Lethal Effects of Apidaecin on Escherichia coliInvolve Sequential Molecular Interactions with Diverse Targets

Abstract

Apidaecins, short proline-arginine-rich peptides from insects, are highly bactericidal through a mechanism that includes stereoselective elements but is completely devoid of any pore-forming activity. The spectrum of antibacterial activity, always limited to Gram-negatives, is further dependent on a small number of variable residues and can be manipulated. We show here that mutations in the evolutionary conserved regions result in a more general loss of function, and we have used such analogs to probe molecular interactions in Escherichia coli. First, an assay was developed to measure selectively chiral association with cellular targets. By using this method, we find that apidaecin uptake is energy-driven and irreversible and yet can be partially competed by proline in a stereospecific fashion, results upholding a model of a permease/transporter-mediated mechanism. This putative transporter is not the end point of apidaecin action, for failure of certain peptide analogs to kill cells after entering indicates the existence of another downstream target. Tetracycline-induced loss of bactericidal activity and dose-dependent in vivo inhibition of translation by apidaecin point at components of the protein synthesis machinery as likely candidates. These findings provide new insights into the antibacterial mechanism of a unique group of peptides and perhaps, by extension, for distant mammalian relatives such as PR-39.

Through the Looking Glass, Mechanistic Insights from Enantiomeric Human Defensins

“…We also noted functional equivalence of L-and D-enantiomers of protegrin-1, in studies performed with bacteria and Candida albicans (64 -66). In contrast, when Tempst and colleagues studied enantiomers of apidaecins, proline-and arginine-rich antimicrobial peptides of insect origin that act preferentially on Gram-negative bacteria, the D-enantiomer proved to be much less potent (67,68). They concluded that the peptides acted via a mechanism that included stereoselective elements but was completely devoid of any pore-forming activity.…”

Primary Structure and in Vitro Antibacterial Properties of the Drosophila melanogaster Attacin C Pro-domain

“…According to recent data (28, After an overnight incubation at 37°C, the number of colony-forming units (CFU) was determined from 1 l of culture medium. 33), pyrrhocoricin and apidaecin, peptides belonging to this family, first bind to an outer membrane component of E. coli and later translocate into the interior of the bacteria where they bind to specific target proteins. For example, pyrrhocoricin was shown to inhibit protein folding in bacteria by inactivating the multihelical lid complex of the 70-kDa heat shock protein DnaK needed for substrate binding and ATPase activity (29).…”

Interaction of protamine with gram‐negative bacteria membranes: possible alternative mechanisms of internalization in Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa

“…The bactericidal effect of many CAPs is thought to be due to the action on the cytoplasmic membrane of the susceptible bacteria, possibly through the formation of pores or destabilization of the membrane bilayer structure leading to lysis of the cell [4,5]. Some CAPs such as buforin 11 [6], indolicidin [7], apidaecin [8], PR 39 [9], PR 26 [10], bactenecin [11] and lactoferricin B [12,13] have been reported to have intracellular targets. In a recent review article, Wimley [14] pointed out that many antimicrobial peptides do not porate or damage bacterial membranes but rather produce transient pores as demonstrated by using unilamellar liposomes loaded with probes.…”