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Electron microscopy images of E. coli DSM 1103 incubated with Api137 (A), Api795 (B), and Api794 (C) at concentrations corresponding to 0.25 × MIC, MIC, and 4 × MIC for 1 h using cell densities of 5 × 10⁸ CFU/mL. Black bars represent 500 nm. Morphological changes are indicated with arrows: dissociated fragments (blue), small vesicle release (green), large vesicle release (red), and relocalized DNA (orange).
Source publication
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 apida...
Citations
... CLSM was used to investigate whether the PrAMPs studied enter fungal cells in a manner similar to bacterial cells (Mattiuzzo et al., 2007;Krizsan et al., 2015a), as opposed to mammalian cells (Hansen et al., 2012;Bluhm et al., 2016). This technique allows investigating whether the peptides are mainly present in the capsule and membrane region, or whether they are able to penetrate the capsule and cell membrane and reach the cytoplasm. ...
... This was confirmed in this publication, including reversed and scrambled sequences. In addition, confocal laser scanning microscopy experiments showed that Api137, among others, is not taken up into HeLa cells (Hansen et al., 2012;Bluhm et al., 2016). Similarly, PrAMPs do not have any immunomodulatory effects and do not affect dendritic cells or macrophages . ...
Background
Cryptococcosis and cryptococcal meningitis, caused by Cryptococcus neoformans infections, lead to approximately 180,000 deaths per year, primarily in developing countries. Individuals with compromised immune systems, e.g., due to HIV infection (AIDS) or chemotherapy, are particularly vulnerable. Conventional treatment options are often limited and can cause severe side effects. Therefore, this study aimed to investigate the antifungal effect of insect-derived proline-rich antimicrobial peptides (PrAMPs) against C. neoformans. These peptides are known for their low toxicity and their high efficacy in murine infection models, making them a promising alternative for treatment.
Results
A preliminary screening of the minimal inhibitory concentrations (MICs) of 20 AMPs, including the well-known PrAMPs Onc112, Api137, and Chex1Arg20 as well as the cathelicidin CRAMP against the C. neoformans strains 1841, H99, and KN99α revealed promising results, with MICs as low as 1.6 μmol/L. Subsequent investigations of selected peptides, determining their influence on fungal colony-forming units, confirmed their strong activity. The antifungal activity was affected by factors such as peptide net charge and sequence, with stronger effects at higher net charges probably due to better intracellular uptake confirmed by confocal laser scanning microscopy. Inactive scrambled peptides suggest a specific intracellular target, although scanning electron microscopy showed that PrAMPs also damaged the cell exterior for a low proportion of the cells. Possible pore formation could facilitate entry into the cytosol.
... In our study, an addition of basic Orn at the N-terminus of N6NH 2 did not improve the stability towards trypsin, serum, and SIF but slightly improved the activity against MDR A. veronii ACCC61732 compared to its parent N6NH 2 ( Figure 1B,C and Table 2), which is consistent with a previous report that found the activity of Api88 against E. coli and Klebsiella pneumoniae was enhanced by 1-fold and 127-fold, respectively, which may be attributed to the addition of Orn at its N-terminus [19]. The basic residue-Orn can increase positive net charges of peptides and most likely allow stronger electrostatic interactions with the negatively charged bacterial surface [19,32]. It has been reported that D-magainin, polybia-CP, and relevant derivatives may be of significant therapeutic potential due to their being highly resistant to proteolysis and nearly identical antibacterial activity [23,24]. ...
... An interesting characteristic was the formation of OMVs at the outer membrane of A. veronii after treatment with V112N6NH 2 (Figure S10), which was similar to the previous reports that sub-MIC Api peptides induced OMVs at the cell membrane of E. coli and P. aeruginosa [32]. Particularly, for membrane permeabilizing peptides, Gram-negative bacteria may form OMVs to remove membrane-interacting drug molecules, even if the membrane is not the final target of these peptides [32]. ...
... An interesting characteristic was the formation of OMVs at the outer membrane of A. veronii after treatment with V112N6NH 2 (Figure S10), which was similar to the previous reports that sub-MIC Api peptides induced OMVs at the cell membrane of E. coli and P. aeruginosa [32]. Particularly, for membrane permeabilizing peptides, Gram-negative bacteria may form OMVs to remove membrane-interacting drug molecules, even if the membrane is not the final target of these peptides [32]. Additionally, OMVs may be related to the overexpressed or misfolded proteins in the periplasmic space, which can trigger similar bacterial cell vesicle formations, including the outer membrane and periplasmic components, and thus help bacteria remove toxic compounds from cell surfaces [32]. ...
