Immune response of histone H2 and antimicrobial activity of its derived peptide in Mytilus coruscus

The research of histones and histone-derived peptides is one of the most important study field of antimicrobial peptides. The genus Mytilus is not only of significant economic importance in aquaculture around the world but also shows strong tolerance to a wide range of environmental factors, including various microbes. Although Mytilus antimicrobial peptides had been studied for more than thirty years, there is no reports for now regarding the immunological effects of histones and their derived peptides in this genus. For understanding the roles of histones and their possible derived peptides in Mytilus immunity, the expression levels of H2A and H2B genes were analyzed by fluorescence quantitative PCR after various microbial stress, including Grand-positive, Grand-negative bacterium, and fungi, respectively. In addition, two peptides, Coruscusin Ⅰ and Coruscusin II, derived from the N-terminal of H2A and H2B, respectively, were synthesized by solid phase chemical synthesis. The special configurations of Coruscusin I and Coruscusin Ⅱ were analyzed by circular dichroism spectrometer, and their antimicrobial activities were studied by growth curve suppression method. The results showed that both H2A and H2B of M. coruscus contain classical histone characteristic domain in their sequences, and the α-helix apparently is the predominant conformation for both H2A and H2B. In addition, both H2A and H2B of M. coruscus presented a high sequence similarity with histones of other bivalves species, indicating the conservation of histones in various species. The results of fluorescence quantitative PCR showed that the genes of histone H2A and H2B of M. coruscus were significantly up-regulated in different tissues under different microbial stresses. H2A gene was significantly up-regulated under Staphylococcus aureus induction in digestive gland and gill tissues, and the highest relative expression level were presented at 4 and 24 h for digestive gland and gill, respectively; in blood cell, the gene of H2A was significantly up-regulated at 8 and 24 h under the induction of Vibrio parahaemolyticus and Candida albicans, respectively. In addition, H2B showed sensitivity to V. parahaemolyticus and C. albicans in both digestive gland and hemocytes, and the expression level of H2B gene was significantly up-regulated at 8 and 24 h after induction. In gill, the H2B gene showed sensitivity to S. aureus induction, and the highest expression level was presented at 24 h. These results indicate a very complex immunological responses of histones in Mytilus, and this observation strongly suggests the existence of different recognition mechanisms or signal transduction pathways in mussels. Moreover, Coruscusin-Ⅰ and Coruscusin-Ⅱwere synthesized successfully with the expected molecular masses. Both Coruscusin-Ⅰ and Coruscusin-Ⅱ showed an increasing helix content in 30% of trifluoroethanol compared to that in pure water and PBS buffer, indicating a conformational change in nonpolar solution for both peptides. In addition, both Coruscusin-Ⅰ and Coruscusin-Ⅱ showed significant antimicrobial activity against tested gram-positive and gram-negative bacteria, as well as fungi. However, Coruscusin-Ⅱ showed stronger antifungal activity than that of Coruscusin-Ⅰ. Further helical structure prediction results indicate that the types and distribution characteristics of basic amino acids in the sequences of the two peptides might be the internal cause of the difference in antimicrobial activity. This study provides a foundation for elucidating the role of histones and their derived peptides in mussel immunity and its mechanism, and also provides a scientific basis for the development of new biological antibiotics derived from mussel histones.