Methicillin-resistant Staphylococcus aureus (MRSA), are the most frequent cause of sepsis, which urgently demanding new drugs for treating infection. Two homologous insect CSαβ peptides-DLP2 and DLP4 from Hermetia illucens were firstly expressed in Pichia pastoris, with the yields of 873.5 and 801.3 mg/l, respectively. DLP2 and DLP4 displayed potent antimicrobial activity against Gram-positive bacteria especially MRSA and had greater potency, faster killing, and a longer postantibiotic effect than vancomycin. A 30-d serial passage of MRSA in the presence of DLP2/DLP4 failed to produce resistant mutants. Macromolecular synthesis showed that DLP2/DLP4 inhibited multi-macromolecular synthesis especially for RNA. Flow cytometry and electron microscopy results showed that the cell cycle was arrested at R-phase; the cytoplasmic membrane and cell wall were broken by DLP2/DLP4; mesosome-like structures were observed in MRSA. At the doses of 3‒7.5 mg/kg DLP2 or DLP4, the survival of mice challenged with MRSA were 80‒100%. DLP2 and DLP4 reduced the bacterial translocation burden over 95% in spleen and kidneys; reduced serum pro-inflammatory cytokines levels; promoted anti-inflammatory cytokines levels; and ameliorated lung and spleen injury. These data suggest that DLP2 and DLP4 may be excellent candidates for novel antimicrobial peptides against staphylococcal infections.

NZ17074 (N1), an arenicin-3 derivative isolated from the lugworm, has potent antibacterial activity and is cytotoxic. To reduce its cytotoxicity, seven N1 analogues with different structures were designed by changing their disulfide bonds, hydrophobicity, or charge. The “rocket” analogue-N2 and the “kite” analogue-N6 have potent activity and showed lower cytotoxicity in RAW264.7 cells than N1. The NMR spectra revealed that N1, N2, and N6 adopt β-sheet structures stabilized by one or two disulfide bonds. N2 and N6 permeabilized the outer/inner membranes of E. coli, but did not permeabilize the inner membranes of S. enteritidis. N2 and N6 induced E. coli and S. enteritidis cell cycle arrest in the I-phase and R-phase, respectively. In E. coli and in S. enteritidis, 18.7–43.8% of DNA/RNA/cell wall synthesis and 5.7–61.8% of DNA/RNA/protein synthesis were inhibited by the two peptides, respectively. Collapsed and filamentous E. coli cells and intact morphologies of S. enteritidis cells were observed after treatment with the two peptides. Body weight doses from 2.5–7.5 mg/kg of N2 and N6 enhanced the survival rate of peritonitis- and endotoxemia-induced mice; reduced the serum IL-6, IL-1β and TNF-α levels; and protected mice from lipopolysaccharide-induced lung injury. These data indicate that N2 and N6, through multiple selective actions, may be promising dual-function candidates as novel antimicrobial and anti-endotoxin peptides.