Proline-rich Antimicrobial Peptides Research Articles

The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

Pseudomonas aeruginosa infections represent a serious threat to worldwide health. Proline-rich antimicrobial peptides (PR-AMPs), a particular group of peptide antibiotics, have demonstrated in vitro activity against P. aeruginosa strains. Here we show that the mammalian PR-AMP Bac7(1-35) is active against some multidrug-resistant cystic fibrosis isolates of P. aeruginosa By confocal microscopy and cytometric analyses, we investigated the mechanism of killing against P. aeruginosa strain PAO1 and three selected isolates, and we observed that the peptide inactivated the target cells by disrupting their cellular membranes. This effect is deeply different from that previously described for PR-AMPs in Escherichia coli and Salmonella enterica serovar Typhimurium, where these peptides act intracellularly after having been internalized by means of the transporter SbmA without membranolytic effects. The heterologous expression of SbmA in PAO1 cells enhanced the internalization of Bac7(1-35) into the cytoplasm, making the bacteria more susceptible to the peptide but at the same time more resistant to the membrane lysis, similarly to what occurs in E. coli The results evidenced a new mechanism of action for PR-AMPs and indicate that Bac7 has multiple and variable modes of action that depend on the characteristics of the different target species and the possibility to be internalized by bacterial transporters. This feature broadens the spectrum of activity of the peptide and makes the development of peptide-resistant bacteria a more difficult process.

In vivo Efficacy and Pharmacokinetics of Optimized Apidaecin Analogs

Proline-rich antimicrobial peptides (PrAMPs) represent promising alternative therapeutic options for the treatment of multidrug-resistant bacterial infections. PrAMPs are predominantly active against Gram-negative bacteria by inhibiting protein expression via at least two different modes of action, i.e., blocking the ribosomal exit tunnel of 70S ribosomes (oncocin-type binding) or inhibiting the assembly of the 50S ribosomal subunit (apidaecin-type binding). The in vivo efficacy and favorable biodistribution of oncocins confirmed the therapeutic potential of short PrAMPs for the first time, whereas the in vivo evaluation of apidaecins is still limited despite the promising efficacy of apidaecin-analog Api88 in an intraperitoneal murine infection model. Here, the in vivo efficacy of apidaecin-analog Api137 was studied, which rescued all NMRI mice from a lethal intraperitoneal infection with E. coli ATCC 25922 when administered three times intraperitoneal at doses of 0.6 mg/kg starting 1 h after infection. When Api88 and Api137 were administered intravenous or intraperitoneal at doses of 5 and 20 mg/kg, their plasma levels were similarly low (<3 μg/mL) and four-fold lower than for oncocin-analog Onc72. This contradicted earlier expectation based on the very low serum stability of Api88 with a half-life time of only ~5 min compared to ~6 and ~3 h for Api137 and Onc72, respectively. Pharmacokinetic data relying on a sensitive mass spectrometry method utilizing multiple reaction monitoring and isotope-labeled peptides revealed that Api88 and Api137 were present in blood, urine, and kidney, and liver homogenates at similar levels accompanied by the same major metabolites comprising residues 1–16 and 1–17. The pretended discrepancy was solved, when all peptides were incubated in peritoneal lavage. Api137 was rapidly degraded at the C-terminus, while Api88 was rather stable despite releasing the same degradation products. Onc72 was very stable explaining its higher plasma levels compared to Api88 and Api137 after intraperitoneal administration illuminating its good in vivo efficacy. The data indicate that the degradation of therapeutic peptides should be studied in serum and further body fluids. Moreover, the high efficacy in murine infection models and the fast clearance of Api88 and Api137 within ~60 min after intravenous and ~90 min after intraperitoneal injections indicate that their in vivo efficacy relates to the maximal peptide concentration achieved in blood.

The Effect of Selective D- or Nα-Methyl Arginine Substitution on the Activity of the Proline-Rich Antimicrobial Peptide, Chex1-Arg20.

