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Antimicrobial Agents and Chemotherapy, August 2006, p. 2602-2607, Vol. 50, No. 8
0066-4804/06/$08.00+0     doi:10.1128/AAC.00331-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Determination of the Antibacterial and Lipopolysaccharide-Neutralizing Regions of Guinea Pig Neutrophil Cathelicidin Peptide CAP11

Daiju Okuda,¹ Shin Yomogida,¹ Hiroshi Tamura,² and Isao Nagaoka^1*

Department of Host Defense and Biochemical Research, Juntendo University School of Medicine, Tokyo 113-8421, Japan,¹ Seikagaku Corporation, Tokyo 100-0005, Japan²

Received 17 March 2006/ Returned for modification 11 May 2006/ Accepted 23 May 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previously, we revealed that a cationic antibacterial polypeptide of 11 kDa (CAP11), a member of the cathelicidins isolated from guinea pig neutrophils, exhibits not only potent antibacterial activity but also lipopolysaccharide (LPS)-neutralizing activity. In this study, to determine the biologically active regions of CAP11, we isolated or synthesized the partial peptides of CAP11 and evaluated their antibacterial and LPS-neutralizing activities. Although CAP11 has a unique homodimeric structure with a disulfide bridge, the biological activities of dimeric and monomeric forms of CAP11 were almost the same. Moreover, the G¹-E³³ peptide of CAP11 showed the same activities as CAP11, whereas the C-terminal region (Y³⁴ to I⁴³) possessed no biological activities. In addition, the three 18-mer peptides (G¹-R¹⁸, T⁹-K²⁶, and L¹⁶-E³³) with overlapping sequences were synthesized, and their activities were determined. The three 18-mer peptides retained the antibacterial activities, and G¹-R¹⁸ was the most potent. In contrast, the LPS-neutralizing activities of these peptides were markedly reduced. Together, these observations indicate that the active region with antibacterial activity is localized at G¹ to R¹⁸ of CAP11, whereas longer sequences (such as G¹ to E³³) would be required for the expression of LPS-neutralizing activity. Furthermore, the C-terminal region (Y³⁴ to I⁴³) and a disulfide bridge are not essential for the antibacterial and LPS-neutralizing activities of CAP11.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peptide antibiotics exhibit potent antimicrobial activities against both gram-positive and gram-negative bacteria, fungi, and viruses, and they form one group of effector components in the innate host defense system (8, 29). The peptide-based defense in mammals against invading microbes relies on the two evolutionarily distinct groups of antimicrobial peptides, defensins and cathelicidins, which have been identified in several epithelial tissues and in the granules of phagocytes (6, 7, 16-18, 20, 35, 39). Defensins contain six conserved cysteine residues in their sequences and exhibit characteristic ß-sheet structures stabilized by three intramolecular disulfide bonds (17, 18, 20). In contrast, cathelicidins are characterized by highly conserved cathelin-like prosequences and variable carboxy-terminal sequences that correspond to the mature antibacterial peptides (6, 7, 16, 39). About 30 cathelicidin peptides from various vertebrates have been identified. Some are -helical, and others are proline/arginine-rich, showing a polyproline-type structure (e.g., porcine PR39 and bactenecins), whereas porcine protegrins form ß-sheet structures (6, 7, 16, 39). Recently, we have characterized two -helical cathelicidins, CAP18 (cationic antibacterial protein of 18 kDa) and CAP11 (cationic antibacterial polypeptide of 11 kDa), isolated from human neutrophils and guinea pig neutrophils, respectively (12, 26, 38). CAP18 is a precursor of cathelicidin, and its carboxy-terminal antibacterial peptide (human CAP18 [hCAP18]/LL-37) is cleaved from its precursor (6, 7, 16, 39). CAP11 is also a carboxy-terminal antibacterial peptide and has a unique homodimeric structure which bridges the two identical 43-amino-acid peptide chains by a disulfide bond (26, 38).

Defensins lose their biological activities in the extracellular milieu containing a physiological NaCl concentration (approximately 150 mM) and also in serum (18, 24). However, the cathelicidin family of antibacterial peptides (hCAP18/LL-37 and CAP11) show antibacterial activities against various bacteria under these physiological conditions (14, 24). In addition, hCAP18/LL-37 and CAP11 exhibit not only antibacterial activities, but also lipopolysaccharide (LPS)-neutralizing activities, by binding with LPS and inhibiting the transfer of LPS to the cell surface membrane receptor CD14 (12, 15, 23, 25, 27). Thus, cathelicidin antibacterial peptide and its related derivatives could be candidates for therapeutic agents that adapt to bacterial infection and/or endotoxin shock (12, 15, 19, 22, 30).

