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

Abstract

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 Y. ruckeri, A. salmonicida, S. Typhimurium and L. lactis 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 S. Typhimurium, Y. ruckeri, A. salmonicida, E. coli, P. aeruginosa, L. lactis, L. monocytogenes and E. faecalis. 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.

The Role of Outer Membrane Proteins and Lipopolysaccharides for the Sensitivity of Escherichia coli to Antimicrobial Peptides

“…Introducing the negatively charged residue aspartic acid either at position I13 or L17 destroys the antimicrobial activity of Cap18 against wild-type and LPS mutants regardless of the inner core-OS structure. This suggests that the changes of Cap18 provoked by the introduction of a negative charged residue at a central hydrophobic position have a dramatic effect on the antimicrobial activity which is in agreement with our previous study showing that Cap18 I13D and L17D lost activity against any of the tested organisms, regardless if Gram-positive or Gram-negative (Ebbensgaard et al, 2018 ). Similarly, the introduction of a proline residue at position L17 will lead to a complete loss of antimicrobial activity.…”

“…In a previous study, we dissected the antimicrobial activity of Cap18 and were able to generate Cap18 derivatives with enhanced species-selective killing (Ebbensgaard et al, 2018 ). Together with our current findings demonstrating that the antimicrobial activity of Cap18 is not or only marginal affected by the presence of the inner core region or by the sugar composition of the outer region of the core-OS, we decided to investigate the mechanism of Cap18 and the role of LPS in more detail.…”

Expeditious synthesis and preliminary antimicrobial activity of deflazacort and its precursors

“…Cytotoxicity was determined spectrophotometrically by measuring the haemoglobin release from horse erythrocytes. 21 Briey, fresh debrinated horse blood (Oxoid) was centrifuged at 500 Â g for 5 min and then washed three times with PBS, pH 7.4. Red blood cells were diluted to 4% in PBS and 190 mL were added to 10 mL of 10 (ranging from 512 mg mL À1 to 1 mg mL À1 ) in a 96-well plate.…”

Correlation between hemolytic activity, cytotoxicity and systemic in vivo toxicity of synthetic antimicrobial peptides

“…Being AMPs and generally positively charged, their cellular selectivity is due to the high affinity to the negatively-charged lipid components of the bacterial membrane 10,11 . The outer layer of the cytoplasmic membrane of mammalian cells is, on the other hand, neutral and consists of mainly zwitterionic phospholipids and cholesterol, the latter has been suggested to further stabilise the membranes to the action of AMPs 3,13,14 . The initial interaction generally occurs through a complex interplay of phenomena including electrostatic and hydrophobic interaction with the bacterial membrane 9 , self-assembly and conformational transitions and induces cell death at sufficiently high concentrations 10 .…”