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Characterization of Cetacean Proline-Rich Antimicrobial Peptides Displaying Activity against ESKAPE Pathogens.

ABSTRACT: Proline-rich antimicrobial peptides (PrAMPs) may be a valuable weapon against multi-drug resistant pathogens, combining potent antimicrobial activity with low cytotoxicity. We have identified novel PrAMPs from five cetacean species (cePrAMPs), and characterized their potency, mechanism of action and in vitro cytotoxicity. Despite the homology between the N-terminal of cePrAMPs and the bovine PrAMP Bac7, some differences emerged in their sequence, activity spectrum and mode of action. CePrAMPs with the highest similarity with the Bac7(1-35) fragment inhibited bacterial protein synthesis without membrane permeabilization, while a second subgroup of cePrAMPs was more membrane-active but less efficient at inhibiting bacterial translation. Such differences may be ascribable to differences in presence and positioning of Trp residues and of a conserved motif seemingly required for translation inhibition. Unlike Bac7(1-35), which requires the peptide transporter SbmA for its uptake, the activity of cePrAMPs was mostly independent of SbmA, regardless of their mechanism of action. Two peptides displayed a promisingly broad spectrum of activity, with minimal inhibiting concentration MIC ? 4 µM against several bacteria of the ESKAPE group, including Pseudomonas aeruginosa and Enterococcus faecium. Our approach has led us to discover several new peptides; correlating their sequences and mechanism of action will provide useful insights for designing optimized future peptide-based antibiotics.

SUBMITTER: Sola R

PROVIDER: S-EPMC7582929 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Characterization of Cetacean Proline-Rich Antimicrobial Peptides Displaying Activity against ESKAPE Pathogens.

Sola Riccardo R Mardirossian Mario M Beckert Bertrand B Sanghez De Luna Laura L Prickett Dennis D Tossi Alessandro A Wilson Daniel N DN Scocchi Marco M

International journal of molecular sciences 20201006 19

Proline-rich antimicrobial peptides (PrAMPs) may be a valuable weapon against multi-drug resistant pathogens, combining potent antimicrobial activity with low cytotoxicity. We have identified novel PrAMPs from five cetacean species (cePrAMPs), and characterized their potency, mechanism of action and <i>in vitro</i> cytotoxicity. Despite the homology between the N-terminal of cePrAMPs and the bovine PrAMP Bac7, some differences emerged in their sequence, activity spectrum and mode of action. CePr ...[more]

PMID: 33036159

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Project description:IntroductionThis study investigated the interaction with membrane mimetic systems (LUVs), bacterial membranes, the CD spectra, and the bactericidal activity of two designed trematocine mutants, named Trem-HK and Trem-HSK. Mutants were constructed from the scaffold of Trematocine (Trem), a natural 22-amino acid AMP from the Antarctic fish Trematomus bernacchii, aiming to increase their positive charge.MethodsThe selectivity of the designed AMPs towards bacterial membranes was improved compared to Trematocine, verified by their interaction with different LUVs and their membranolytic activity. Additionally, their α-helical conformation was not influenced by the amino acid substitutions. Our findings revealed a significant enhancement in antibacterial efficacy against ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae family) pathogens for both Trem-HK and Trem-HSK.ResultsFirstly, we showed that the selectivity of the two new designed AMPs towards bacterial membranes was greatly improved compared to Trematocine, verifying their interaction with different LUVs and their membranolytic activity. We determined that their α-helical conformation was not influenced by the amino acid substitutions. We characterized the tested bacterial collection for resistance traits to different classes of antibiotics. The minimum inhibitory and bactericidal concentration (MIC and MBC) values of the ESKAPE collection were reduced by up to 80% compared to Trematocine. The bactericidal concentrations of Trematocine mutants showed important membranolytic action, evident by scanning electron microscopy, on all tested species. We further evaluated the cytotoxicity and hemolytic activity of the mutants. At 2.5 μM concentration, both mutants demonstrated low cytotoxicity and hemolysis, indicating selectivity towards bacterial cells. However, these effects increased at higher concentrations.DiscussionAssessment of in vivo toxicity using the Galleria mellonella model revealed no adverse effects in larvae treated with both mutants, even at concentrations up to 20 times higher than the lowest MIC observed for Acinetobacter baumannii, suggesting a high potential safety profile for the mutants. This study highlights the significant improvement in antibacterial efficacy achieved by increasing the positive charge of Trem-HK and Trem-HSK. This improvement was reached at the cost of reduced biocompatibility. Further research is necessary to optimize the balance between efficacy and safety for these promising AMPs.