Although antibacterial peptides are diverse in their sizes, structures, and activities, they are mostly amphipathic, retaining both cationic (positively charged) and hydrophobic surfaces (6, 8, 18, 29). These characteristic features facilitate interactions with negatively charged microbial surface membranes, followed by insertion into the microbial lipid membrane, resulting in the disruption of the bacterial membrane and killing of bacteria (6, 8, 18, 29). Moreover, the amphipathic structures of cathelicidins (such as hCAP18/LL-37 and CAP11) are also assumed to be essential for interaction with an amphipathic bacterial LPS (10, 28, 32). Interestingly, however, it has been revealed that the two distinct activities of hCAP18/LL-37 (antibacterial and cytokine-producing activities) are localized in different regions of the molecule (1). Thus, we hypothesized that the antibacterial and LPS-neutralizing activities of cathelicidins do not necessarily reside in the common structures (regions). To confirm this, in this study, we determined the regions responsible for the antibacterial and LPS-neutralizing activities, and the involvement of a disulfide-bond and dimeric structure in these activities, using CAP11 with a unique dimeric structure.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents. Mueller-Hinton broth was purchased from Difco Laboratories (Detroit, MI); heart infusion broth was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan); RPMI 1640 with 2.05 mM L-glutamine with or without phenol red was purchased from Sigma (St. Louis, MO) or Gibco Invitrogen Corporation (Grand Island, NY), respectively; endoprotease Glu-C was purchased from Boehringer Mannheim (Marburg, Germany); alamarBlue was purchased from Biosource International, Inc. (Camarillo, CA); and fluorescein isothiocyanate-conjugated and unconjugated LPSs (from Escherichia coli O111:B4) were purchased from Sigma. Tissue culture supplies were obtained from Corning Inc. (Acton, MA).

Synthesis and isolation of CAP11-derived peptides. A 43-mer peptide of CAP11, G¹-I⁴³ (G¹LRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI⁴³), and its partial peptides, G¹-E³³ (G¹LRKKFRKTRKRIQKLGRKIGKTGRKVWKAWRE³³), Y³⁴-I⁴³ (Y³⁴GQIPYPCRI⁴³), G¹-R¹⁸ (G¹LRKKFRKTRKRIQKLGR¹⁸), T⁹-K²⁶ (T⁹RKRIQKLGRKIGKTGRK²⁶), and L¹⁶-E³³ (L¹⁶GRKIGKTGRKVWKAWRE³³), (Fig. 1), were synthesized by the solid-phase method on a peptide synthesizer (model PSSM-8; Shimadzu, Kyoto, Japan) by fluorenylmethoxycarbonyl chemistry. The peptides were eluted from the resin and purified to homogeneity by reversed-phase high-performance liquid chromatography (RP-HPLC) on a Cosmosil 5 C₁₈ column (Nacalai Tesque, Kyoto, Japan) by use of a 0 to 70% acetonitrile gradient in 0.1% trifluoroacetic acid. The molecular masses of the synthesized peptides were confirmed on a mass spectrometer (model TSQ 700; Thermo Quest Finnigan, Manchester, United Kingdom). A dimer form of CAP11 [(G¹-I⁴³)₂] was prepared by oxidation of G¹-I⁴³ using glutathione (the oxidized form) to form a disulfide bridge at position Cys⁴¹. S-pyridylethylated CAP11 (Pe-CAP11) was prepared by reduction and alkylation of G¹-I⁴³ with dithiothreitol and 4-vinylpyridine. (G¹-I⁴³)₂ and Pe-CAP11 were purified by RP-HPLC on a CAPCELL PAK C₁₈ column (Shiseido Fine Chemicals, Tokyo, Japan). The G¹-E³³ and Y³⁴-I⁴³ peptides were prepared by the enzymatic digestion of Pe-CAP11 using endoproteinase Glu-C (with a substrate/enzyme ratio of 50:1) at room temperature for 6 h in 25 mM ammonium carbonate buffer (pH 7.8). The digested samples were fractionated and purified using RP-HPLC. Their molecular weights were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. The G¹-I⁴³ and Y³⁴-I⁴³ peptides were always freshly dissolved in 0.01% HCl and used immediately, to avoid autodimerization.