In vivo pharmacokinetics studies have shown that the proline-rich antimicrobial peptide, A3-APO, which is a discontinuous dimer of the peptide, Chex1-Arg20, undergoes degradation to small fragments at positions Pro6-Arg7 and Val19-Arg20. With the aim of minimizing or abolishing this degradation, a series of Chex1-Arg20 analogs were prepared via Fmoc/tBu solid phase peptide synthesis with D-arginine or, in some cases, peptide backbone Nα-methylated arginine, substitution at these sites. All the peptides were tested for antibacterial activity against the Gram-negative bacterium Klebsiella pneumoniae. The resulting activity of position-7 substitution of Chex1-Arg20 analogs showed that arginine-7 is a crucial residue for maintaining activity against K. pneumoniae. However, arginine-20 substitution had a much less deleterious effect on the antibacterial activity of the peptide. Moreover, none of these peptides displayed any cytotoxicity to HEK and H-4-II-E mammalian cells. These results will aid the development of more effective and stable PrAMPs via judicious amino acid substitutions.

Proline-rich antimicrobial peptides targeting protein synthesis

Covering: up to 2017The innate immune system employs a broad array of antimicrobial peptides (AMPs) to attack invading microorganisms. While most AMPs act by permeabilizing the bacterial membrane, specific subclasses of AMPs have been identified that pass through membranes and inhibit bacterial growth by targeting fundamental intracellular processes. One such subclass is the proline-rich antimicrobial peptides (PrAMPs) that bind to the ribosome and interfere with the process of protein synthesis. A diverse range of PrAMPs have been identified in insects, such as bees, wasps and beetles, and crustaceans, such as crabs, as well as in mammals, such as cows, sheep, goats and pigs. Mechanistically, the best-characterized PrAMPs are the insect oncocins, such as Onc112, and bovine bactenecins, such as Bac7. Biochemical and structural studies have revealed that these PrAMPs bind within the ribosomal exit tunnel with a reverse orientation compared to a nascent polypeptide chain. The PrAMPs allow initiation but prevent the transition into the elongation phase of translation. Insight into the interactions of PrAMPs with their ribosomal target provides the opportunity to further develop these peptides as novel antimicrobial agents.

Methods for Elucidating the Mechanism of Action of Proline-Rich and Other Non-lytic Antimicrobial Peptides.

A distinct group of antimicrobial peptides kills bacteria by interfering with internal cellular functions and without concurrent lytic effects on cell membranes. Here we describe some methods to investigate the mechanisms of action of these antimicrobial peptides. They include assays to detect the possible temporal separation between membrane permeabilization and bacterial killing events, to assess the capacity of antimicrobial peptides to cross the bacterial membranes and reside in the cytoplasm, and later to inhibit vital cell functions such as DNA transcription and protein translation.

Fluorescent Ion Efflux Screening Assay for Determining Membrane-Active Peptides

A major global health threat is the emergence of antibiotic-resistant microbes. Coupled with a lack of development of modified antibiotics, there is a need to develop new antimicrobial molecules and screening assays for them. In this study, we provide proof of concept that a large unilamellar vesicle (LUV) method used to study chloride ion efflux facilitated by ionophores and surfactant-like molecules that disrupt membrane integrity can be adapted to identify membrane-interactive antimicrobial peptides (AMPs) and to screen relative activity of AMPs. Lucigenin was encapsulated in LUVs in the presence of Cl– ion (NaCl), which quenches fluorescence, and then incubated with AMPs in 100 mM NaNO3 buffer. Upon AMP membrane interaction or disruption, the Cl– ion is exchanged with the NO3– ion, and the resultant lucigenin fluorescence is indicative of relative AMP activity. Seven AMPs were synthesized by solid-phase peptide chemistry and incubated with LUVs of different phospholipid compositions. Each AMP resulted in lucigenin fluorescence, which was dose dependent, and the relative fluorescence correlated with the minimum inhibitory concentration and minimum bactericidal concentration values for the corresponding peptide. Furthermore, using mammalian model phospholipid LUVs, lucigenin-induced fluorescence also correlated with the AMP cytotoxicity half-maximal inhibitory concentration values. The proline-rich AMP, Chex1-Arg20, which is non-lytic but interacts with the bacterial membrane resulted in lucigenin fluorescence of bacterial membrane model LUVs but not of mammalian membrane model LUVs. The fluorescent ion efflux assay developed here should have applicability for most AMPs and could be tailored to target particular bacterial species membrane composition, potentially leading to the identification of novel membrane-interactive AMPs. The rapid high-throughput method also allows for screening of relative AMP activity and toxicity before biological testing.

