that was ATP-dependent. Among the
various classes of antimicrobial agents, quinolones
show different in vivo and in vitro effects that are
Fig. 4. Effect of the incubation time on the LD
50
of H4-(86–100) (A)
and ciprofloxacin (B). E. coli bacteria were incubated with the
bactericidal agent in phosphate buffer for the indicated time, and
the LD
50
values were measured as described in Experimental
procedures. LD
50
is expressed in lgÆmL
)1
.*P £ 0.05 as compared
to the 2 h incubation.
Antimicrobial histone H4 peptides S. Lemaire et al.
5292 FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS
ATP-dependent [28]. Quinolones represent a class of

antimicrobial agents that act by an inhibition of DNA
gyrase. Their antimicrobial effects in vivo are blocked
by drugs such as DNP and fluoroacetate, which are
known to lower cell levels of ATP [23]. Similarly,
in vitro, their inhibitory effects on gyrase-mediated
DNA catenation are potentiated by the presence of
ATP [28]. In order to verify whether H4-(86–100) and
related compounds affect bacterial cell growth by a
mechanism that involves modulation of DNA gyrase
activity, we first verified whether the antimicrobial
activities of the peptides were affected by the presence
of DNP or fluoroacetate. The two agents significantly
blocked the antimicrobial activity of H4-(86–100)
(Fig. 3A,C), whereas the presence of coumermycin A1,
an inhibitor of DNA gyrase-linked ATPase, enhanced
its antimicrobial activity (Fig. 3E). Thus, agents that
decrease cellular levels of ATP inhibit the antimicro-
bial activity of HN-like peptides, whereas coumermy-
cin A1, which may increase cellular levels of ATP by
its inhibition of DNA gyrase-linked ATPase, enhances
their activity. Interestingly, similar observations were
obtained with the potent quinolone antibiotic cipro-
floxacin (Fig. 3B,D,F).
More direct support for the involvement of DNA
gyrase in the antimicrobial effects of H4-(86–100) and
related compounds was obtained in vitro by the mea-
surement of their inhibitory activity on the supercoiling
of pBR322 plasmid induced by the commercially avail-
able DNA gyrase holoenzyme (Topogen) (Fig. 6). In
this assay, the inhibitory effects of the potent anti-

microbial compounds HNr, H4-(86–100), compound 3
and compound 8 were very pronounced (Fig. 6A,B),
whereas those of the inactive antimicrobial fragments
HNb-(1–13) and HNb-(3–13) (Table 2) were not signifi-
cant (Fig. 6B).
The antimicrobial and anti-DNA gyrase potencies of
HNr, H4-(86–100), compound 3 and compound 8 may
depend upon their particular structural arrangements,
displaying basic and aliphatic or neutral groups at
opposite sides of the molecule. Such molecules were
demonstrated to present their positively charged side
to the external anionic sites of bacterial membranes,
first inducing neutralization of some external groups of
the membrane, and then allowing the aliphatic or
neutral groups of the peptide to attach to internal
hydrophobic sites for rapid penetration and action
inside the cell [26]. Like the potent quinolone antibiotic
ciprofloxacin, HNr and H4-(86–100) did not affect the
electrophoretic mobility of supercoiled and relaxed
pBR322 DNAs (Fig. 6D), suggesting that the effects of
these compounds on DNA gyrase activity most likely
Fig. 5. Antimicrobial activity of ciprofloxacin (0.2 lg), H4-(86–100) and some related compounds (30 lg) against B. subtilis in the radial
diffusion assay. Samples were applied to paper disks and placed onto an agarose plate inoculated with bacteria as described in Experimental
procedures. After overnight incubation at 37 °C, plates were photographed and clear zone diameters were measured.
S. Lemaire et al. Antimicrobial histone H4 peptides
FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS 5293
do not result from their direct interaction with DNA.
On the other hand, the marked difference between the
doses of histone H4 and HN peptides necessary to
inhibit DNA gyrase (1–5 lgÆ20 lL