Cells. The murine macrophage cell line RAW 264.7 was obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Equitech-Bio, Kerreville, TX; the endotoxin concentration was <0.03 ng/ml) and penicillin (50 IU/ml)-streptomycin (50 µg/ml) (Sigma) at 37°C under 5% CO₂. Confluent RAW 264.7 cells were detached by washing them with 0.05% EDTA in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.4) and suspended in RPMI 1640 medium containing 10% FBS.

Assay for antibacterial activity. To determine the antibacterial activities of peptides, alamarBlue was used as a metabolic indicator. As a consequence of bacterial growth, the color of the oxidation-reduction indicator alamarBlue is changed from blue to pink. It has been confirmed that the classical colony formation assay and the alamarBlue assay using a redox indicator are comparable to evaluate bacterial viability; the results of the two methods significantly correlate, and the bacterial concentrations generated by the two assays show good agreement (4, 33, 37). In fact, we confirmed that CAP11 completely kills E. coli at 189 nM (1 µg/ml) but hardly affects bacterial growth at 18.9 nM (0.1 µg/ml) by the classical colony formation assay (24) and the alamarBlue assay (Fig. 2). Thus, we evaluated the antibacterial activities of CAP11 and its derived peptide by using alamarBlue as a metabolic indicator.

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FIG. 2. Effects of peptide dimerization on the biological activities of CAP11. (A) Antibacterial activity was assayed with the alamarBlue reagent using E. coli as a target. (B) LPS-neutralizing activity was assayed by inhibition of the binding of Alexa 488-LPS to RAW264.7 cells. Dimer and Pe-monomer represent a homodimeric (native) form of CAP11 and S-pyridylethylated CAP11, respectively. The data are the means ± standard deviations of four independent experiments.

As target organisms, Escherichia coli (NIHJ JC-2) and Staphylococcus aureus (NIHJ JC-1) were utilized. Both bacterial clones were supplied by Keiichi Hiramatsu (Department of Bacteriology, Juntendo University School of Medicine). The cells were cultured in Mueller-Hinton broth at 37°C for 14 h with shaking. The cells were centrifuged and washed twice with RPMI 1640 medium without phenol red and diluted in the same medium. Bacteria were incubated in the dark at 37°C for 4 to 6 h at the indicated concentrations (1 x 10⁷ CFU/ml, E. coli; 5 x 10⁶ CFU/ml, S. aureus) in RPMI 1640 medium containing 20 µl alamarBlue in the absence or presence of antibacterial peptides dissolved in 0.01% HCl in a total volume of 200 µl in a 96-well microplate. Aliquots containing all assay reagents except bacteria were used as blanks. After incubation, the absorbances at 550 and 590 nm were measured using a microplate reader (Model 680; Bio-Rad Laboratories, Inc., Hercules, CA) and expressed as bacterial growth. Fifty-percent effective concentrations (EC₅₀s) of antibacterial activities were determined as the concentrations of peptides that were required for 50% inhibition of maximum bacterial growth (absorbances at 550 and 590 nm) in the absence of antibacterial peptides. In preliminary experiments, a standard curve for each bacterial clone was obtained by performing an alamarBlue assay using serially diluted bacterial suspensions, and the optimal concentration of each bacterial species, described above, were determined for the quantifications.

Since all the peptides used in this study exhibited essentially the same antibacterial activities against E. coli and S. aureus, only the results using E. coli are presented.

Assay for binding of LPS to RAW 264.7 cells. The LPS-neutralizing activities of CAP11-derived peptides were assessed by the inhibition of LPS binding to CD14⁺ cells (RAW 264.7 cells), as previously described (22). RAW 264.7 cells (5 x 10⁵/ml) were incubated with Alexa 488-labeled LPS (50 ng/ml) in the absence or presence of antibacterial peptide (CAP11 or its partial peptide) in RPMI 1640 medium containing 10% FBS for 15 min at 37°C. After the cells were washed with phosphate-buffered saline, the binding of Alexa 488-labeled LPS was analyzed by flow cytometry (FACScan; Becton Dickinson, Rutherford, N.J.), and the mean fluorescence intensity was determined. LPS binding was expressed as a percentage of LPS binding, compared with control cells that were incubated with Alexa 488-labeled LPS in the absence of antibacterial peptide. EC₅₀s of LPS-neutralizing activities were determined as the concentrations of antibacterial peptides that were required for 50% inhibition of the maximum LPS binding in the absence of antibacterial peptides.