Project description:As monotherapy, modified proline-rich antimicrobial peptides (PrAMPs) protect animals from experimental bacteremia in a dose-dependent manner. We evaluated the in vitro synergy of a modified PrAMP, A3-APO, a dimer, previously shown to inhibit the 70 kDa bacterial heat shock protein DnaK, with imipenem or colistin against two antibiotic-resistant pathogens; a carbapenemase-expressing Klebsiella pneumoniae strain K97/09 and Acinetobacter baumannii (ATCC BAA-1605). Combining antimicrobials resulted in synergy for PrAMP/colistin combination against both K. pneumoniae and A. baumannii (ΣFIC = 0.08 both) and additive activity for the A3-APO/imipenem combination against K. pneumoniae (ΣFIC = 0.53). Chex1-Arg20, (designated as ARV-1502 in preclinical development), the single chain PrAMP monomer of A3-APO, showed synergy with meropenem against a carbapenem-resistant uropathogenic Escherichia coli strain (ΣFIC = 0.38). In a murine bacteremia model using K97/09, A3-APO at 1 mg/kg demonstrated improved survival when co-administered with standard (10 mg/kg) or subtherapeutic (1 mg/kg) doses of colistin at 36 h (p < 0.05). Surprisingly, the survival benefit of A3-APO was augmented when the A3-APO dose was decreased by 50% to 0.5 mg/kg (p < 0.02) in conjunction with a subtherapeutic colistin dose (1 mg/kg). ARV-1502, as monotherapy demonstrated prolonged (>24 h) activity in a mouse Escherichia coli infection assay. Co-treatment with ARV-1502 and subtherapeutic doses of ceftazidime (150 mg/kg) was studied in a mouse model of melioidosis. ARV-1502 provided a 50% improvement in long-term (62 days) survival, but only at the lowest of 3 administered doses; survival advantage was demonstrated at 2.5 mg/kg but not at 5 or 10 mg/kg. The mortality benefit of combination therapies was not routinely accompanied by a parallel decline in blood or tissue bacterial counts in surviving animals, suggesting that the anti-infective activity of the host defense peptides (HDP) is broader than simply bacterial eradication. In fact, the hormetic effect observed in either animal models suggest that low dose HDP treatment may change the dominant mode of action in experimental bacteremia.

Project description:ESKAPE bacteria are a major cause of multidrug-resistant infections, and new drugs are urgently needed to combat these pathogens. Given the importance of iron in bacterial physiology and pathogenicity, iron uptake and metabolism have become attractive targets for the development of new antibacterial drugs. In this scenario, the FDA-approved iron mimetic metal Gallium [Ga(III)] has been successfully repurposed as an antimicrobial drug. Ga(III) disrupts ferric iron-dependent metabolic pathways, thereby inhibiting microbial growth. This work provides the first comparative assessment of the antibacterial activity of Ga(NO3)3 (GaN), Ga(III)-maltolate (GaM), and Ga(III)-protoporphyrin IX (GaPPIX), belonging to the first-, second- and third-generation of Ga(III) formulations, respectively, on ESKAPE species, including reference strains and multidrug-resistant (MDR) clinical isolates. In addition to the standard culture medium Mueller Hinton broth (MHB), iron-depleted MHB (DMHB) and RPMI-1640 supplemented with 10% human serum (HS) (RPMI-HS) were also included in Ga(III)-susceptibility tests, because of their different nutrient and iron contents. All ESKAPE species were resistant to all Ga(III) compounds in MHB and DMHB (MIC > 32 μM), except Staphylococcus aureus and Acinetobacter baumannii, which were susceptible to GaPPIX. Conversely, the antibacterial activity of GaN and GaM was very evident in RPMI-HS, in which the low iron content and the presence of HS better mimic the in vivo environment. In RPMI-HS about 50% of the strains were sensitive (MIC < 32) to GaN and GaM, both compounds showing a similar spectrum of activity, although GaM was more effective than GaN. In contrast, GaPPIX lost its antibacterial activity in RPMI-HS likely due to the presence of albumin, which binds GaPPIX and counteracts its inhibitory effect. We also demonstrated that the presence of multiple heme-uptake systems strongly influences GaPPIX susceptibility in A. baumannii. Interestingly, GaN and GaM showed only a bacteriostatic effect, whereas GaPPIX exerted a bactericidal activity on susceptible strains. Altogether, our findings raise hope for the future development of Ga(III)-based compounds in the treatment of infections caused by multidrug-resistant ESKAPE pathogens.

Dataset Information

Characterization of Cetacean Proline-Rich Antimicrobial Peptides Displaying Activity against ESKAPE Pathogens.

Publications

Characterization of Cetacean Proline-Rich Antimicrobial Peptides Displaying Activity against ESKAPE Pathogens.

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