)1
) and to kill bacte-
ria (3–4 lgÆmL
)1
) may be due to the greater ability of
antimicrobial peptides to adopt the a-helical conforma-
tion in a hydrophobic cell-containing milieu than in an
aqueous cell-free DNA gyrase assay [30,31].
Histones are known to possess domains that bind to
other histones and other nuclear proteins, and domains
that bind to DNA to form the nucleosome. In his-
tone H4, the C-terminal segment that corresponds to
H4-(86–100) or HN is the portion of the molecule that
binds to other histones and ⁄ or proteins, whereas the
N-terminal segment has the ability to bind to DNA
according to its acetylation and methylation status
[29]. The possibility that HN and H4-(86–100) bind to
DNA gyrase to inhibit its activity is a subject that
merits our attention. The site of action of quinolone
antibiotics has been determined to be on the gyrA sub-
unit of the enzyme, due to the ability of the mutation
of Ser83 to Trp to induce resistance and affect quino-
lone binding to the gyrase–DNA complex [35]. How-
ever, histone H4-derived peptides and HN would be
expected to be devoid of resistance induction, due to
the evolutionary stability of histones, and more partic-
ularly histone H4 [32]. Interestingly, mutations at
codon 751 of the E. coli gyrB gene conferred resistance
to the antimicrobial peptide microcin B17 [36]. As
H4-(86–100) and HN are peptides that display

quinolone-like antibiotic activity, it will be particularly
interesting to verify whether mutations in the gyrA
and gyrB subunits that confer resistance to quinolone
antibiotics and microcin B17, respectively, affect the
bactericidal activities of C-terminal histone H4
peptides. Further studies will be necessary to determine
the mechanism of action of C-terminal histone H4
peptides on DNA gyrase, but the close structure–activ-
ity relationship established between their antimicrobial
(Table 1) and DNA gyrase antagonist activities
(Fig. 6) strongly suggests that both effects are related
and depend upon the structures of the pentadecapep-
tides H4-(86–100) and HNr, the precursors [H4-(84–
102) and pro-HNr] or fragments [OGP, HNb-(1–13),
HNb-(3–13)] being either much less potent or inactive.
Experimental procedures
Materials
Coumermycin A1, DNP and fluoroacetic acid were pur-
chased from Sigma Chemical Co. E. coli D31, S. aureus
ATCC 6538, B. subtilis ATCC 19659 and P. aeruginosa
ATCC 15442 were obtained from M. Hincke (University of
Ottawa). Growth kinetics in the 96-well microplates were
monitored (as turbidity) with a computer-controlled Spec-
tramax M5 plate reader (Molecular Devices, Sunnyvale,
CA) running softmax pro software (version 3.1) and a
650 nm filter. Growth of the seed cultures was monitored
on a Beckman DU-640 spectrophotometer by measuring
the attenuance (D) at 650 nm of 1 mL samples. Synthetic
HN and related peptides and nonpeptides were prepared by
a solid-phase procedure as previously described [22,23]. The

compounds were cleaved from the resin and purified by
passage through Sephadex G-10, Sep-Pak cartridges
(Waters) and semipreparative HPLC columns (l-bondapak
C18; Waters). The purity of the products was verified by
TLC (one spot, ninhydrin detection) and analytical HPLC
on l-bondapak C-18 columns (Waters). Their identity was
A
B
C
D
Fig. 6. Inhibitory effects of various concentrations of HNr (A),
7 lgÆ20 lL
)1
of H4-(86–100), OGP, compound 3, compound 8,
HNb-(3–13) and HNb-(1–13) (B) and 3 lgÆ20 lL
)1
of HNr, coumer-
mycin A1, ciprofloxacin, LL-37 and ampicillin (C) on DNA gyrase-
mediated pBR322 supercoiling. The DNA gyrase supercoiling assay
was carried out with the Topogen assay kit as described in Experi-
mental procedures using relaxed pBR322 plasmid as the substrate.
R, relaxed pBR322; S, supercoiled pBR322; SS, standard super-
coiled pBR322. In order to verify whether the various compounds
interacted directly with the DNA, the effects of 3 lgÆ20 lL
)1
of H4-
(86–100), HNr, coumermycin A1 (Cou), ciprofloxacin (Cip), LL-37
and ampicillin (Amp) on the migration of SS in the absence of the
enzyme was also monitored (D).
Antimicrobial histone H4 peptides S. Lemaire et al.