Aeromonas veronii is one of the main pathogens causing various diseases in humans and animals. It is currently difficult to eradicate drug-resistant A. veronii due to the biofilm formation by conventional antibiotic treatments. In this study, a marine peptide-N6NH2 and its analogs were generated by introducing Orn or replacing with D-amino acids, Val and Pro; their enzymic stability and antibacterial/antibiofilm ability against multi-drug resistant (MDR) A. veronii ACCC61732 were detected in vitro and in vivo, respectively. The results showed that DN6NH2 more rapidly killed A. veronii ACCC61732 and had higher stability in trypsin, simulated gastric/intestinal fluid, proteinase K, and mouse serum than the parent peptide-N6NH2. DN6NH2 and other analogs significantly improved the ability of N6NH2 to penetrate the outer membrane of A. veronii ACCC61732. DN6NH2, N6PNH2 and V112N6NH2 protected mice from catheter-associated biofilm infection with MDR A. veronii ACCC61732, superior to N6NH2 and CIP. DN6NH2 had more potent efficacy at a dose of 5 μmol/kg (100% survival) in a mouse peritonitis model than other analogs (50–66.67%) and CIP (83.33%), and it inhibited the bacterial translocation, downregulated pro-inflammatory cytokines, upregulated the anti-inflammatory cytokine, and ameliorated multiple-organ injuries (including the liver, spleen, lung, and kidney). These data suggest that the analogs of N6NH2 may be a candidate for novel antimicrobial and antibiofilm agents against MDR A. veronii infections.
... N-terminal mutation of apidaecins not only reinforces the interaction with unidentified intracellular target(s), but also promotes the cell-penetration efficiency [94]. Structure N-terminal Ile-Orn-and Trp-Orn-motif repeats increases the antimicrobial activity against Pseudomonas aeruginosa [95]. ...
Antimicrobial peptides (AMPs) are crucial effectors of the innate immune system. They provide the first line of defense against a variety of pathogens. AMPs display synergistic effects with conventional antibiotics, and thus present the potential for combined therapies. Insects are extremely resistant to bacterial infections. Insect AMPs are cationic and comprise less than 100 amino acids. These insect peptides exhibit an antimicrobial effect by disrupting the microbial membrane and do not easily allow microbes to develop drug resistance. Currently, membrane mechanisms underlying the antimicrobial effects of AMPs are proposed by different modes: the barrel-stave mode, toroidal-pore, carpet, and disordered toroidal-pore are the typical modes. Positive charge quantity, hydrophobic property and the secondary structure of the peptide are important for the antibacterial activity of AMPs. At present, several structural families of AMPs from insects are known (defensins, cecropins, drosocins, attacins, diptericins, ponericins, metchnikowins, and melittin), but new AMPs are frequently discovered. We reviewed the biological effects of the major insect AMPs. This review will provide further information that facilitates the study of insect AMPs and shed some light on novel microbicides.
... 12 Still, optimized analogues of the short PrAMPs apidaecin and oncocin are active against P. aeruginosa without disrupting the bacterial membrane. 13,14 This selective bacterial uptake usually prevents the translocation of short PrAMPs into mammalian cells, providing higher margins of safety that are further increased by their specific intracellular targets. 15 Chaperone DnaK and the bacterial 70S ribosome are known targets of all PrAMPs, such as apidaecin analogue Api88 16 and oncocin analogue Onc112. ...
... In contrast, the literature relies on much higher cell densities of 2%10 7 to 2%10 8 cells/mL, which results also in higher MIC values when measured for these conditions. 13 Table S1. . PAEs obtained for P. aeruginosa ATCC 27853 (7.5%10 6 cells/mL; 1 h, 37 C, 600 rpm) when incubated with PrAMPs and antibiotic comparators at concentrations of 0.1 %, 0.4 %, 1 % and 4 % MIC (white, light grey, dark grey and black bars, respectively). ...
