Citrullination Alters the Antibacterial and Anti-Inflammatory Functions of the Host Defense Peptide Canine Cathelicidin K9CATH In Vitro

Abstract

K9CATH is the sole cathelicidin in canines (dogs) and exhibits broad antimicrobial activity against both Gram-positive and Gram-negative bacteria. K9CATH also modulates inflammatory responses and binds to LPS. These activities depend on the secondary structure and a net-positive charge of the peptide. Peptidylarginine deiminases (PAD) convert cationic peptidyl arginine to neutral citrulline. Thus, we hypothesized that citrullination is a biologically relevant modification of the peptide that would reduce the antibacterial and LPS-binding activities of K9CATH. Recombinant PAD2 and PAD4 citrullinated K9CATH to various extents and circular dichroism spectroscopy revealed that both native and citrullinated K9CATH exhibited similar a-helical secondary structures. Notably, citrullination of K9CATH reduced its bactericidal activity, abolished its ability to permeabilize the membrane of Gram-negative bacteria and reduced the hemolytic capacity. Electron microscopy showed that citrullinated K9CATH did not cause any morphological changes of Gram-negative bacteria, whereas the native peptide caused clear alterations of membrane integrity, concordant with a rapid bactericidal effect. Finally, citrullination of K9CATH impaired its capacity to inhibit LPS-mediated release of proinflammatory molecules from mouse and canine macrophages. In conclusion, citrullination attenuates the antibacterial and the LPS-binding properties of K9CATH, demonstrating the importance of a net positive charge for antibacterial lysis of bacteria and LPS-binding effects and suggests that citrullination is a means to regulate cathelicidin activities.

Antimicrobial peptide MPX alleviates the lethal attack of Escherichia coli in mice

“…Their small molecular weight, good water solubility, and resistance to resistance are considered the best alternative to antibiotics and have become a research hotspot in recent years (Santos et al, 2021). Antimicrobial peptides have various biological functions such as antibacterial, antivirus, anti-parasitic, anti-inflammatory, anti-cancer, improving animal performance and immunity (Xie et al, 2020;Al Adwani et al, 2021;Piyadasa et al, 2021;Gong et al, 2021). MPX was extracted from wasp venom consisting of 14 amino acids and had four positive charges, which had good bactericidal activity against both Gram-positive and Gram-negative bacteria (Zhao et al, 2021).…”