The antimicrobial peptide Brevinin-2ISb enhances the innate immune response against methicillin-resistant Staphylococcus aureus by activating DAF-2/DAF-16 signaling in Caenorhabditis elegans, as determined by in vivo imaging

Abstract

Abstract Objective: In order to study the important role and molecular mechanism of Brevinin-2 family antimicrobial peptide Brevinin-2ISb in methicillin-resistant Staphylococcus aureus (MRSA) infection of Caenorhabditis (C.) elegans, and to find the optimal therapeutic concentration of Brevinin-2ISb. Methods: By using a C. elegans model and MRSA infection modelto study the therapeutic effect of different concentrations of Brevinin-2ISb on C. elegans. Real-time PCR was used for investigating the effect of Brevinin-2ISb on the downstream gene expression of DAF-2/DAF-16 innate immune pathway and the major virulence factor gene expression of MRSA. With protein activity tests to study the inhibitory effect of Brevinin-2ISb on MRSA virulence factor protein activity. Finally, laser confocal imaging was carried out to observe real-time expression and distribution of downstream antimicrobial proteins to further prove the effect of Brevinin-2ISb on the activation of DAF-2/DAF-16 pathway by in vivo imaging. All animal study procedures were approved by the Academic Committee at Xidian University and Xi’an Jiaotong University Animal Care and Use Committee, China (approval No. JGC201207) on July 15, 2017. Results: Host immunity was largely enhanced by Brevinin-2ISb, and the expression of staphylococcal enterotoxin genes, as well as virulence factors, was suppressed by Brevinin-2ISb. Indeed, the expression of many C. elegans innate immune genes, including lys-7, spp-1, K05D8.5 and C29F3.7, was induced by Brevinin-2ISb. In particular, robust, sustained expression of the antibacterial gene lys-7 was observed after Brevinin-2ISb treatment, resulting in increased protein levels. These effects correlated with a reduction in the MRSA-mediated death of the C. elegans host. Low concentrations of Brevinin-2ISb exhibited very low hemolytic activity, and may play a positive role in host innate immunity. Specifically, activation of the DAF-2/DAF-16 pathway appears to be essential for immune activation in C. elegans treated with Brevinin-2ISb. Based on the evolutionary conservation of innate immune pathways, our results suggest that Brevinin-2ISb not only has strong antibacterial activity, but may also enhance the innate immune response in humans. This study demonstrates that Brevinin-2ISb-related peptides are potential candidates for the development of novel anti-inflammatory or anti-microbial drugs. Conclusion: Antimicrobial peptide Brevinin-2ISb effectively inhibits MRSA at low concentration. This antimicrobial peptide can prolong the life of MRSA-infected C. elegans, has very low hemolytic activity and inhibits the activity and expression of various MRSA virulence factors. More importantly, Brevinin-2ISb activated the expression of antimicrobial genes downstream of DAF-2/DAF-16, which enhanced the MRSA resistance of C. elegans. This peptide could be used as the basis for developing new drugs to replace antibiotics.