Dataset Information

(Re)Defining the Proline-Rich Antimicrobial Peptide Family and the Identification of Putative New Members.

ABSTRACT: As we rapidly approach a post-antibiotic era in which multi-drug resistant bacteria are ever-pervasive, antimicrobial peptides (AMPs) represent a promising class of compounds to help address this global issue. AMPs are best-known for their membrane-disruptive mode of action leading to bacteria cell lysis and death. However, many AMPs are also known to be non-lytic and have intracellular modes of action. Proline-rich AMPs (PrAMPs) are one such class, that are generally membrane permeable and inhibit protein synthesis leading to a bactericidal outcome. PrAMPs are highly effective against Gram-negative bacteria and yet show very low toxicity against eukaryotic cells. Here, we review both the PrAMP family and the past and current definitions for this class of peptides. Computational analysis of known AMPs within the DRAMP database (http://dramp.cpu-bioinfor.org/) and assessment of their PrAMP-like properties have led us to develop a revised definition of the PrAMP class. As a result, we subsequently identified a number of unknown and unclassified peptides containing motifs of striking similarity to known PrAMP-based DnaK inhibitors and propose a series of new sequences for experimental evaluation and subsequent addition to the PrAMP family.

SUBMITTER: Welch NG

PROVIDER: S-EPMC7736402 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

(Re)Defining the Proline-Rich Antimicrobial Peptide Family and the Identification of Putative New Members.

Welch Nicholas G NG Li Wenyi W Hossain Mohammed Akhter MA Separovic Frances F O'Brien-Simpson Neil M NM Wade John D JD

Frontiers in chemistry 20201201

As we rapidly approach a post-antibiotic era in which multi-drug resistant bacteria are ever-pervasive, antimicrobial peptides (AMPs) represent a promising class of compounds to help address this global issue. AMPs are best-known for their membrane-disruptive mode of action leading to bacteria cell lysis and death. However, many AMPs are also known to be non-lytic and have intracellular modes of action. Proline-rich AMPs (PrAMPs) are one such class, that are generally membrane permeable and inhi ...[more]

PMID: 33335890

Similar Datasets

Project description:Proline-rich antimicrobial peptides (PR-AMPs) are a group of cationic host defense peptides that are characterized by a high content of proline residues. Up to now, they have been reported in some insects, vertebrate and invertebrate animals, but are not found in plants. In this study, we performed an in silico screening of antimicrobial peptides, which led to discovery of a Brassica napus gene encoding a novel PR-AMP. This gene encodes a 35-amino acid peptide with 13 proline residues, designated BnPRP1. BnPRP1 has 40.5% identity with a known proline-rich antimicrobial peptide SP-B from the pig. BnPRP1 was artificially synthetized and cloned into the prokaryotic expression vector pET30a/His-EDDIE-GFP. Recombinant BnPRP1 was produced in Escherichia coli and has a predicted molecular mass of 3.8 kDa. Analysis of its activity demonstrated that BnPRP1 exhibited strong antimicrobial activity against Gram-positive bacterium, Gram-negative bacterium, yeast and also had strong antifungal activity against several pathogenic fungi, such as Sclerotinia sclerotiorum, Mucor sp., Magnaporthe oryzae and Botrytis cinerea. Circular dichroism (CD) revealed the main secondary structure of BnPRP1 was the random coil. BnPRP1 gene expression detected by qRT-PCR is responsive to pathogen inoculation. At 48 hours after S. sclerotiorum inoculation, the expression of BnPRP1 increased significantly in the susceptible lines while slight decrease occurred in resistant lines. These suggested that BnPRP1 might play a role in the plant defense response against S. sclerotiorum. BnPRP1 isolated from B. napus was the first PR-AMP member that was characterized in plants, and its homology sequences were found in some other Brassicaceae plants by the genome sequences analysis. Compared with the known PR-AMPs, BnPRP1 has the different primary sequences and antimicrobial activity. Above all, this study gives a chance to cast a new light on further understanding about the AMPs' mechanism and application.

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.

Project description:Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif D3KPRPYLPRP12 with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluorescein-labeled (Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant (Kd) of 201 ± 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and ΔsbmA and ΔmdtM knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake.

Dataset Information

(Re)Defining the Proline-Rich Antimicrobial Peptide Family and the Identification of Putative New Members.

Publications

(Re)Defining the Proline-Rich Antimicrobial Peptide Family and the Identification of Putative New Members.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets