LHH1, a novel antimicrobial peptide with anti-cancer cell activity identified from Lactobacillus casei HZ1

Abstract

Antimicrobial peptides have been attracting increasing attention for their multiple beneficial effects. In present study, a novel AMP with a molecular weight of 1875.5 Da, was identified from the genome of Lactobacillus casei HZ1. The peptide, which was named as LHH1 was comprised of 16 amino acid residues, and its α-helix content was 95.34% when dissolved in 30 mM SDS. LHH1 exhibited a broad range of antimicrobial activities against Gram-positive bacteria and fungus. It could effectively inhibit Staphylococcus aureus with a minimum inhibitory concentration of 3.5 μM and showed a low hemolytic activity. The scanning electron microscope, confocal laser scanning microscope and flow cytometry results showed that LHH1 exerted its antibacterial activity by damaging the cell membrane of Staphylococcus aureus. Meanwhile, LHH1 also exhibited anti-cancer cell activities against several cancer cells via breaking the cell membrane of MGC803, HCT116 and C666-1 cancer cells.

Keywords: Anti-cancer; Antimicrobial peptide; Lactobacillus casei; Pathogenic bacteria; Staphylococcus aureus.

Fig. 1

Inhibition zones of the four AMPs against several pathogenic bacteria

Fig. 2

Hemolytic activity of LHH1. The goat red blood cell was treated by a serial concentration of LHH1 or Melittin from 1 µM to 512 µM

Fig. 3

Release of calcein from liposomes. The concentration of LHH1 was from 1 μM to 128 μM

Fig. 4

CD spectrum of LHH1. LHH1 was dissolved in water, 10 mM PBS (pH 7.4), 30 mM SDS, 25% TFE, 50% TFE and 500 μM POPG:CL (58:42) to reach a final concentration of 100 μM, respectively. Three scans were averaged for each sample

Fig. 5

CLSM observation of S. aureus treated with LHH1. S. aureus was co-incubated with 10 μM FITC and 0, 5 μM or 10 μM FITC-labeled LHH1 for 1 h, and was observed with CLSM respectively

Fig. 6

Morphology of S. aureus treated with different concentrations of LHH1. a S. aureus was not treated with LHH1; b S. aureus treated with LHH1 in 0.5 × MIC; c S. aureus treated with LHH1 in 0.75 × MIC; d S. aureus treated with LHH1 in 1.0 × MIC

Fig. 7

Flow cytometric analysis of the membrane integrity of S. aureus. S. aureus was treated with LHH1 at different concentrations at 4 °C for 30 min. a S. aureus of control group was treated with 10 mM PBS; b S. aureus was treated with LHH1 of 0.5 × MIC; c S. aureus was treated with LHH1 of 1.0 × MIC; d S. aureus was treated with LHH1 of 2.0 × MIC

Fig. 8

Effect of AMPs on cell viability of several cancer cells. MGC803 cells (a), HCT116 cells (b) and C666-1 cells (c) were treated with different concentrations of LHH1, respectively. Melittin was uused as a positive control

Fig. 9

Toxicity of LHH1 to RAW264.7 cells. The human macrophage cell RAW264.7 was treated with a serial concentration of LHH1 or melittin

Fig. 10

CLSM analysis of membrane destroying effects of LHH1 on cancer cells. The cancer cells were treated with FITC-labeled LHH1 or not and cell nucleus were stained with DAPI

Fig. 11

Flow cytometry analysis of apoptosis-promoting effect of LHH1 on cancer cells. The apoptosis rate of the three cancer cells treated with different concentrations of LHH1 (0 μM, 5 μM, 10 μM and 20 μM) was detected with flow cytometry using Annexin V-FITC/PI staining

Fig. 12

The sequence similarity alignment. The red ones are hydrophobic amino acids; The green ones are hydrophilic amino acid; * indicates highest similarity