5294 FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS
determined by ESI MS. All peptides and nonpeptides were
96–100% pure and displayed the expected molecular
masses. H4-(86–100), HNr and compound 3 were 99.5%,
97% and 98% pure, respectively, as evaluated by analytical
HPLC. ESI MS [M +H
+
] values were 1771.4 [H4-(86–
100), C
83
H
132
N
23
O
20
, calculated 1771.1], 1802.8 (HNr,
C
84
H
134
N
23
O
21
, calculated 1802.1) and 559.4 (compound 3,
C
26
H
33

ClN
7
O
5
, calculated 559.0).
Bactericidal assays
Microwell turbidimetric procedure
Cultures were grown overnight in LB media with shaking
(250 r.p.m.). On the next day, the cultures were diluted 1 : 50
in the same medium and incubated for an additional 2 h to
obtain the cells in the midlogarithmic phase. They were then
diluted in 10 mm sodium phosphate buffer (pH 7.4) at
6.1 million virtual colony-forming units (CFUv)ÆmL
)1
(vir-
tual colony counting) [25]. Cells (35 lL) were preincubated
for 2 h or (as indicated in Fig. 4) at 37 °C with shaking
(250 r.p.m.) in microwells in the absence or presence of vari-
ous concentrations (from 0.5 to 300 lgÆmL
)1
) of HN and
related compounds in a total volume of 50 lL. In some
experiments, the inhibitory effects of H4-(86–100), HNr or
compound 3 on cell growth were modulated by the addition
to the preincubation media of ATP (1 mm), DNP (an uncou-
pler of oxidative phosphorylation; 2 mm), fluoroacetic acid
(an aconitase inhibitor; 4 mm) or coumermycin A1 (an ATP-
antagonizing gyrase poison; 2 lm). Serial dilutions of the
cells (1.0, 0.5, 0.1, 0.05, 0.01 and 0.005 · 10
6

CFUvÆmL
)1
)
were also made in the absence of drug for standardization of
the growth assay. After preincubation, 150 lL of LB media
was added to each well, and the cells were grown for 6–8 h at
37 °C in the rotating incubator. D readings at 650 nm were
monitored every 30 min. The generated growth curves
allowed the evaluation of the number of living cells after the
phosphate buffer preincubation procedure. Growth curve
data were processed as described below.
The D measurements from the plate reader run were
imported into microsoft excel software and corrected by
subtracting each well’s initial reading from the subsequent
data for that well [25]. The times at which each growth curve
crossed the threshold change in D
650
nm of 0.02 absorbance
units were plotted against log(C_0) to generate a calibration
curve that related the threshold times to cell concentration
at incubation time of zero (C_0). The rate of survival was
calculated as the number of CFUv of HN-like compound-
treated cells ⁄ number of CFUv of control cells. The virtual
50% and 90% lethal doses (vLD
50
and vLD
90
) were
reported as the compound concentrations that resulted in
survival rates of 0.5 and 0.1, respectively. Results represent

the mean ± SEM of three duplicated sets of experiments.
Statistical significance was determined using one-way analy-
sis of variance followed by a Bonferonni comparison test.
P £ 0.05 is considered as significant.
Radial diffusion assay
The radial diffusion assay of Steinberg & Lehrer [37] was
used to confirm the antimicrobial activity of H4-(86–100)
and related compounds. B. subtilis bacteria were grown to
log phase in LB broth, centrifuged at 1000 g for 15 min,
and resuspended in 10 mm sodium phosphate buffer
(pH 7.3) (1 · 10
5
CFUÆmL
)1
). Molten culture medium
(1.5% low-electroendosmosis agarose, 1% biotryptone,
0.5% yeast extract; Bioshop, Burlington, Canada) was
prepared in 10 mm sodium phosphate buffer. The medium
was distributed into culture Petri dishes and allowed to
solidify. The gel surface was inoculated by the addition of
5 mL of the bacterial preparation, which was immediately
removed. Test compounds (50 lgin10lL of phosphate
buffer) were applied to Whatman No. 1 paper disks (6 mm
in diameter). After the solvent had been allowed to evapo-
rate for 1 h at room temperature, disks were applied to
bacterial plates. Disks treated with the phosphate buffer or
0.2 lg of ciprofloxacin in the phosphate buffer were used as
negative and positive controls. Following the application of
sample disks, plates were incubated at 37 °C for 18 h. The
antimicrobial activities of the compounds were assessed by

measurement of the clear zones around the disks.
DNA gyrase supercoiling assays
DNA gyrase supercoiling assays were carried out as previ-
ously described [38], using 5 nm gyrase (Topogen), 3.5 nm
relaxed pBR322, and 0.5–7.0 lg of HNr or H4-(86–100) as
indicated, and incubated at 37 °C for 1 h. DNA products
were analyzed on 1% agarose gels.
Acknowledgements
This work was supported by the Faculty of Medicine,
University of Ottawa.
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