Helical-wheel prediction. The -helical wheel structures of CAP11-derived peptides were predicted by using a Genetyx-Win computer system (Software Development, Tokyo, Japan) and a Java applet (http://cti.itc.virginia.edu/cmg/Demo/wheel/wheelApp.html).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of the dimerization of CAP11 on its biological activities. Among the members of the cathelicidin family of antibacterial peptides, CAP11 has a unique homodimeric structure with a disulfide bridge. Thus, we looked at the involvement of dimerization in the biological actions of CAP11. We prepared both the dimer (native) form of CAP11 and the monomer form of Pe-CAP11 and compared their activities. As shown in Fig. 2A and B, the dimer and monomer forms of CAP11 exhibited essentially the same antibacterial and LPS-neutralizing activities: the EC₅₀s of antibacterial activities were 82 nM for the dimer and 101 nM for the monomer; the EC₅₀s of LPS-neutralizing activities were 53 nM for the dimer and 98 nM for the monomer. In addition, we confirmed that the antibacterial and LPS-neutralizing activities of freshly dissolved CAP11 peptide (monomer) were identical to those of Pe-CAP11 (data not shown).

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FIG. 1. Amino acid sequences of guinea pig CAP11 and its partial peptides. Native CAP11 forms a homodimeric structure with a disulfide bridge at the C41 residue. Pe-CAP11 represents a modified CAP11 with S-pyridylethylation (Pe) at C41.

Biologically active regions of CAP11. Next, we tried to identify the active regions of CAP11. Of note, CAP11 contains only one glutamic acid residue at position 33 (Fig. 1). Thus, we digested CAP11 with endoproteinase Glu-C and examined the activities of the digested peptides. The two peptides (G¹-E³³ and Y³⁴-I⁴³) were isolated by the enzyme digestion. The C-terminal peptide G³⁴-I⁴³ exhibited no antibacterial or LPS-neutralizing activities, even at 10,000 nM, whereas G¹-E³³ retained almost the same activities as G¹-I⁴³: the EC₅₀s of antibacterial activities were 109 nM for G¹-E³³ and 115 nM for G¹-I⁴³ (Fig. 3A); the EC₅₀s of LPS-neutralizing activities were 108 nM for G¹-E³³ and 98 nM for G¹-I⁴³ (Fig. 3B).

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FIG. 3. Biological activities of a truncated peptide (G1-E33) and the C-terminal region (Pe-Y34 to I43) of CAP11. (A) Antibacterial activity was assayed with the alamarBlue reagent using E. coli as a target. (B) LPS-neutralizing activity was assayed by inhibition of the binding of Alexa 488-LPS to RAW264.7 cells. The data represent the means ± standard deviations of three independent experiments.

Further, we evaluated the activities of smaller peptides (18-mers G¹-R¹⁸, T⁹-K²⁶, and L¹⁶-E³³) of G¹-E³³ with overlapping sequences. Although the antibacterial activities of the 18-mer peptides were lower than those of G¹-I⁴³, G¹-R¹⁸ exhibited the most potent antibacterial activity: the EC₅₀s were 2.8 µM for G¹-R¹⁸, 19.3 µM for L¹⁶-E³³, >100 µM for T⁹-K²⁶, and 0.11 µM for G¹-I⁴³ (Fig. 4A). Unexpectedly, the LPS-neutralizing activities of 18-mer peptides were almost completely lost; however, G¹-R¹⁸ was the most potent of the 18-mer peptides (Fig. 4B).

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FIG. 4. Biological activities of CAP11 and its 18-mer partial peptides. (A) Antibacterial activity was assayed with the alamarBlue reagent using E. coli as a target. (B) LPS-neutralizing activity was assayed by inhibition of the binding of Alexa 488-LPS to RAW264.7 cells. The data represent the means ± standard deviations of four independent experiments.

