Potent intracellular antibacterial activity of a marine peptide-N6NH2 and its D-enantiomer against multidrug-resistant Aeromonas veronii.

Aeromonas veronii can cause a variety of diseases such as sepsis in humans and animals. However, there has been no effective way to eradicate A. veronii. In this study, the intracellular antibacterial activities of the C-terminal aminated marine peptide N6 (N6NH2) and its D-enantiomer (DN6NH2) against A. veronii were investigated in macrophages and in mice, respectively. The result showed that DN6NH2 with the minimum inhibitory concentration (MIC) of 1.62 μM is more resistant to cathepsin B than N6NH2 (3.23 μM). The penetration percentages of the cells treated with 4-200 μg/mL fluorescein isothiocyanate (FITC)-DN6NH2 were 52.5-99.6%, higher than those of FITC-N6NH2 (27.0-99.1%). Both N6NH2 and DN6NH2 entered macrophages by macropinocytosis and an energy-dependent manner. DN6NH2 reduced intracellular A. veronii by 34.57%, superior to N6NH2 (19.52%). After treatment with 100 μg/mL DN6NH2, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β were reduced by 53.45%, 58.54%, and 44.62%, respectively, lower than those of N6NH2 (15.65%, 12.88%, and 14.10%, respectively); DN6NH2 increased the IL-10 level (42.94%), higher than N6NH2 (7.67%). In the mice peritonitis model, 5 μmol/kg DN6NH2 reduced intracellular A. veronii colonization by 73.22%, which was superior to N6NH2 (32.45%) or ciprofloxacin (45.67%). This suggests that DN6NH2 may be used as the candidate for treating intracellular multidrug-resistant (MDR) A. veronii. KEY POINTS: • DN6NH2 improved intracellular antibacterial activity against MDR A. veronii. • DN6NH2 entered macrophages by micropinocytosis and enhanced the internalization rates. • DN6NH2 effectively protected the mice from infection with A. veronii.