Sphistin Synthetic Peptide(12-38)

Analysis of a selected set of antimicrobial peptides: The rapid emergence of resistance to classical antibiotics has increased the interest in novel antimicrobial compounds. Antimicrobial peptides (AMPs) represent an attractive alternative to classical antibiotics and a number of different studies have reported antimicrobial activity data of various AMPs, but there is only limited comparative data available. The mode of action for many AMPs is largely unknown even though several models have suggested that the lipopolysaccharides (LPS) play a crucial role in the attraction and attachment of the AMP to the bacterial membrane in Gram-negative bacteria. We compared the potency of Cap18, Cap11, Cap11-1-18m2, Cecropin P1, Cecropin B, Bac2A, Bac2A-NH2, Sub5-NH2, Indolicidin, Melittin, Myxinidin, Myxinidin-NH2, Pyrrhocoricin, Apidaecin and Metalnikowin I towards Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Aeromonas salmonicida, Listeria monocytogenes, Campylobacter jejuni, Flavobacterium psychrophilum, Salmonella typhimurium and Yersinia ruckeri by minimal inhibitory concentration (MIC) determinations. Additional characteristics such as cytotoxicity, thermo and protease stability were measured and compared among the different peptides. Further, the antimicrobial activity of a selection of cationic AMPs was investigated in various E. coli LPS mutants. Cap18 shows a high broad spectrum antimicrobial activity: Of all the tested AMPs, Cap18 showed the most efficient antimicrobial activity, in particular against Gram-negative bacteria. In addition, Cap18 is highly thermostable and showed no cytotoxic effect in a hemolytic assay, measured at the concentration used. However, Cap18 is, as most of the tested AMPs, sensitive to proteolytic digestion in vitro. Thus, Cap18 is an excellent candidate for further development into practical use; however, modifications that should reduce the protease sensitivity would be needed. In addition, our findings from analyzing LPS mutant strains suggest that the core oligosaccharide of the LPS molecule is not essential for the antimicrobial activity of cationic AMPs, but in fact has a protective role against AMPs.

Histone H2A is known to participate in host immune defense through generating special antimicrobial peptides (AMPs), for which it has been an interesting research focus to characterize this kind of peptides in vertebrates and invertebrates. Although thousands of AMPs have been reported in variety of life species, only several AMPs are known in crabs and in particular no H2A-derived AMP has yet been reported. In the present study, a 38-amino acid peptide with antimicrobial activity was determined based on the sequence analysis of a histone H2A identified from the mud crab Scylla paramamosain. The histone H2A derived peptide was an AMP-like molecule and designated as Sphistin. Sphistin showed typical features of AMPs such as amphiphilic α-helical second structrue and positive charge net. The synthetic Sphistin exerted high antimicrobial activity against Gram-positive, Gram-negative bacteria and yeast, among which Aeromonas hydrophila, Pseudomonas fluorescens and Pseudomonas stutzeri are important aquatic pathogens. Leakage of the cell content and disruption of the cell surface were observed in bacterial cells treated with Sphistin using scanning electron microscopy. It was proved that the increasing cytoplasmic membrane permeability of Escherichia coli was caused by Sphistin. Further observation under confocal microscopy showed that Sphistin could combine onto the membrane of Staphylococcus aureus, E. coli MC1061 and Pichia pastoris but not translocate into the cytoplasm. Moreover, the affinity of Sphistin with either LPS or LTA was also testified that there was an interaction between Sphistin and cell membrane. Thus, the antimicrobial mechanism of this peptide likely exerted via adsorption and subsequently permeabilization of the bacterial cell membranes other than penetrating cell membrane. In addition, synthetic Sphistin exhibited no cytotoxicity to primary cultured crab haemolymphs and mammalian cells even at a high concentration of 100 μg/mL for 24 h. This is the first report of a histone-derived Sphistin identified from S. paramamosain with a specific antimicrobial activity and mechanism, which could be a new candidate for future application in aquaculture and veterinary medicine.

Overuse or abuse of antibiotics has undoubtedly accelerated the increasing prevalence of global antibiotic resistance crisis, and thus, people have been trying to explore approaches to decrease dosage of antibiotics or find new antibacterial agents for many years. Antimicrobial peptides (AMPs) are the ideal candidates that could kill pathogens and multidrug-resistant bacteria either alone or in combination with conventional antibiotics. In the study, the antimicrobial efficacy of mud crab Scylla paramamosain AMPs Sphistin and Sph12-38 in combination with eight selected antibiotics was evaluated using a clinical pathogen, Pseudomonas aeruginosa. It was interesting to note that the in vitro combination of rifampicin and azithromycin with Sphistin and Sph12-38 showed significant synergistic activity against P. aeruginosa. Moreover, an in vivo study was carried out using a mouse model challenged with P. aeruginosa, and the result showed that the combination of Sph12-38 with either rifampicin or azithromycin could significantly promote the healing of wounds and had the healing time shortened to 4-5 days compared with 7-8 days in control. The underlying mechanism might be due to the binding of Sphistin and Sph12-38 with P. aeruginosa lipopolysaccharides (LPS) and subsequent promotion of the intracellular uptake of rifampicin and azithromycin. Taken together, the significant synergistic antibacterial effect on P. aeruginosa in vitro and in vivo conferred by the combination of low dose of Sphistin and Sph12-38 with low dose of rifampicin and azithromycin would be beneficial for the control of antibiotic resistance and effective treatment of P. aeruginosa-infected diseases in the future.