Proline-rich Antimicrobial Peptides Optimized for Binding to Escherichia coli Chaperone DnaK

The bacterial protein DnaK promotes folding of newly synthesized polypeptide chains, refolding of misfolded proteins, and protein trafficking. Assisted refolding is especially important under stress conditions induced by antibiotic therapies reducing the desired bactericidal effects. DnaK is supposedly targeted by proline-rich antimicrobial peptides (PrAMPs), but Escherichia coli ΔdnaK mutants and wild type strains are equally susceptible indicating further intracellular targets, such as the 70S ribosome. Crystal structures of PrAMPDnaK- complexes revealed forward and reverse binding modes at the substrate binding domain. Here, we used these ligand-target structures for the first time to rationally optimize peptides using molecular modeling and docking leading to the prediction of four-residue long sequences for improved binding to DnaK. When these sequences were used to replace the original sequence stretch in Onc72, most peptides showed significantly reduced dissociation constants (Kd) determined by fluorescence polarization. In a second approach, the X-ray structures of Api88 and Onc72 bound to DnaK were examined to predict substitutions prone to stronger interactions. Among the 36 peptides obtained from both approaches, six derivatives bound to DnaK with more than 10-fold higher affinities (Kd values in the low micromolar to nanomolar range). Peptides binding stronger to DnaK showed the same minimal inhibitory concentrations against wild type E. coli as the original peptide, but were slightly less active for ΔdnaK mutants. However, one peptide was able to overcome the resistance in an E. coli mutant lacking the SbmA transporter obligatory for the uptake of PrAMPs including Api88 and Onc72. Thus, it´s tempting to speculate that DnaK might be involved in the translocation of PrAMPs into E. coli.

C-Terminal Modification and Multimerization Increase the Efficacy of a Proline-Rich Antimicrobial Peptide.

Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 μm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL-1 ) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C-terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action.

Minibactenecins ChBac7.Nα and ChBac7. Nβ - Antimicrobial Peptides from Leukocytes of the Goat Capra hircus.

Antimicrobial peptides (AMPs) of neutrophils play an important role in the animal and human host defenses. We have isolated two AMPs (average molecular masses of 2895.5 and 2739.3 Da), with potent antimicrobial activity from neutrophils of the domestic goat (Capra hircus). A structural analysis of the obtained peptides revealed that they encompass N-terminal fragments (1-21 and 1-22) of the proline-rich peptide bactenecin 7.5. The primary structure of caprine bactenecin 7.5 had been previously deduced from the nucleotide sequence, but the corresponding protein had not been isolated from leukocytes until now. The obtained caprine AMPs were designated as mini-batenecins (mini-ChBac7.5Nα and mini-ChBac7.5Nβ), analogously to the reported C-terminal fragment of the ovine bactenecin 7.5 named Bac7.5mini [Anderson, Yu, 2003]. Caprine mini-ChBac7.5Nα and mini-ChBac7.5Nβ exhibit significant antimicrobial activity against Gram-negative bacteria, including drug-resistant strains of Pseudomonas aeruginosa, Klebsiella spp., Acinetobacter baumannii at a range of concentrations of 0.5-4 μM, as well as against some species of Gram-positive bacteria (Listeria monocytogenes EGD, Micrococcus luteus). The peptides demonstrate lipopolysaccharide-binding activity. Similarly to most proline-rich AMPs, caprine peptides inactivate bacteria without appreciable damage of their membranes. Mini-ChBac7.5Nα and mini-ChBac7.5Nβ have no hemolytic effect on human red blood cells and are nontoxic to various cultured human cells. Therefore, they might be considered as promising templates for the development of novel antibiotic pharmaceuticals. Isolation of highly active fragments of the antimicrobial peptide from goat neutrophils supports the hypothesis that fragmentation of cathelicidin-related AMPs is an important process that results in the generation of potent effector molecules, which are in some cases more active than full-size AMPs. These truncated AMPs may play a crucial role in host defense reactions.

Ribosomal binding and antibacterial activity of ethylene glycol-bridged apidaecin Api137 and oncocin Onc112 conjugates.

