Investigation into the antimicrobial action and mechanism of a novel endogenous peptide β-casein 197 from human milk

Abstract

A novel endogenous peptide cleaved from 197-213 AA of β-casein, named β-casein 197, was identified by tandem mass spectrometry. β-casein 197 constituted a significant proportion of the peptide content in preterm milk. This study investigated the antibacterial effects and mechanisms against common pathogenic bacteria. Six bacterial strains were selected for this study: Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, Listeria monocytogenes, Klebsiella pneumonia and Bacillus subtilis. After synthesis, serial twofold dilutions of β-casein 197 were added to select for sensitive bacteria. The disk diffusion method and analysis of bacterial staining were used to identify antibacterial effect, while DNA-binding, scanning electron microscopy and transmission electron microscopy were used to explore antimicrobial mechanisms. Disk diffusion showed that E. coli, S. aureus and Y. enterocolitica were sensitive to the β-casein 197. In addition, live/dead fluorescent staining also confirmed antibacterial effects. Scanning electron and transmission electron microscopy revealed affected extracellular and intracellular structure for three species of bacteria, while a DNA-binding assay showed that the antimicrobial activity did not occur through DNA binding. This study suggests that β-casein 197 has antimicrobial activity against common pathogenic bacteria in newborns with infection. The peptide induced membrane permeabilization but did not bind to genomic DNA. Based on our findings, β-casein 197 has potential clinical value for preventing infections of premature infants.

Keywords: Antibacterial effect; Common pathogenic bacteria; Human milk; β-Casein 197.

Fig. 1

Characteristics of β-casein 197. a β-Casein 197 is a 17-amino-acid antimicrobial peptide that corresponds to amino acids 197–213 of β-casein. b The biochemical information of β-casein 197. c The helical wheel plot of β-casein 197. The output presents the hydrophilic residues as circles, hydrophobic residues as diamonds, potentially negatively charged residues as triangles, and potentially positively charged residues as pentagons. The most hydrophobic residue is green, and the amount of green decreases proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are coded red with pure red being the most hydrophilic residue, and the amount of red decreasing proportionally to the hydrophilicity

Fig. 2

Antimicrobial activity of β-casein 197. a After incubation at 37 °C for 24 h, bacterial viability of E. coli, S. aureus, Y. enterocolitica, L. monocytogenes, K. pneumonia and B. subtilis after treatment with different concentrations of β-casein 197. b Inhibition zone diameters of E. coli, S. aureus and Y. enterocolitica. NC native control

Fig. 3

Microscopic assessment of E. coli, S. aureus and Y. enterocolitica, after treatment with β-casein 197. Fluorescence microscopy pictures of E. coli, S. aureus and Y. enterocolitica, control (left two column figures) and β-casein 197 treated (right two column figures). Green cells intact cells; red cells cells with a damaged membrane. PI propidium iodide, NC native control

Fig. 4

Morphology of E. coli, S. aureus and Y. enterocolitica cells were observed with scanning electron microscopy (SEM). A–C Control E. coli, S. aureus and Y. enterocolitica SEM images; a–c β-casein 197 treated samples. NC native control. Arrows indicate damage points

Fig. 5

Morphosis of E. coli, S. aureus and Y. enterocolitica cells were observed under transmission electron microscopy (TEM). A–C Normal E. coli, S. aureus and Y. enterocolitica TEM images; a–c β-casein 197 treated samples. NC native control. Arrows indicate damage points

Fig. 6

The DNA-binding properties of β-Casein 197. a ProtParam analysis of predicted binding residues of β-casein 197, predicted binding residues was labeled with a red “+”. b Gel retardation assays. Binding was assayed by the migration of pDNA. Various concentrations of peptides were incubated with 5 µL of 25 µg mL⁻¹ pBR322 vector from E. coli at 37 °C for 1 h, and then the reaction mixtures were applied to 0.7% agarose gel electrophoresis. Lane M DNA marker DL 10,000; Lane 1 5 µL pDNA as control; Lanes 2–6 mixture of pDNA and various concentrations of peptides (12.5, 25, 50, 100 and 500 µg mL⁻¹)