Dissection of the antimicrobial and hemolytic activity of Cap18: Generation of Cap18 derivatives with enhanced specificity

<div><p>Due to the rapid emergence of resistance to classical antibiotics, novel antimicrobial compounds are needed. It is desirable to selectively kill pathogenic bacteria without targeting other beneficial bacteria in order to prevent the negative clinical consequences caused by many broad-spectrum antibiotics as well as reducing the development of antibiotic resistance. Antimicrobial peptides (AMPs) represent an alternative to classical antibiotics and it has been previously demonstrated that Cap18 has high antimicrobial activity against a broad range of bacterial species. In this study we report the design of a positional scanning library consisting of 696 Cap18 derivatives and the subsequent screening for antimicrobial activity against <i>Y</i>. <i>ruckeri</i>, <i>A</i>. <i>salmonicida</i>, <i>S</i>. Typhimurium and <i>L</i>. <i>lactis</i> as well as for hemolytic activity measuring the hemoglobin release of horse erythrocytes. We show that the hydrophobic face of Cap18, in particular I13, L17 and I24, is essential for its antimicrobial activity against <i>S</i>. Typhimurium, <i>Y</i>. <i>ruckeri</i>, <i>A</i>. <i>salmonicida</i>, <i>E</i>. <i>coli</i>, <i>P</i>. <i>aeruginosa</i>, <i>L</i>. <i>lactis</i>, <i>L</i>. <i>monocytogenes</i> and <i>E</i>. <i>faecalis</i>. In particular, Cap18 derivatives harboring a I13D, L17D, L17P, I24D or I24N substitution lost their antimicrobial activity against any of the tested bacterial strains. In addition, we were able to generate species-specific Cap18 derivatives by particular amino acid substitutions either in the hydrophobic face at positions L6, L17, I20, and I27, or in the hydrophilic face at positions K16 and K18. Finally, our data showed the proline residue at position 29 to be essential for the inherent low hemolytic activity of Cap18 and that substitution of the residues K16, K23, or G21 by any hydrophobic residues enhances the hemolytic activity. This study demonstrates the potential of generating species-specific AMPs for the selective elimination of bacterial pathogens.</p></div

Multilingual signs for Latin American residents in a local town in Japan : Findings based on fieldwork in Minokamo city

Analysis of a Selected Set of Antimicrobial Peptides The rapid emergence of resistance to classical antibiotics has increased the interest in novel antimicrobial compounds. Antimicrobial peptides (AMPs) represent an attractive alter-native to classical antibiotics and a number of different studies have reported antimicrobial activity data of various AMPs, but there is only limited comparative data available. The mode of action for many AMPs is largely unknown even though several models have sug-gested that the lipopolysaccharides (LPS) play a crucial role in the attraction and attach-ment of the AMP to the bacterial membrane in Gram-negative bacteria. We compared th

Cap18 derivatives identified in the screening with lost antimicrobial activity or changed species-specificity.

<p>Cap18 derivatives identified in the screening with lost antimicrobial activity or changed species-specificity.</p

Complete Substitution Analysis of Cap18 measuring the antimicrobial activity against <i>Aeromonas salmonicida</i>.

<p>The original Cap18 sequence (GLRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY) and amino acid assignments are presented in the first two rows. The second column identifies the amino acid substitution at each position (A-Y). Each box in the matrix represents a Cap18 peptide harboring one single amino acid substitution compared to the original Cap18 sequence. For example, the amino acid sequence of the peptide in column 1/row 1 is </p><p><u>A</u>LRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY</p>, the sequence of the peptide in column 1/row 2 is <p><u>C</u>LRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY</p>, the sequence of peptide in column 2/row 1 is <p>G<u>A</u>RKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY</p> etc.; the values within each box represent a MIC value (μg/ml) against <i>Aeromonas salmonicida</i>. Grey boxes represent the original Cap18 sequence. NS = no MIC value determination possible due to the insolubility of the peptide in DMSO.<p></p

Complete Substitution Analysis of Cap18 measuring the antimicrobial activity against <i>Lactococcus lactis</i>.

<p>The original Cap sequence is (GLRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY) and amino acid assignments are presented in the first two rows. The second column identifies the amino acid substitution at each position (A-Y). Each box in the matrix represents a Cap18 peptide harboring one single amino acid substitution compared to the original Cap18 sequence. For example, the amino acid sequence of the peptide in column 1/row 1 is </p><p><u>A</u>LRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY</p>, the sequence of the peptide in column 1/row 2 is <p><u>C</u>LRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY</p>, the sequence of peptide in column 2/row 1 is <p>G<u>A</u>RKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY</p> etc.; the values within each box represent a MIC value (μg/ml) against <i>Lactococcus lactis</i>. Grey boxes represent the original Cap18 sequence. NS = no MIC value determination possible due to the insolubility of the peptide in DMSO.<p></p

Antimicrobial activity of Cap18 and selected antibiotics.

<p>Antimicrobial activity of Cap18 and selected antibiotics.</p

Complete Substitution Analysis of Cap18 measuring the antimicrobial activity against <i>Yersinia ruckeri</i>.

<p>The original Cap18 sequence (GLRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY) and amino acid assignments are presented in the first two rows. The second column identifies the amino acid substitution at each position (A-Y). Each box in the matrix represents a Cap18 peptide harboring one single amino acid substitution compared to the original Cap18 sequence. For example, the amino acid sequence of the peptide in column 1/row 1 is </p><p><u>A</u>LRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY</p>, the sequence of the peptide in column 1/row 2 is <p><u>C</u>LRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY</p>, the sequence of peptide in column 2/row 1 is <p>G<u>A</u>RKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY</p> etc.; the values within each box represent a MIC value (μg/ml) against <i>Yersinia ruckeri</i>. Grey boxes represent the original Cap18 sequence. NS = no MIC value determination possible due to the insolubility of the peptide in DMSO.<p></p