Antimicrobial effects of H4-(86–100), histogranin and related compounds – possible involvement of DNA gyrase ˆ Simon Lemaire, Thuy-Tien 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 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: slemaire@uottawa.ca (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 antimicrobial 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 (LD50: 3.48 and 4.34 lgỈmL)1) are comparable to that of the antimicrobial peptide LL-37 (LD50: 4.10 lgỈmL)1) The antimicrobial activities of H4-(86–100) and HNr depend upon the integrity of the molecules, as precursors [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 compound 3, like those of quinolone antibiotics acting as DNA gyrase poisons, are potentiated by ATP (1 mm) and coumermycin A1 (a DNA gyraselinked 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, compound 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 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 extranuclear and extracellular functions [2] Their antimicrobial 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 mixture of histones H3 and H4) displayed potent bactericidal 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 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 S Lemaire et al 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 histone H2A fragment buforin-I formed a dense immunoreactive 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 thereafter 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 gastrointestinal tract [7] Their finding of buforin-I in porcine, bovine and human stomach lavage fluids supported 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 neutrophils 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-terminal histone H4 peptide homologous to H4-(86–100), was first isolated in our laboratory from bovine adrenal 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 Antimicrobial histone H4 peptides 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 histone 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 lipopolysaccharide 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 Grampositive bacteria (Table 1) The bactericidal activities of HNr and H4-(86–100) were comparable, except for Staphylococcus aureus, against which H4-(86–100) displayed 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) Nonetheless, their antimicrobial potency was 1.19 to > 17.14 times higher in Gram-negative (Escherichia coli, Pseudomonas aeruginosa) than Gram-positive (S aureus, Bacillus subtilis) bacteria, with LD50 values of 1.75– 4.34 lgỈmL)1 and 5.16 to > 30 lgỈmL)1, respectively At the LD90, 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 compared with those of the cationic peptides protegrin and LL-37 (Table 2) Among the various HN-related FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS 5287 Antimicrobial histone H4 peptides S Lemaire et al A B C D E Fig Structures of HN and related peptides and nonpeptides (A) Amino acid sequences of naturally occurring HN-like peptides (B) Theoretical amphipathic a-helical conformation of HNr (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi) (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 peptides tested, H4-(86–100) displayed the highest potency, with an LD50 comparable to that of LL-37 (3.48 versus 4.1 lgỈmL)1) Protegrin was somewhat more potent, with an LD50 of 0.70 lgỈmL)1 H4-(86– 100) and HNr had close LD50 values (4.34 and 3.48 lgỈmL)1) All fragments of HN and H4-(86–100), 5288 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 FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS S Lemaire et al Antimicrobial histone H4 peptides Table Bactericidal activity of HNr and H4-(86–100) in Gram-negative and Gram-positive bacteria Structures of HNr and H4-(86–100) are illustrated in Fig Compound LD50 ± SEM (lgỈmL)1) Escherichia coli (Gram-negative) HNr 4.34 ± 0.39 H4-(86–100) 3.48 ± 0.15 Pseudomonas aeruginosa (Gram-negative) HNr 3.50 ± 0.86 H4-(86–100) 1.75 ± 0.03 Bacillus subtilis (Gram-positive) HNr 5.16 ± 0.84 H4-(86–100) 5.16 ± 0.61 Staphylococcus aureus (Gram-positive) HNr > 30 H4-(86–100) 5.83 ± 0.03 LD90 ± SEM (lgỈmL)1) 10.33 ± 0.78 6.68 ± 0.73 8.70 ± 1.32 4.80 ± 0.41 14.06 ± 0.84 17.10 ± 2.43 > 30 14.66 ± 0.24 Table Relative bactericidal activity of HN and related compounds in E coli Structures of HN and related compounds are illustrated in Fig Significance is expressed as P £ 0.05 as compared to HNr* or H4-(86–100)** Compound LD50 ± SEM (lgỈmL)1) HN-like peptides HNr 4.34 ± Pro-HNr 22.50 ± HNb 88.3 ± HNb-(1–13) > 300* HNb-(3–13) > 300* HNb-(7–15) 16.25 ± HNb-(8–15) 150 ± HNb-amide 10.6 ± HNb-Gly-amide 15.3 ± H4-(86–100) 3.48 ± H4-(86–102) 4.94 ± H4-(84–100) 26.25 ± H4-(84–102) 55.25 ± OGP or H4-(89–102) 14.40 ± H4-(92–100) > 150** HN-like cyclic tetrapeptides Compound 11.30 ± Compound 30.25 ± Compound 3.08 ± HN-like nonpeptides Compound > 300* Compound 119 ± Compound > 300* Compound 117 ± Compound 13.90 ± Compound 180 ± Reference peptides LL-37 4.10 ± Protegrin 0.70 ± 0.39 4.47* 5.36* LD90 ± SEM (lgỈmL)1) 10.3 ± 0.78 59.7 ± 2.7* 155 ± 17* 0.21* 20* 1.7* 2.8* 0.15* 0.94 5.45** 1.77** 2.77** > 30* > 150* 21.2 ± 22.5 ± 6.68 ± 17.7 ± 76.0 ± 144 ± 67.2 ± 2.05* 5.07* 0.42* 21.00 ± 6.83 59.25 ± 4.71* 4.66 ± 0.23* 19* 24* 0.30* 34* 0.44 0.10* 2.8 4.5 0.73* 5.8 17.2** 6** 15.2** > 300* 220 ± 22* > 300* 264 ± 18* 19.63 ± 2.25 > 300* 6.93 ± 0.07* 1.00 ± 0.12* better tolerated for antimicrobial activity (LD50: 4.94 lgỈmL)1) than the addition of two extra N-terminal amino acids in H4-(84–100) and H4-(84–102) (LD50: 26.25 and 55.25 lgỈmL)1, respectively) Arg in position 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 (LD50: 88.3 lgỈmL)1) Interestingly, the antimicrobial potency of HNb could be improved by amidation of its C-terminal amino acid (LD50: 10.6 lgỈmL)1) or addition of Gly-amide (LD50: 15.3 lgỈmL)1) HN-like cyclic tetrapeptides and nonpeptides were recently designed to mimic the antinociceptive effects of parent HN [22,23] Among these compounds, compounds 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 orientation of basic and hydrophobic amino acids in linear amphipathic a-helical model peptides was shown to be an important determinant for their antimicrobial activity [24], it was of interest to verify whether HN-like compounds with similar structural arrangements display antimicrobial activity Table indicates that among the HN-like cyclic tetrapeptides, compound [Fig 1C: cyclo-(Gly-pCl-Phe-Tyr-d-Arg)] [22] is the most potent E coli-killing agent, with an LD50 of 3.08 lgỈmL)1 With the exception of compound [Fig 1D: N-5-guanidinopentanamide-(2R)-yl-2-N(p-hydroxyphenylacetyl)-4-(p-chlorobenzoyl)-phenylenediamine] [23], the HN-like nonpeptides were much less potent, with LD50 values varying between 117 and > 300 lgỈmL)1 However, the relatively high bactericidal potency of compound (LD50: 13.9 lgỈmL)1) makes this structure attractive for further consideration 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 possibly 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 FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS 5289 Antimicrobial histone H4 peptides S Lemaire et al Fig ATP potentiation of the bactericidal activity of H4-(86–100) (A), HNr (B), compound (C) and ciprofloxacin (D) against E coli LL-37 (E) and protegrin (F) were used as negative controls LD50 and LD90 values are expressed in lgỈmL)1 *P £ 0.05 as compared to control short-lived and 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 presumed 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 and the potent quinolone antibiotic ciprofloxacin (Fig 2) ATP (10)3 m) alone did not affect E coli growth, but it significantly potentiated the antimicrobial activities of HN and related compounds as well as that of ciprofloxacin Thus, their LD50 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 LD90 values were observed in the pres5290 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 mechanism of action of H4-(86–100) and ciprofloxacin was strengthened by the observations that their antimicrobial 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 histone H4 peptides and compare it with that of the quinolone antibiotic ciprofloxacin, the LD50 values of FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS S Lemaire et al Antimicrobial histone H4 peptides Fig 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 LD50 is expressed in lgỈmL)1 *P £ 0.05 as compared with control H4-(86–100) and ciprofloxacin were measured after different periods (15 min, 30 and 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 LD50 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 LD50 values (3.55-fold and 1.73fold higher, respectively; Fig 4) Radial diffusion assay The antimicrobial activities of H4-(86–100) and some related compounds were also assessed by the radial diffusion 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 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 (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 dosedependent manner, total inhibition being observed with 5–7 lgỈ20 lL)1 assay Inhibition of DNA gyrase supercoiling activity was also observed with the potent antimicrobial compounds and 8, but not bovine histogranin (HNb)-(1–13) or HNb-(3–13) (Fig 6B), two inactive HN fragments, in the antimicrobial assay FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS 5291 Antimicrobial histone H4 peptides S Lemaire et al electrophoretic mobility of standard supercoiled pBR322 DNA (SS sample from Topogen) The migration of the small amount of relaxed DNA contained within this sample was also not affected by any one of these agents (Fig 6D) Discussion Fig Effect of the incubation time on the LD50 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 LD50 values were measured as described in Experimental procedures LD50 is expressed in lgỈmL)1 *P £ 0.05 as compared to the h incubation (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 ampicillin (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 supercoiled 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 ciprofloxacin, LL-37 and ampicillin, not change the 5292 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 structures in water [27] In this regard, H4-(86–102) dissolved 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] Histone 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 possibility 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 surfaces 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 comparable to those observed with the potent antimicrobial peptides protegrin and LL-37 (Table 2) The bactericidal activity of the peptides was assessed by both turbidimetric 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 by ATP led us to believe that the peptides might be acting at an intracellular level that was ATP-dependent Among the various classes of antimicrobial agents, quinolones show different in vivo and in vitro effects that are FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS S Lemaire et al Antimicrobial histone H4 peptides Fig 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 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 antimicrobial activity of HN-like peptides, whereas coumermycin 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 ciprofloxacin (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 