Recent surveillance data on antimicrobial resistance predict the beginning of the post-antibiotic era with pan-resistant bacteria even overcoming polymyxin as the last available treatment option. Thus, new substances using novel modes of antimicrobial action are urgently needed to reduce this health threat. Antimicrobial peptides are part of the innate immune system of most vertebrates and invertebrates and accepted as valid substances for antibiotic drug development efforts. Especially, short proline-rich antimicrobial peptides (PrAMP) of insect origin have been optimized for activity against Gram-negative strains. They inhibit protein expression in bacteria by blocking the 70S ribosome exit tunnel (oncocin-type) or the assembly of the 50S subunit (apidaecin-type binding). Thus, apidaecin analog Api137 and oncocin analog Onc112 supposedly bind to different nearby or possibly partially overlapping binding sites. Here, we synthesized Api137/Onc112-conjugates bridged by ethylene glycol spacers of different length to probe synergistic activities and binding modes. Indeed, the antimicrobial activities against Escherichia coli and Pseudomonas aeruginosa improved for some constructs, although the conjugates did not bind better to the 70S ribosome of E. coli than Api137 and Onc112 using 5(6)-carboxyfluorescein-labelled Api137 and Onc112 in a competitive fluorescence polarization assay. In conclusion, Api137/Onc112-conjugates showed increased antimicrobial activities against P. aeruginosa and PrAMP-susceptible and -resistant E. coli most likely because of improved membrane interactions, whereas the interaction to the 70S ribosome was most likely not improved relying still on the independent apidaecin- and oncocin-type binding modes. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Identification of New Resistance Mechanisms in Escherichia coli against Apidaecin 1b Using Quantitative Gel- and LC-MS-Based Proteomics.

Bacteria have acquired resistance mechanisms to overcome antibiotic treatments, triggering major concerns about the return of epidemic infections. Antimicrobial peptides identified in insects, animals, and plants represent a huge pool of promising lead structures that can be further developed for medical applications. Short proline-rich antimicrobial peptides (PrAMPs) have gained much attention due to their clinically interesting activity spectrum, serum protease stability, efficacy in murine infection models, and low adverse effects. Here we induced resistances by incubating Escherichia coli with increasing concentrations of apidaecin 1b, a PrAMP isolated from honeybees, and quantitatively evaluated the proteomes between wild-type and resistant strains. Surprisingly, 2D differential gel electrophoresis did not reveal differences, indicating that the expression levels of dominant proteins were very similar. Reversed-phase chromatography coupled online to a mass spectrometer identified 2131 proteins in the soluble fraction (cytosolic fraction) and 1296 proteins in the nonsolubilized pellet (membrane fraction). Overall 29 proteins showed a statistically significant upregulation in the resistant E. coli strain, whereas 18 proteins were downregulated. Interestingly, periplasmic chaperone FimC, fimbrial protein FimA, and mannose-binding domain protein FimH, which are part of the fimbrial complex, were not detected in the resistant strain that was also unable to form biofilms. Furthermore, the expression of a few other proteins known as virulence factors was downregulated. Additionally, the expression level of isochorismatase hydrolase (YcaC) decreased in the membrane fraction of the resistant strain to 35%, and the corresponding knockout mutant of E. coli BW25113 was eight times less susceptible to apidaecin 1b and the related designer peptide Api88.

Probiotic Lactobacillus casei expressing porcine antimicrobial peptide PR39 elevates antibacterial activity in the gastrointestinal tract.

PR39, a 4.7 kDa proline-rich antimicrobial peptide, acts as a cationic host defense peptide. In addition to killing bacteria, PR39 mediates inflammatory reactions, including cell proliferation, migration, wound healing, and angiogenesis. Here, we examined the antibacterial effects of this peptide. The synthetic gene fragment PR39 was inserted into the secretory expression vector plasmid pPG:612 of Lactobacillus casei, yielding the recombinant strain pPG:612-PR39/L. casei 393. In vitro antibacterial tests showed that expression of the PR39 peptide in recombinant L. casei resulted in antibacterial activity against Escherichia coli and Salmonella but had only minor antibacterial effects in Staphylococcus aureus. In addition, BALB/c mice fed the recombinant pPG:612-PR39/L. casei 393 grew better and had increased peripheral blood lymphocyte percentages, white blood cell numbers, and spleen indices than mice in the control group. Scanning electron microscopy showed that jejunum and duodenum villus height, crypt depth, and the ratio of villus height/crypt depth in the intestinal villi also increased. Moreover, mice fed the recombinant strain showed significantly lower mortality rates than the control group mice when challenged with the enterotoxigenic E. coli K88+. Thus, this recombinant expression system had the beneficial characteristics of both L. casei and PR39, supporting its potential as an animal feed additive.