Top Abstract Introduction Materials and Methods Results Discussion References

 
For prevention of bacterial infections and their related symptoms (e.g., gram-negative-bacterial septic shock), much attention has been focused on the low-molecular-weight cationic antibacterial peptides that possess both antibacterial and LPS-neutralizing activities (2, 9, 11, 12, 14, 19, 22, 30, 36). The bacterial membrane contains abundant negatively charged phospholipids, such as phosphatidylglycerol and cardiolipin. Therefore, the cationic amphipathic antibacterial peptides possess high affinities for bacterial-membrane components and kill bacteria by permeabilization and/or disruption of the membrane (18, 21, 34). In contrast, the mammalian cell membrane is mainly composed of uncharged phospholipids, such as phosphatidylcholine and sphingomyelin, resulting in a lower affinity of antibacterial peptides for mammalian cells (21). Moreover, the existence of membrane-stabilizing cholesterol protects mammalian host cells from the toxicity of the antibacterial peptides (21).

Previously, we determined the biologically active region of hCAP18/LL-37, an -helical cathelicidin, and modified its region (K¹⁵ to V³²) to enhance the antibacterial activity by replacement of amino acid residues (25). In this study, we have characterized the active regions of CAP11, another member of the -helical cathelicidin peptides, which possess the antibacterial and LPS-neutralizing activities. CAP11 has a unique homodimeric structure of 43-amino-acid peptides with a disulfide bridge (Fig. 1). Most cathelicidin peptides have a monomeric structure, except CAP11 and PMAP36 (the 36-residue C-terminal region of pig myeloid antibacterial peptide), a recently characterized cathelicidin (31). Thus, we evaluated the effect of dimerization or the presence of a disulfide bond on antibacterial and LPS-neutralizing activity. As shown in Fig. 2, the dimeric (native) form of CAP11 exhibited almost the same antibacterial and LPS-neutralizing activities as the S-pyridylethylated monomer. Consistent with our findings, Scocchi et al. reported that the monomeric and dimeric forms of PMAP36 have the same antimicrobial activity against various microbes (31). These results suggest that the dimerization or disulfide bonding of the -helical cathelicidin peptides has no effect on their biological activities. In addition, it has been reported that disulfide bridges are not required for the antibacterial activities of human ß-defensin 3 (13).

To identify the active regions of CAP11, we digested a monomer of CAP11 at residue E³³ with endoproteinase Glu-C and evaluated the antibacterial and LPS-neutralizing activities of the two peptides (G¹-E³³ and Y³⁴-I⁴³). The truncated peptide G¹-E³³ showed antibacterial and LPS-neutralizing activities identical to those of a full-length peptide, G¹-I⁴³. In contrast, the C-terminal peptide Y³⁴-I⁴³ did not show any activities, even at 10 µM (Fig. 3). These data indicate that the C-terminal region of CAP11 (Y³⁴ to I⁴³) does not contribute to the biological activities of CAP11 (G¹-I⁴³).

To further investigate the active regions, we synthesized the three 18-mer peptides, G¹-R¹⁸, T⁹-K²⁶, and L¹⁶-E³³, derived from G¹-E³³. As shown in Fig. 4, both the antibacterial and LPS-neutralizing activities of the three 18-mer peptides were much lower than those of G¹-E³³. Of note, the LPS-neutralizing activities of the 18-mer peptides were almost abolished. In contrast, the antibacterial activities of the peptides were retained, and G¹-R¹⁸ exhibited the most potent antibacterial activity among three peptides. Thus, the active region with antibacterial activity is assumed to be localized at G¹ to R¹⁸ of CAP11. In contrast, G¹-R¹⁸ was not enough for the LPS-neutralizing activity, although it was the most potent of the 18-mer peptides. For LPS-neutralizing activity, longer sequences (such as G¹ to E³³), which form the amphipathic structure (balance), would be expected to be required (as described below). The deduced pIs of 18-mer peptides were almost the same as those of G¹-E³³ and G¹-I⁴³ (the pIs were 13.1 for G¹-R¹⁸, 12.9 for T⁹-K²⁶, 12.6 for L¹⁶-E³³, 12.9 for G¹-E³³, and 12.3 for G¹-I⁴³). These observations suggest that the antibacterial and LPS-neutralizing activities of CAP11 and its related peptides cannot be determined simply by the basic (cationic) features of the molecules. The cathelicidin family of antibacterial peptides with -helix structure, such as hCAP18/LL-37, have been shown to interact with the negatively charged phospholipids on the bacterial membrane via their positively charged surfaces to disrupt the bacterial membrane and kill bacteria (6, 8, 29). The G¹-R¹⁸ peptide, with the largest hydrophilic (positively charged) sector, demonstrated the most potent antibacterial activity among the three 18-mer peptides. Thus, the interaction of basic surfaces of the peptides with the bacterial membrane is likely to be important for the expression of bactericidal activity.

