Anionic antimicrobial peptide 2, partial

Scygonadin is a novel antimicrobial peptide, which was originally isolated from the seminal plasma of the mud crab, Scylla serrata. Based on the partial 20-residue NH(2)-terminal sequence of the peptide, H-Gly-Gln-Ala-Leu-Asn-Lys-Leu-Met-Pro-Lys-Ile-Val-Ser-Ala-Ile-Ile-Tyr-Met-Val-Gly-OH, scygonadin was cloned from the gonads of S. serrata using a degenerated reverse transcriptase (RT)-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA sequence contains an open reading frame of 539 bases (excluding polyA) with a coding capacity of 126 amino acids, which constitutes a putative NH(2)-terminal signal sequence (1-24) and a mature peptide (25-126). Analysis of the genomic DNA sequence revealed that scygonadin consists of 2300 bp containing two introns (1569 and 120 bp) and three exons (187, 131 and 218 bp) and this sequence is different from any other reported antimicrobial peptide. The theoretical pI of the mature peptide is 6.09, which suggests that it is an anionic molecule. The sex and tissue-specific expression of the scygonadin gene was revealed using RT-PCR and Northern-blot analysis of multiple tissues of S. serrata males and females and this demonstrated that the scygonadin gene was predominantly expressed in the male reproductive tract of S. serrata and was restricted to the ejaculatory duct. This suggests that scygonadin might be one of the antibacterial peptides responsible for protection of the male crab reproductive tract from invading pathogenic microorganisms, so as to maintain a sterile environment leading to successful fertilization.

Antimicrobial peptides (AMPs) represent crucial components of the natural immune defense machinery of different organisms. Generally, they are short and positively charged, and bind to and destabilize bacterial cytoplasmic membranes, ultimately leading to cell death. Natural proteolytic cleavage of αs2-casein in bovine milk generates the antimicrobial peptides casocidin I and II. In the current study, we report for the first time on a detailed structure characterization of casocidins in solution by means of Nuclear Magnetic Resonance spectroscopy (NMR). Structural studies were conducted in H2O and different membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE) and lipid anionic and zwitterionic vesicles. For both peptides, results indicate a mainly disordered conformation in H2O, with a few residues in a partial helical structure. No wide increase of order occurs upon interaction with lipid vesicles. Conversely, peptide conformation becomes highly ordered in presence of TFE, with both casocidins presenting a large helical content. Our data point out a preference of casocidins to interact with model anionic membranes. These results are compatible with possible mechanisms of action underlying the antimicrobial activity of casocidins that ultimately may affect membrane bilayer stability.

Anionic antimicrobial peptides constitute an integral component of animal innate immunity, however the mechanisms of their antifungal activity are still poorly understood. The action of a unique Galleria mellonella anionic peptide 2 (AP2) against fungal pathogen Candida albicans was examined using different microscopic techniques and Fourier transform infrared (FTIR) spectroscopy. Although the exposure to AP2 decreased the survival rate of C. albicans cells, the viability of protoplasts was not affected, suggesting an important role of the fungal cell wall in the peptide action. Atomic force microscopy showed that the AP2-treated cells became decorated with numerous small clods and exhibited increased adhesion forces. Intensified lomasome formation, vacuolization, and partial distortion of the cell wall was also observed. FTIR spectroscopy suggested AP2 interactions with the cell surface proteins, leading to destabilization of protein secondary structures. Regardless of the anionic character of the whole AP2 molecule, bioinformatics analyses revealed the presence of amphipathic α-helices with exposed positively charged lysine residues. High content of the α-helical structure was confirmed after deconvolution of the IR absorption spectrum and during circular dichroism measurements. Our results indicated that the antimicrobial properties of G. mellonella AP2 rely on the same general characteristics found in cationic defense peptides.