measurement of their inhibitory activity on the supercoiling of pBR322 plasmid induced by the commercially available DNA gyrase holoenzyme (Topogen) (Fig 6) In this assay, the inhibitory effects of the potent antimicrobial compounds HNr, H4-(86–100), compound and compound were very pronounced (Fig 6A,B), whereas those of the inactive antimicrobial fragments HNb-(1–13) and HNb-(3–13) (Table 2) were not significant (Fig 6B) The antimicrobial and anti-DNA gyrase potencies of HNr, H4-(86–100), compound and compound 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 FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS 5293 Antimicrobial histone H4 peptides S Lemaire et al A B C D Fig Inhibitory effects of various concentrations of HNr (A), lgỈ20 lL)1 of H4-(86–100), OGP, compound 3, compound 8, HNb-(3–13) and HNb-(1–13) (B) and lgỈ20 lL)1 of HNr, coumermycin A1, ciprofloxacin, LL-37 and ampicillin (C) on DNA gyrasemediated pBR322 supercoiling The DNA gyrase supercoiling assay was carried out with the Topogen assay kit as described in Experimental procedures using relaxed pBR322 plasmid as the substrate R, relaxed pBR322; S, supercoiled pBR322; SS, standard supercoiled pBR322 In order to verify whether the various compounds interacted directly with the DNA, the effects of lgỈ20 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) 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 lgỈ20 lL)1) and to kill bacteria (3–4 lgỈmL)1) may be due to the greater ability of antimicrobial peptides to adopt the a-helical conformation 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 histone 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 5294 [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 subunit of the enzyme, due to the ability of the mutation of Ser83 to Trp to induce resistance and affect quinolone binding to the gyrase–DNA complex [35] However, histone H4-derived peptides and HN would be expected to be devoid of resistance induction, due to the evolutionary stability of histones, and more particularly 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–activity 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 pentadecapeptides 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 purchased 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 Spectramax 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 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 FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS S Lemaire et al determined by ESI MS All peptides and nonpeptides were 96–100% pure and displayed the expected molecular masses H4-(86–100), HNr and compound were 99.5%, 97% and 98% pure, respectively, as evaluated by analytical HPLC ESI MS [M + H+] values were 1771.4 [H4-(86– 100), C83H132N23O20, calculated 1771.1], 1802.8 (HNr, C84H134N23O21, calculated 1802.1) and 559.4 (compound 3, C26H33ClN7O5, 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 : 50 in the same medium and incubated for an additional 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)ỈmL)1 (virtual colony counting) [25] Cells (35 lL) were preincubated for h or (as indicated in Fig 4) at 37 °C with shaking (250 r.p.m.) in microwells in the absence or presence of various concentrations (from 0.5 to 300 lgỈmL)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 on cell growth were modulated by the addition to the preincubation media of ATP (1 mm), DNP (an uncoupler of oxidative phosphorylation; mm), fluoroacetic acid (an aconitase inhibitor; mm) or coumermycin A1 (an ATPantagonizing gyrase poison; lm) Serial dilutions of the cells (1.0, 0.5, 0.1, 0.05, 0.01 and 0.005 · 106 CFUvỈmL)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 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 D650 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 compoundtreated cells ⁄ number of CFUv of control cells The virtual 50% and 90% lethal doses (vLD50 and vLD90) 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 analysis of variance followed by a Bonferonni comparison test P £ 0.05 is considered as significant Antimicrobial histone H4 peptides 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 · 105 CFmL)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 mL of the bacterial preparation, which was immediately removed Test compounds (50 lg in 10 lL of phosphate buffer) were applied to Whatman No paper disks (6 mm in diameter) After the solvent had been allowed to evaporate for 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 previously described [38], using 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 h DNA products were analyzed on 1% agarose gels Acknowledgements This work was supported by the Faculty of Medicine, University of Ottawa References Zhao J (2004) 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Mizuuchi M, O’Ddea MH & Gellert M (1984) Cloning and simplified purification of Escherichia coli DNA gyrase A and B proteins J Biol Chem 259, 9199–9201 FEBS Journal 275 (2008) 5286–5297 ª 2008 The Authors Journal compilation ª 2008 FEBS 5297 ... the involvement of DNA gyrase in the antimicrobial effects of H4-(8 6–1 00) and related compounds was obtained in vitro by the measurement of their inhibitory activity on the supercoiling of pBR322... those of the inactive antimicrobial fragments HNb-( 1–1 3) and HNb-( 3–1 3) (Table 2) were not significant (Fig 6B) The antimicrobial and anti -DNA gyrase potencies of HNr, H4-(8 6–1 00), compound and. .. effects on DNA gyrase Results Antimicrobial effects of HNr and related peptides and nonpeptides H4-(8 6–1 00) and HNr (Fig 1) were tested for their bactericidal activity against Gram-negative and Grampositive