Fragments of the Nonlytic Proline-Rich Antimicrobial Peptide Bac5 Kill Escherichia coli Cells by Inhibiting Protein Synthesis

Abstract

Unlike most antimicrobial peptides (AMPs), the main mode of action of the subclass of proline-rich antimicrobial peptides (PrAMPs) is not based on disruption of the bacterial membrane. Instead, PrAMPs exploit the inner membrane transporters SbmA and YjiL/MdtM to pass through the bacterial membrane and enter the cytosol of specific Gram-negative bacteria, where they exert an inhibitory effect on protein synthesis. Despite sharing a high proline and arginine content with other characterized PrAMPs, the PrAMP Bac5 has a low sequence identity with them. Here we investigated the mode of action of three N-terminal Bac5 fragments, Bac5(1-15), Bac5(1-25), and Bac5(1-31). We show that Bac5(1-25) and Bac5(1-31) retained excellent antimicrobial activity toward and low toxicity toward eukaryotic cells, whereas Bac5(1-15) was inactive. Bac5(1-25) and Bac5(1-31) inhibited bacterial protein synthesis and Competition assays suggested that the binding site of Bac5 is within the ribosomal tunnel, where it prevents the transition from the initiation to the elongation phase of translation, as reported for other PrAMPs, such as the bovine PrAMP Bac7. Surprisingly, unlike Bac7, Bac5(1-25) exhibited species-specific inhibition, being an excellent inhibitor of protein synthesis on ribosomes but a poor inhibitor on ribosomes. This indicates that while Bac5 most likely has an overlapping binding site with Bac7, the mode of interaction is distinct, suggesting that Bac5 fragments may be interesting alternative lead compounds for the development of new antimicrobial agents.

Search for Shorter Portions of the Proline‐Rich Antimicrobial Peptide Fragment Bac5(1–25) That Retain Antimicrobial Activity by Blocking Protein Synthesis

“…The insect-derived peptides apidaecin and oncocin have been modified by sequence substitutions to increase their antimicrobial potential and to broaden their spectrumo fa ctivity. [9][10]15] The presents tudyh ighlights that further shortening of Bac5(1-25) gives rise to small peptides that maintain antimicrobial activity,a cting by inhibition of bacterial protein synthesis. [13] New PrAMPss uch as A3-APO have even been rationally designed.…”

“…This suggests that all the Bac5(1-25) fragments endowed with antimicrobial potential exploit the same internalization route via SbmA used by the parent peptide [5,10] for its nonlytic mode of action. This suggests that all the Bac5(1-25) fragments endowed with antimicrobial potential exploit the same internalization route via SbmA used by the parent peptide [5,10] for its nonlytic mode of action.…”

“…[4b, 5] Once in the cytosol, these peptidesi nhibit protein synthesis by blockingt he prokaryotic ribosome. Some N-terminal fragments of Bac5 have been shown to have high antimicrobialp otential, [9][10] and it has been demonstrated that the main bactericidal mechanism of action of Bac5(1-25) and Bac5(1-31) is the inhibition of protein synthesis, as the peptides inhibitt he transition of ribosomes from the initiation to the elongation step during translation. [7] The low cytotoxicity of PrAMPsa nd their antimicrobial potential have made these peptides very appealing as lead compounds to design new antibiotics.…”

“…[6] Moreover, PrAMPsa lso have significant antimicrobiala ctivity againstp athogens that lack SbmA, such as Pseudomonas aeruginosa,e xploiting as econdary lytic mechanism of action. [10] Moreover,i th as been demonstrated that Bac5(1-25) is the most active Bac5 fragment against E. coli,w hile the lack of antimicrobial activity of the shorter Bac5 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) [9][10] is due to poor inhibitory activity toward protein synthesis. Native PrAMPs have been modified to expand their therapeutic index andr ange of activity,toi ncrease their stability and permanencei nt he body,o rt oo btain smaller and therefore less expensive molecules whilestill retaining the antimicrobial potential of the original peptide.…”

“…[7] The low cytotoxicity of PrAMPsa nd their antimicrobial potential have made these peptides very appealing as lead compounds to design new antibiotics. [10] This suggests that blocking this biosynthetic pathway is the discriminating factor for the efficacy of the main Bac5 fragment's antimicrobial mechanism. These modifications consist mainly of substitution of amino acid residues (also with non-proteinogenic residues) throughout the PrAMPs equence, in the linking of other molecules to the native peptides, or simply in shortening the original peptide sequence.…”