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ABSTRACT: A truncated version of host defense peptide chicken cathelicidin-2, C1-15, possesses potent, broad spectrum antibacterial activity. A variant of this peptide, F(2,5,12)W, which contains 3 phenylalanine to tryptophan substitutions, possesses improved antibacterial activity and lipopolysaccharide (LPS) neutralizing activity compared to C1-15. In order to improve the proteolytic resistance of both peptides we engineered novel chicken cathelicidin-2 analogs by substitution of l- with D-amino acids and head-to-tail cyclization. Both cyclic and D-amino acid variants showed enhanced stability in human serum compared to C1-15 and F(2,5,12)W. The D-amino acid variants were fully resistant to proteolysis by trypsin and bacterial proteases. Head-to-tail cyclization of peptide F(2,5,12)W resulted in a 3.5-fold lower cytotoxicity toward peripheral blood mononuclear cells. In general, these modifications did not influence antibacterial and LPS neutralization activities. It is concluded that for the development of novel therapeutic compounds based on chicken cathelicidin-2 D-amino acid substitutions and cyclization must be considered. These modifications increase the stability and lower cytotoxicity of the peptides without altering their antimicrobial potency.
Peptides 03/2011; 32(5):875-80. · 2.43 Impact Factor
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ABSTRACT: Staphylococcus epidermidis is a major cause of nosocomial infections owing to its ability to form biofilms on the surface of medical devices. Biofilms are surface-adhered bacterial communities. In mature biofilms these communities are encased in an extracellular matrix composed of bacterial polysaccharides, proteins and DNA. The antibiotic resistance of bacteria present in biofilms can be up to 1000-fold higher compared with the planktonic phenotype. Host defence peptides (HDPs) are considered to be excellent candidates for the development of novel antibiotics. Recently, we demonstrated that a short variant of the HDP chicken cathelicidin-2, peptide F(2,5,12)W, has potent antibacterial and lipopolysaccharide-neutralising activities. This study reports on the antibiofilm activity of peptide F(2,5,12)W against two strains of S. epidermidis, including a multiresistant strain. Peptide F(2,5,12)W potently inhibited the formation of bacterial biofilms in vitro at a low concentration of 2.5 μM, which is below the concentration required to kill or inhibit growth (minimal inhibitory concentration=10 μM). Moreover, peptide F(2,5,12)W also impaired existing S. epidermidis biofilms. A 4-h challenge of pre-grown biofilms with 40 μM F(2,5,12)W reduced the metabolic activity of the wild-type strain biofilm completely and reduced that of the multiresistant strain biofilm by >50%. It is concluded that F(2,5,12)W prevents biofilm formation and impairs mature S. epidermidis biofilms.
International journal of antimicrobial agents 03/2011; 37(5):476-9. · 3.03 Impact Factor
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ABSTRACT: Host defence peptides (HDPs) are considered to be excellent candidates for the development of novel therapeutic agents. Recently, it was demonstrated that the peptide C1-15, an N-terminal segment of chicken HDP cathelicidin-2, exhibits potent antibacterial activity while lacking cytotoxicity towards eukaryotic cells. In the present study, we report that C1-15 is active against bacteria such as Bacillus anthracis and Yersinia pestis that may potentially be used by bioterrorists. Substitution of single and multiple phenylalanine (Phe) residues to tryptophan (Trp) in C1-15 resulted in variants with improved antibacterial activity against B. anthracis and Y. pestis as well as decreased salt sensitivity. In addition, these peptides exhibited enhanced neutralisation of lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs). The antibacterial and LPS-neutralising activities of these C1-15-derived peptides are exerted at concentrations far below the concentrations that are toxic to human PBMCs. Taken together, we show that Phe-->Trp substitutions in C1-15 variants enhances the antibacterial and LPS-neutralising activities against pathogenic bacteria, including those that may potentially be used as biological warfare agents.