Insect-derived short proline-rich and murine cathelicidin-related antimicrobial peptides act synergistically on Gram-negative bacteria in vitro.

Organisms express a set of antimicrobial peptides (AMP) to protect the host against invading microbes by targeting their membranes or intracellular structures. Structurally optimized proline-rich AMPs (PrAMPs) are substantially more efficient in murine infection models than previously assumed from in vitro activities. Thus, we hypothesized that PrAMPs act synergistically with lytic AMPs intrinsically produced in hosts in response to an infection. Synergistic effects between lytic murine cathelicidin-related AMP (CRAMP) and apidaecin- and oncocin-derivatives were studied in chequerboard assays. Evaluation of fractional inhibitory concentration indices revealed synergies against Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. CRAMP synergistically enhances the activity of proline-rich AMPs, which will allow evaluating their therapeutic potential more precisely in vitro.

N-Terminal Ile-Orn- and Trp-Orn-Motif Repeats Enhance Membrane Interaction and Increase the Antimicrobial Activity of Apidaecins against Pseudomonas aeruginosa.

The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N′,N′-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8–16 and 8–32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared with Api795. The uptake was reduced for both peptides by 50 and 80%, respectively, after inhibiting endocytotic uptake with dynasore. In summary, Api794 and Api795 were highly active against P. aeruginosa in vitro. Both peptides passed across the bacterial membrane efficiently, most likely then disturbing the ribosome assembly, and resulting in further intracellular damage. Api795 with its IOIO-motif, which was particularly active and only slightly toxic in vitro, appears to represent a promising third generation lead compound for the development of novel antibiotics against P. aeruginosa.

Readily adaptable release kinetics of prodrugs using protease-dependent reversible PEGylation

Protein and peptide therapeutics with good in vitro activities often fail due to poor bioavailability, circulation lifetime, and immunogenicity. PEGylation, i.e. conjugation of polyethylene glycol (PEG), significantly improves serum stability and renal clearance besides reducing the immunogenicity and thus enhances pharmacokinetics and tolerance in vivo. Several PEGylated drugs are marketed including several top-selling blockbusters. However, PEGylation can mask the binding site, especially in peptides, and thereby reduce the activity drastically, which is only rarely compensated by the improved bioavailability. Prodrug strategies using temporary PEGylation, i.e. the authentic drug is released from a PEG-linked precursor by hydrolysis or enzymatic degradation, can overcome these weaknesses. Recently, we reported a strategy coupling PEG via a peptide linker cleaved C-terminally by trypsin-like proteases in blood to release the unmasked therapeutic peptide. Here, we designed twelve short peptide linkers (four or five residues) to tune the release-rates of oncocin Onc112, a proline-rich antimicrobial peptide. In 25% aqueous mouse serum, Onc112 was released with half-life times from 0.5 to 12h. When elongated N-terminally with 5kDa ɑ-methoxy-ω-mercapto PEG as thioether, the half-life times of the prodrugs ranged from 7 to 42h in full mouse serum. Conjugation of a 20kDa instead of the 5kDa PEG increased the half-life times more than twofold, whereas longer peptide linkers up to twelve residues increased them only slightly. In all cases, Onc112 was released continuously providing stable peptide levels for at least 16h. The kinetics will allow the specific design of PEG-linker-drug-combinations for optimizing the pharmacokinetics of promising peptide therapeutics.

Inner membrane proteins YgdD and SbmA are required for the complete susceptibility of Escherichia coli to the proline-rich antimicrobial peptide arasin 1(1-25).

Arasin 1 from the spider crab Hyas araneus is a proline-rich antimicrobial peptide (PR-AMP), which kills target bacteria by a non-membranolytic mechanism. By using a fluorescent derivative of the peptide, we showed that arasin 1 rapidly penetrates into Escherichia coli cells without membrane damage. To unravel its mode of action, a knockout gene library of E. coli was screened and two types of mutants with a less susceptible phenotype to the arasin 1 fragment (1-23) were found. The first bore the mutation of sbmA, a gene coding for an inner membrane protein involved in the uptake of different antibiotic peptides. The second mutation was located in the ygdD gene, coding for a conserved inner membrane protein of unknown function. Functional studies showed that YgdD is required for the full susceptibility to arasin 1(1-25), possibly by supporting its uptake and/or intracellular action. These results indicated that different bacterial proteins are exploited by arasin 1(1-25) to exert its antibacterial activity and add new insights on the complex mode of action of PR-AMPs.

