Evaluation of Antimicrobial and Lipopolysaccharide-Neutralizing Effects of a Synthetic CAP18 Fragment againstPseudomonas aeruginosain a Mouse Model

Abstract

CAP18 (cationic antimicrobial protein; 18 kDa) is a neutrophil-derived protein that can bind to and inhibit various activities of lipopolysaccharide (LPS). The 37 C-terminal amino acids of CAP18 make up the LPSbinding domain. A truncated 32-amino-acid C-terminal fragment of CAP18 had potent activity against Pseudomonas aeruginosa in vitro. We studied the antimicrobial and LPS-neutralizing effects of this synthetic truncated CAP18 peptide (CAP18 106-137 ) on lung injury in mice infected with cytotoxic P. aeruginosa. To determine its maximal effect, the CAP18 106-137 peptide was mixed with bacteria just prior to tracheal instillation, and lung injury was evaluated by determining the amount of leakage of an alveolar protein tracer ( 125 I-albumin) into the circulation and by the quantification of lung edema. The lung injury caused by the instillation of 5 ؋ 10 5 CFU of P. aeruginosa was significantly reduced by the concomitant instillation of CAP18 106-137 . However, the administration of CAP18 106-137 alone, without bacteria, induced lung edema, suggesting that it has some toxicity. Also, the peptide did not significantly reduce the number of bacteria that had been simultaneously instilled, nor did it significantly improve the survival of the infected mice. The addition of CAP18 106-137 to aztreonam along with the bacteria did decrease the level of antibiotic-induced release of inflammatory mediators including tumor necrosis factor alpha, interleukin-6, and nitric oxide and also improved the survival of the mice. Therefore, more investigations are needed to confirm the toxicities and the therapeutic benefits of CAP18 106-137 as an adjunctive therapy to antibiotics in the treatment of infections caused by gram-negative bacteria.

Structural features and biological activities of the cathelicidin-derived antimicrobial peptides

“…In contrast, the mortality rate is greatly improved when the peptide is instilled in association with aztreonam, an antibiotic that on its own increases lung injury and edema due to lipopolysaccharide release from killed gram-negative bacteria. 93 This result suggests that the peptide-induced improvement results from its ability to neutralize the released lipopolysaccharide.…”

“…92 The LPS-neutralizing effect of the CAP18 106 -137 fragment from rabbit has also been studied on lung injury in mice infected with cytotoxic P. aeruginosa. 93 Although the injury caused by the instillation of bacteria is significantly reduced by the concomitant instillation of the peptide, this does not significantly reduce the number of bacteria, nor does it improve the survival of infected mice. In contrast, the mortality rate is greatly improved when the peptide is instilled in association with aztreonam, an antibiotic that on its own increases lung injury and edema due to lipopolysaccharide release from killed gram-negative bacteria.…”

Augmentation of the bactericidal activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by amino acid substitutions

“…In this context, it has been demonstrated that high concentrations of cationic antimicrobial peptides are occasionally toxic to the host cells [2,3,37,38], and that the cytotoxicity is correlated with the extent of the hydrophobic regions in the peptides [19]. Actually, 18-mer LLKKK with the increased hydrophobicity was toxic to mammalian cells such as human mononuclear cells and mouse RAW264.7 cells, and the viabilities were markedly reduced after incubation with 8.8 mM (20 mg/ml) 18-mer LLKKK; however, it exerted strong antimicrobial actions at < 0.43 mM (1 mg/ml) against S. aureus, S. pneumoniae, S. pyogenes and E. coli, and at < 2.2 mM (5 mg/ml) against P. aeruginosa without substantial cytotoxic effect on these cells.…”

“…Mueller Hinton broth and tryptic soy broth were purchased from Difco Laboratories (Detroit, MI); heart infusion broth from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan); RPMI medium 1640 with 2.05 mM L-glutamine with or without phenol red from Gibco' Invitrogen Corporation (Grand Island, NY); alamarBlue' from BioSource International, Inc. (Camarillo, CA); 7-(thienyl-2-acetamido)-3-[2-(4-N,N- 1 LGDF-FRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES 37 ) and its 18-mer derivatives (18-mer K 15 -V 32 , 18-mer LL and 18-mer LLKKK) were synthesized by the solid phase method on a peptide synthesizer (model PSSM-8, Shimadzu, Kyoto, Japan) by fluorenylmethoxycarbonyl chemistry as described before [15]. The peptides were eluted from the resin, and purified to homogeneity by reversed phase-HPLC on a Cosmosil 5C18 column (Nacalai Tesque, Kyoto, Japan), using a 0-70 % acetonitrile gradient in 0.1% trifluoroacetic acid.…”

“…In contrast, cathelicidins are characterized by the highly conserved cathelin-like pro-sequences and variable carboxyl-terminal sequences that correspond to the mature antibacterial peptides [6][7][8][9]. About 30 cathelicidin members have been identified from various mammalian species; however, only one cathelicidin hCAP18 (human cationic antibacterial protein of 18 kDa) has been found in humans, and its carboxylterminal antibacterial peptide called LL-37 comprising 37 amino acid residues (L 1 LGDFFRKSKEKIGKEFKRIVQR-IKDFLRNLVPRTES 37 ) has been recently identified [6][7][8][9].…”