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Activity, structure, and diversity of Type II proline-rich antimicrobial peptides from insects

ABSTRACT: Apidaecin 1b (Api), the first characterized Type II Proline-rich antimicrobial peptide (PrAMP), is encoded in the honeybee genome. It inhibits bacterial growth by binding in the nascent peptide exit tunnel of the ribosome after the release of the completed protein and trapping the release factors. By genome mining, we have identified 71 PrAMPs encoded in insect genomes as pre-pro-polyproteins. Having chemically synthesized and tested the activity of 26 peptides we demonstrate that despite significant sequence variation in the N-terminal sequence, the majority of the PrAMPs that retain the conserved C-terminal sequence of Api are able to trap the ribosome at the stop codons and induce stop codon readthrough - all hallmarks of Type II PrAMP mode of action. Some of the characterized PrAMPs exhibit superior antibacterial activity in comparison with Api. The newly solved crystallographic structures of the ribosome complexed with Api and a more active peptide Fva1 from the stingless bee demonstrate the universal placement of the PrAMPs' C-terminal pharmacophore in the post-release ribosome despite variations in their N-terminal sequence.

SUBMITTER: Dr. Weiping Huang

PROVIDER: S-SCDT-10_1038-S44319-024-00277-5 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:Proline-rich antimicrobial peptides (PrAMPs) kill bacteria via a nonlytic mechanism in which they permeate through the outer membrane, utilize protein-mediated transport across the inner membrane, and target the ribosome to inhibit protein synthesis. We previously reported that substitutions of oncocin ( VDKPPYLPRPRPPRRIYNR-NH2 ) with a pair of cationic residues improved the antimicrobial activity. In this study, we applied the design protocol to three other PrAMPs: apidaecin-1b, pyrrhocoricin, and bactenecin 7(1-16) and found that the substitutions (R4K and I8K/R) for apidaecin-1b improve the activity by twofold (p?<?.05) against nonpathogenic Escherichia coli. Moreover, the substitutions (L7K/R and R14K) for pyrrhocoricin improve the activity by 2-10-fold (p?<?.05) against some strains of E. coli and Salmonella Typhimurium. We also performed activity tests against inner membrane protein (SbmA or YgdD) knockout strains. The result is consistent with previous studies that SbmA is the major transporter for apidaecin-1b and pyrrhocoricin derivatives. However, bactenecin 7(1-16) functions independently of these transporters. In addition, several apidaecin-1b derivatives exhibit enhanced activity relative to wild-type only in the absence of SbmA, which is consistent with mutations that enhance transport across the inner membrane. A high performance liquid chromatography-based kinetic assay for cellular association and internalization demonstrates that the selected cationic mutations can improve cellular association in minimal media, but this enhanced association is not required for increased activity, which suggests the importance of inner membrane transport. These functional studies on cationic mutants of PrAMPs advance understanding of potency and mechanism and advance the ability to engineer improved antimicrobials as evidenced by the identification of the pyrrhocoricin mutant (L7R and R14K) with 10-fold elevated potency against pathogenic E. coli.

Project description:The health threat caused by multiresistant bacteria has continuously increased and recently peaked with pathogens resistant to all current drugs. This has triggered intense research efforts to develop novel compounds to overcome the resistance mechanisms. Thus, antimicrobial peptides (AMPs) have been intensively studied, especially the family of proline-rich AMPs (PrAMPs) that was successfully tested very recently in murine infection models. PrAMPs enter bacteria and inhibit chaperone DnaK. Here, we studied the toxicity of intracellular PrAMPs in HeLa and SH-SY5Y cells. As PrAMPs cannot enter most mammalian cells, we coupled the PrAMPs with penetratin (residues 43 to 58 in the antennapedia homeodomain) via a C-terminally added cysteine utilizing a thioether bridge. The resulting construct could transport the covalently linked PrAMP into mammalian cells. Penetratin ligation reduced the MIC for Gram-negative Escherichia coli only slightly (1 to 8 ?mol/liter) but increased the activity against the Gram-positive Micrococcus luteus up to 32-fold (MIC ? 1 ?mol/liter), most likely due to more effective penetration through the bacterial membrane. In contrast to native PrAMPs, the penetratin-PrAMP constructs entered the mammalian cells, aligned around the nucleus, and associated with the Golgi apparatus. At higher concentrations, the constructs reduced the cell viability (50% inhibitory concentration [IC(50)] ? 40 ?mol/liter) and changed the morphology of the cells. No toxic effects or morphological changes were observed at concentrations of 10 ?mol/liter or below. Thus, the IC(50) values were around 5 to 40 times higher than the MIC values. In conclusion, PrAMPs are in general not toxic to mammalian cells, as they do not pass through the membrane. When shuttled into mammalian cells, however, PrAMPs are only slightly cross-reactive to mammalian chaperones or other intracellular mammalian proteins, providing a second layer of safety for in vivo applications, even if they can enter some human cells.

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Activity, structure, and diversity of Type II proline-rich antimicrobial peptides from insects

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