Hypothetical -helical wheel structures of G¹-E³³ and three 18-mer peptides are shown in Fig. 5. The helical-wheel regions of G¹-E³³ are clearly amphipathic and subtended by the hydrophilic (positively charged) and hydrophobic sectors. The 18-mer peptides also adopt an -helical amphipathic conformation; however, the hydrophobic sectors of G¹-R¹⁸ and T⁹-K²⁶ and the hydrophilic sector of L¹⁶-E³³ are relatively reduced compared with those of G¹-E³³. Interestingly, structure-activity relationship studies using different kinds of natural and synthetic model peptides have revealed that the potencies of the antibacterial activities of amphipathic -helical antimicrobial peptides can be influenced by the interrelated structural and physicochemical parameters, such as charge (cationicity), hydrophobicity, and amphipathicity. David also revealed, by using synthetic peptides, that the amphiphilic property, which is determined by cationic and hydrophobic structures, is necessary for the LPS-neutralizing activities of the peptides (3). Moreover, the presence of helix-stabilizing hydrophobic residues (e.g., Leu and/or Ala) has been shown to be important for the activities of antibacterial peptides (34). Thus, the decreased antibacterial and LPS-neutralizing activities of the 18-mer peptides seem to be due to the reduction of hydrophilic (positively charged) and hydrophobic sectors in their helical structures. Supporting this, we confirmed that a modified G¹-R¹⁸ peptide with increased hydrophobicity due to replacement of K⁵, T⁹, R¹⁰, R¹², and G¹⁷ by leucine exhibited augmented antibacterial and LPS-neutralizing activities (more than 100-fold) compared with those of a parent G¹-R¹⁸ peptide (data not shown).

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FIG. 5. Helical-wheel projections for 18-mer peptides and G1-E33 of CAP11. The sequences of CAP11-derived -helical peptides, G1-E33, G1-R18, T9-Ky, and L16-E33, are presented according to the Shiffer-Edmundson wheel projection analysis. Positively charged residues are in white circles, hydrophobic residues are in black circles, neutral hydrophilic residues are in gray circles, and negatively charged residues are boxed.

Recently, a human cathelicidin, LL-37, has been demonstrated to promote the processing and release of interleukin-1ß from monocytes and to suppress neutrophil apoptosis via the activation of the cell surface receptors FPRL1 (formyl-peptide receptor-like 1) and P2X₇ (a nucleotide receptor) (5). Thus, it is tempting to speculate that the guinea pig cathelicidin CAP11 also has some biological activities against host defense cells.

Human CAP18/LL-37 and guinea pig CAP11 peptides can exhibit antibacterial activities against gram-negative and gram-positive bacteria in the extracellular milieu under physiological conditions. Moreover, CAP18/LL-37 and CAP11 are able to neutralize the activities of LPS. Thus, CAP18/LL-37, CAP11, and their derivatives could be attractive candidates for adjunctive therapy of gram-negative bacterial sepsis. Based on the findings of this study, we are now preparing 18-mer peptides with augmented bactericidal and LPS-neutralizing activities by amino acid substitutions using G¹-R¹⁸ as a template.

 
This work was supported in part by grants from Grant-in-Aid for 21st Century COE research and the Institute for Environmental and Gender-Specific Medicine, Juntendo University School of Medicine.

We thank Tsutomu Fujimura (Division of Proteomics and BioMolecular Science) for synthesizing CAP11 and its partial peptides.

 
* Corresponding author. Mailing address: Department of Host Defense and Biochemical Research, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81-3-5802-1033. Fax: 81-3-3813-3157. E-mail: nagaokai{at}med.juntendo.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, August 2006, p. 2602-2607, Vol. 50, No. 8
0066-4804/06/$08.00+0     doi:10.1128/AAC.00331-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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