Membrane interactions of proline-rich antimicrobial peptide, Chex1-Arg20, multimers

The increasing prevalence of antibiotic-resistant pathogens requires the development of new antibiotics. Proline-rich antimicrobial peptides (PrAMPs), including native apidaecins, Bac7, and oncocins or designed A3APO, show multi-modal actions against pathogens together with immunostimulatory activities. The interactions of the designed PrAMP, Chex1-Arg20, and its dimeric and tetrameric oligomers with different model membranes were investigated by circular dichroism spectroscopy, dynamic light scattering, zeta potential, differential scanning calorimetry, and dye leakage. Chex1-Arg20 oligomers showed stronger affinity and preferential binding to negatively charged phospholipid bilayers and led to lipid aggregation and neutralization. Fluorescence microscopy of negatively charged giant unilamellar vesicles with AlexFluor-647-labeled Chex1-Arg20 dimers and tetramers displayed aggregation at a peptide/lipid low ratio of 1:200 and at higher peptide concentrations (1:100/1:50) for Chex1-Arg20 monomer. Such interactions, aggregation, and neutralization of PrAMP oligomers additionally showed the importance of interactions of PrAMPs with negatively charged membranes.

The functional interaction between abaecin and pore-forming peptides indicates a general mechanism of antibacterial potentiation

Long-chain proline-rich antimicrobial peptides such as bumblebee abaecin show minimal activity against Gram-negative bacteria despite binding efficiently to specific intracellular targets. We recently reported that bumblebee abaecin interacts with Escherichia coli DnaK but shows negligible antibacterial activity unless it is combined with sublethal doses of the pore-forming peptide hymenoptaecin. These two bumblebee peptides are co-expressed in vivo in response to a bacterial challenge. Here we investigated whether abaecin interacts similarly with pore-forming peptides from other organisms by replacing hymenoptaecin with sublethal concentrations of cecropin A (0.3μM) or stomoxyn (0.05μM). We found that abaecin increased the membrane permeabilization effects of both peptides, confirming that it can reduce the minimal inhibitory concentrations of pore-forming peptides from other species. We also used atomic force microscopy to show that 20μM abaecin combined with sublethal concentrations of cecropin A or stomoxyn causes profound structural changes to the bacterial cell surface. Our data indicate that the potentiating functional interaction between abaecin and pore-forming peptides is not restricted to specific co-expressed peptides from the same species but is likely to be a general mechanism. Combination therapies based on diverse insect-derived peptides could therefore be used to tackle bacteria that are recalcitrant to current antibiotics.

Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition.

With bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac71–35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac71–35, Pyrrhocoricin, Metalnikowin and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibiotic-binding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics.

Structure of the mammalian antimicrobial peptide Bac7(1-16) bound within the exit tunnel of a bacterial ribosome.

Proline-rich antimicrobial peptides (PrAMPs) produced as part of the innate immune response of animals, insects and plants represent a vast, untapped resource for the treatment of multidrug-resistant bacterial infections. PrAMPs such as oncocin or bactenecin-7 (Bac7) interact with the bacterial ribosome to inhibit translation, but their supposed specificity as inhibitors of bacterial rather than mammalian protein synthesis remains unclear, despite being key to developing drugs with low toxicity. Here, we present crystal structures of the Thermus thermophilus 70S ribosome in complex with the first 16 residues of mammalian Bac7, as well as the insect-derived PrAMPs metalnikowin I and pyrrhocoricin. The structures reveal that the mammalian Bac7 interacts with a similar region of the ribosome as insect-derived PrAMPs. Consistently, Bac7 and the oncocin derivative Onc112 compete effectively with antibiotics, such as erythromycin, which target the ribosomal exit tunnel. Moreover, we demonstrate that Bac7 allows initiation complex formation but prevents entry into the elongation phase of translation, and show that it inhibits translation on both mammalian and bacterial ribosomes, explaining why this peptide needs to be stored as an inactive pro-peptide. These findings highlight the need to consider the specificity of PrAMP derivatives for the bacterial ribosome in future drug development efforts.