Killing of Staphylococcus aureus and Salmonella enteritidis and neutralization of lipopolysaccharide by 17-residue bovine lactoferricins: improved activity of Trp/Ala-containing molecules

Ya Hao,Na Yang,Da Teng,Jianhua Wang,Ruoyu Mao,Xiumin Wang,Zhanzhan Li,Xiao Wang

doi:10.1038/srep44278

Abstract

Bovine lactoferricin (LfcinB) has potent antibacterial, antifungal and antiparasitic activities but is also hemolytic. Our objective was to identify LfcinB17-31 derivatives with reduced hemolysis and improved antimicrobial activity via substituting Cys3, Arg4, Gln7, Met10, and Gly14 with more hydrophobic residues. Two peptides, Lfcin4 and Lfcin5, showed higher activity against Staphylococcus aureus and Salmonella enteritidis and lower hemolytic activity than the parent peptide LfcinB17-31. These peptides permeabilized the outer and inner membranes of S. enteritidis; however, Lfcin5 did not permeabilize the inner membrane of S. aureus. Gel retardation and circular dichroism spectra showed that Lfcin4 and Lfcin5 bound to bacterial genomic DNA. Lfcin4 inhibited DNA, RNA and protein synthesis. Both peptides induced the peeling of membranes and the lysis of S. enteritidis. At doses of 10 and 15 mg/kg, Lfcin4 and Lfcin5 reduced the bacterial counts in infected thigh muscles by 0.03‒0.10 and 0.05‒0.63 log10 CFU/g of tissue, respectively, within 10 h. Lfcin4 and Lfcin5 enhanced the survival rate of endotoxemic mice; reduced serum IL-6, IL-1β and TNF-α levels; and protected mice from lipopolysaccharide-induced lung injury. These data suggest that Lfcin4 and Lfcin5 may be antimicrobial and anti-endotoxin peptides that could serve as the basis for the development of dual-function agents.

Highlights

Immunological activities[8,9,10,11,12,13,14,15,16,17]
The Boman index, which is a measure of peptide affinity to proteins and the ability to establish bio-interactions, of LfcinB17-31 derivatives was in the range of 1.45‒3.02 kcal/mol
We designed five 17-residue LfcinB17-31 derivatives (Trp-rich and Ala-rich) and first studied their antibacterial activities; Lfcin[4] and Lfcin[5] were found to be the most active peptides among those tested against both S. aureus and S. enteritidis, displaying more than 2- to 8-fold increases in antibacterial activity over the parent peptide, Lfcin[1] (LfcinB17-31) (Table 2)

Summary

Introduction

Immunological activities[8,9,10,11,12,13,14,15,16,17]. A shorter version of LfcinB containing amino acids 17 to 31 (LfcinB17–31) was found to have slightly less antimicrobial activity against S. aureus and Escherichia coli than LfcinB17-4118. Ho et al identified the intracellular targets of LfcinB using E. coli K12 proteome microarrays and found that LfcinB binds to two response regulators, BasR and CreB, members of the two-component system family They further showed that LfcinB inhibited the growth of bacteria by directly influencing the phosphorylation of a two-component system protein[26]. On the basis of previous reports wherein some residues of Phe[1], Lys[2], Trp[6], Trp[8], Arg[9], Lys[11], Lys[12], and Ala[15] are found to be essential for antibacterial activity[18,19,20,22,27], we performed a systematic analysis of residues in the 3rd, 4th, 7th, 10th, and 14th positions of LfcinB17–31 by substituting the hydrophobic amino acids (Ala, Phe and Trp) at each position. The in vivo antibacterial activities of LfcinB derivatives were examined in a mouse thigh model of S. aureus infection, as well as their endotoxin-neutralizing activities in C57BL/6 mice challenged with LPS

Objectives

Methods

Results

Conclusion