International journal of antimicrobial agents 09/2010; 36(3):271-4. · 3.03 Impact Factor
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ABSTRACT: Chicken host defense peptide cathelicidin-2 (CATH-2) is known to exert antimicrobial and immunomodulatory activities and consists of two alpha-helices connected by a hinge region. Here we report the biological properties of the separate alpha-helical segments and the importance of the proline residue in the hinge region. Substitution of proline-14 in the CATH-2 hinge region by leucine, but not by glycine, strongly reduced antibacterial and hemolytic activity. Furthermore, substitution by leucine strongly reduced the neutralization of LPS-induced cytokine production and peptide-induced monocyte chemotactic protein-1 (MCP-1) production by human peripheral blood mononuclear cells (PBMCs). This indicates that the hinge region is important for rapid penetration of the bacterial membrane as well as indirect and direct immunomodulatory activities. The highly cationic and amphipathic N-terminal segment (C1-15) exhibited very potent antibacterial activity and fast killing kinetics, while displaying low cytotoxicity towards chicken erythrocytes and PBMCs. The N-terminal and, to a lesser extent, the C-terminal helical regions potently neutralized LPS-induced release of TNFalpha, IL-6 and IL-10 by PBMCs, while IL-8 production was only moderately affected. These results indicate that core elements within mature CATH-2 can be identified that are linked to antibacterial and/or immunomodulatory activities. Further studies may lead to the development of peptide antibiotics with specific properties that can be used for prophylactic and/or therapeutic applications.
Molecular Immunology 07/2009; 46(13):2465-73. · 2.90 Impact Factor
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ABSTRACT: Cathelicidins are effector molecules of the innate host defense system that establish an antimicrobial barrier at epithelial interfaces. The human cathelicidin LL-37, in addition to its antimicrobial activity, also exhibits immunomodulatory effects, such as inhibition of pro-inflammatory responses to bacterial LPS in human monocytic cells. In this report, we demonstrate that LL-37 almost completely prevents the pro-inflammatory cytokine release by human peripheral blood mononuclear cells (PBMCs) following stimulation with Toll-like receptor (TLR)4 and TLR2/1 agonists while leaving TLR2/6, TLR5, TLR7 and TLR8 responses unchanged. Modulation of the TLR response by LL-37 occurred at least partly through the MAP kinase pathway via inhibition of p38 phosphorylation. By using an LL-37 library with overlapping sequences, we identified the mid-region of LL-37, comprising amino acids 13-31, as the active domain for the modulation of TLR responses. The mechanism of immunomodulation of LL-37 and LL-37 fragments is lipopoly-saccharide binding. Correlations between the capacity of LL-37 fragments to modulate TLR responses and their physico-chemical properties revealed that cationicity and hydrophobicity are essential for the modulation of LL-37-mediated TLR responses.
Biological Chemistry 02/2009; 390(4):295-303. · 2.96 Impact Factor
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ABSTRACT: Chicken host defense peptide cathelicidin-2 (CATH-2) is known to exert antimicrobial and immunomodulatory activities and consists of two α-helices connected by a hinge region. Here we report the biological properties of the separate α-helical segments and the importance of the proline residue in the hinge region. Substitution of proline-14 in the CATH-2 hinge region by leucine, but not by glycine, strongly reduced antibacterial and hemolytic activity. Furthermore, substitution by leucine strongly reduced the neutralization of LPS-induced cytokine production and peptide-induced monocyte chemotactic protein-1 (MCP-1) production by human peripheral blood mononuclear cells (PBMCs). This indicates that the hinge region is important for rapid penetration of the bacterial membrane as well as indirect and direct immunomodulatory activities. The highly cationic and amphipathic N-terminal segment (C1-15) exhibited very potent antibacterial activity and fast killing kinetics, while displaying low cytotoxicity towards chicken erythrocytes and PBMCs. The N-terminal and, to a lesser extent, the C-terminal helical regions potently neutralized LPS-induced release of TNFα, IL-6 and IL-10 by PBMCs, while IL-8 production was only moderately affected. These results indicate that core elements within mature CATH-2 can be identified that are linked to antibacterial and/or immunomodulatory activities. Further studies may lead to the development of peptide antibiotics with specific properties that can be used for prophylactic and/or therapeutic applications.
Molecular Immunology.
