A chemically synthesized version of the insect antibacterial glycopeptide,diptericin, disrupts bacterial membrane integrity

Insects protect themselves against bacterial infection by secreting a batte ry of antimicrobial peptides into the hemolymph. Despite recent progress, i mportant mechanistic questions, such as the precise bacterial targets, the nature of any cooperation that occurs between peptides, and the purpose of multiple peptide isoforms, remain largely unanswered. We report herein the chemical synthesis and preliminary mechanistic investigation of diptericin, an 82 residue glycopeptide that contains regions similar to two different types of antibacterial peptides. A revised, highly practical synthesis of t he precursor N-alpha-Fmoc-Thr(Ac-3-alpha-D-GalNAc) allowed us to produce su fficient quantities of the glycopeptide for mechanistic assays. The synthet ic, full-length polypeptide proved to be active in growth inhibition assays with an IC50 of approximately 250 nM, a concentration similar to that foun d in the insect hemolymph. Biological analysis of diptericin fragments indi cated that the main determinant of antibacterial activity lay in the C-term inal region that is similar to the attacin peptides, although the N-termina l segment, related to the proline-rich family of antibacterial peptides, au gmented that activity by 100-fold. In all assays, activity appeared glycosy lation independent. Circular dichroism of unglycosylated diptericin indicat ed that the peptide lacked structure both in plain buffer and in the presen ce of liposomes. Diptericin increased the permeability of the outer and inn er membranes of Escherichia coli D22 cells, suggesting possible mechanisms of action. The ability to access glycopeptides of this type through chemica l synthesis will facilitate further mechanistic studies.