MDAP-2 is a new AMP with high inhibitory activity on Salmonella gallinarum, which may be developed as an antimicrobial agent in the agricultural industry and food preservation. To investigate the underlying the action mechanism of MDAP-2 on Salmonella gallinarum, impacts of MDAP-2 on the growth curve and bacterial morphology of Salmonella gallinarum were studied. iTRAQ-based proteomics analysis was also performed on proteins extracted from treated and untreated Salmonella gallinarum cells. The differentially expressed proteins were then analyzed using the KEGG and GO databases. Finally, the function of some differentially expressed proteins was verified. The results showed that 150 proteins (41 up-regulated and 109 down-regulated) were found differentially expressed (fold > 1.8, p<0.05). The results indi- cate that MDAP-2 kills Salmonella gallinarum mainly through two mechanisms: (i) direct inhibi- tion of cell wall/ membrane/ envelope biogenesis, energy production/ conversion, carbohydrate transport/ metabolism, and DNA transcription/ translation through regulation of special protein levels; (ii) indirect effects on the same pathway through the accumulation of Reactive oxygen species (O2 ▪-, H2O2 and OH▪-).

MDAP-2 is a new antibacterial peptide with a unique structure that was isolated from house- flies. However, its biological characteristics and antibacterial mechanisms against bacteria are still poorly understood. To study the biological characteristics, antibacterial activity, hemolytic activi- ty, cytotoxicity to mammalian cells, and the secondary structure of MDAP-2 were detected; the results showed that MDAP-2 displayed high antibacterial activity against all of the tested Gram-negative bacteria. MDAP-2 had lower hemolytic activity to rabbit red blood cells; only 3.4% hemolytic activity was observed at a concentration of 800μg/ml. MDAP-2 also had lower cytotoxicity to mammalian cells; IC50 values for HEK-293 cells, VERO cells, and IPEC-J2 cells were greater than 1000 μg/ml. The circular dichroism (CD) spectra showed that the peptide most- ly has α-helical properties and some β-fold structure in water and in membrane-like conditions. MDAP-2 is therefore a promising antibacterial agent against Gram-negative bacteria. To deter- mine the antibacterial mechanism(s) of action, fluorescent probes, flow cytometry, and transmis- sion electron microscopy (TEM) were used to study the effects of MDAP-2 on membrane perme- ability, polarization ability, and integrity of Gram-negative bacteria. The results indicated that the peptide caused membrane depolarization, increased membrane permeability, and destroyed membrane integrity. In conclusion, MDAP-2 is a broad-spectrum, lower hemolytic activity, and lower cytotoxicity antibacterial peptide, which is mainly effective on Gram-negative bacteria. It exerts its antimicrobial effects by causing bacterial cytoplasm membrane depolarization, increas- ing cell membrane permeability and disturbing the membrane integrity of Gram-negative bacte- ria. MDAP-2 may offer a new strategy to for defense against Gram-negative bacteria.