Cecropin A1

Cecropin A1 (CecA1) promoter from Bombyx mori was cloned and characterized to provide insight into the transcriptional control of this antimicrobial peptide gene upon immune challenges. Reporter gene assays demonstrated that both Escherichia coli and lipopolysaccharide could induce expression in BmE cells but B. bombyseptieus or peptidoglycan failed, and the induction pattern of the reporter gene was coincident with the endogenous CecA1. Analysis of deletion and mutation constructs revealed that the regulatory region was the κB motif located between -176 and -166, and no other predicted elements on CecA1 promoter affected its inducibility. Insertion of additional κB motifs increased the activity of CecA1 promoter. Furthermore, binding of Relish to κB motif was confirmed by electrophoretic mobility shift assay. These findings indicate the regulatory mechanism of CecA1 expression in IMD pathway and suggest an approach of engineering antimicrobial peptide promoter with enhanced activities that may lead to broad applications.

Due to emergence of multidrug resistance in pathogens, the attention of the scientific community is now directed towards strengthening the reservoir of antimicrobial compounds. Prior to in vivo studies, the interaction and penetration of a hybrid peptide K11 in bacterial cells using confocal microscopy was assessed which was observed as early as 10 min after incubation with the peptide. Cell lysis along with leakage of cytoplasmic content was confirmed by electron microscopy. To evaluate the in vivo performance of the peptide, it was contained in carbopol hydrogel. Efficacy of the hydrogel formulation was then evaluated against Acinetobacter baumannii-infected wounds using a murine excision model. Treatment resulted in restoration of body weight, complete clearance of infection from the wound by day 7 and 99% wound enclosure by day 21, in contrast to the persistence of infection and 70% wound enclosure in the infected group. Further, this treatment resulted in a 2.6-fold decrease in the levels of malondialdehyde along with a 4.5-fold increase in the levels of catalase on day 3. Appearance of normal histo-architecture was observed in the treatment group. Based on these results, the peptide hydrogel can be exploited in future as one of the strategies for developing a topical anti-infective therapeutic agent.

Regulation of the Aedes albopictus cecropin A1 promoter was studied to provide insight into the transcriptional control of this antimicrobial peptide (AMP) gene in mosquitoes. Gene expression levels of cecropin A1 increased in A. albopictus C6/36 cells in response to heat-killed Escherichiacoli. Reporter gene assays incorporating -757 to +32 of the A. albopictus cecropin A1 promoter revealed that E. coli could induce expression in these cells with more pronounced expression than that seen with lipopolysaccharide (LPS). Analysis of deletion constructs demonstrated that the 5' boundary of the regulatory region for the activation of this AMP was located between -173 and -64. Western blotting with anti-phospho-specific antibodies demonstrated that p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) were activated by LPS, whereas only p38 MAPK was activated by E. coli. Moreover, pharmacological experiments revealed that pre-incubation of cells with the p38 MAPK inhibitor SB203580 resulted in a striking activation of the cecropin A1 promoter following immune challenge, demonstrating that p38 MAPK negatively regulates cecropin A1 promoter activity. Finally the region required for the negative regulation by p38 MAPK was identified as being between -173 and -64. This report is the first to show involvement of the p38 MAPK pathway in the negative regulation of AMP production in a mosquito.