Background:
Proline-rich antimicrobial peptides (PrAMPs) represent a promising class of potential therapeutics to treat multiresistant infections. They inhibit bacterial protein translation at the 70S ribosome by either blocking the peptide-exit tunnel (oncocin type) or trapping release factors (apidaecin type).
Objectives:
Besides direct concentration-dependent antibacterial effects, the post-antibiotic effect (PAE) is the second most important criterion of antimicrobial pharmacodynamics to be determined in vitro. Here, PAEs of 10 PrAMPs and three antibiotics against three Escherichia coli strains, Klebsiella pneumoniae ATCC 10031 and Pseudomonas aeruginosa ATCC 27853 were studied after 1 h of exposure.
Methods:
A robust high-throughput screening to determine PAEs was established, i.e. liquid handling by a 96-channel pipetting system and continuous incubation and absorbance measurement in a microplate reader.
Results:
Prolonged PAEs (≥4 h) were detected for all peptides at their MIC values against all strains; PAEs were even >10 h for Api88, Api137, Bac7(1-60) and A3-APO. The PAEs increased further at 4 × MIC. Aminoglycosides gentamicin and kanamycin usually showed lower PAEs (≤4 h) at MIC, but PAEs increased to > 10 h at 4 × MIC. Bacteriostatic chloramphenicol exhibited the shortest PAEs (<4 h).
Conclusions:
The PAEs of PrAMPs studied against Enterobacteriaceae and P. aeruginosa for the first time were typically 4-fold stronger than for conventional antibiotics. Together with their fast and irreversible uptake by bacteria, the observed prolonged PAE of PrAMPs helps to explain their high in vivo efficacy despite unfavourable pharmacokinetics.
... It enters bacterial cells through a nonpore-forming mechanism with stereospecificity 4,5 . Although the antimicrobial activity of apidaecin is relatively low compared to that of conventional antibiotics, several studies have reported the engineering of apidaecin for enhanced antimicrobial activity [6][7][8] . Attempts to identify its intracellular targets have been made using Escherichia coli as a test strain. ...
Identifying the target molecules of antimicrobial agents is essential for assessing their mode of action. Here, we propose Acquired Resistance induced by Gene Overexpression (ARGO) as a novel in vivo approach for exploring target proteins of antimicrobial agents. The principle of the method is based on the fact that overexpression of the expected target protein leads to reduced sensitivity to the antimicrobial agent. We applied this approach to identify target proteins of the antimicrobial peptide apidaecin, which is specifically effective against Gram-negative bacteria. To this end, a set of overexpression Escherichia coli clones was tested, and peptide chain release factor 1, which directs the termination of translation, was found as a candidate, suggesting that apidaecin inhibits the termination step of translation. This finding was confirmed in vivo and in vitro by evaluating the inhibitory activity of apidaecin towards lacZ reporter gene expression, which is tightly dependent on its stop codon. The results of this study demonstrate that apidaecin exerts its antimicrobial effects partly by inhibiting release factors.
... Myticalins A5, A8, C9 and D2 displayed a broad spectrum of activity, being active against Gram-positive and -negative bacteria, although their potential activity in the natural marine environment remains to be ascertained. The spectrum of antibacterial activity of the Pro-rich myticalins A5, A8 and D2 interestingly matches that of well characterized linear Pro-rich peptides (PR-AMPs) previously identified in insects, crustacean and mammals [59], which are mainly active against Gram-negative species, especially Escherichia coli, Acinetobacter baumannii and, to a lesser extent, Pseudomonas aeruginosa [60,61], but also displayed a significant activity against the Gram-positive bacteria Bacillus subtilis. This selectivity derives from the fact that PR-AMPs act internally, and require a specific cytoplasmic membrane protein transport system to translocate to the cytoplasm, which is present in some Gram-negative bacterial species (e.g., E. coli and A. baumannii), but not in others (e.g., P. aeruginosa) or Gram-positive ones [60,61]. ...
... The spectrum of antibacterial activity of the Pro-rich myticalins A5, A8 and D2 interestingly matches that of well characterized linear Pro-rich peptides (PR-AMPs) previously identified in insects, crustacean and mammals [59], which are mainly active against Gram-negative species, especially Escherichia coli, Acinetobacter baumannii and, to a lesser extent, Pseudomonas aeruginosa [60,61], but also displayed a significant activity against the Gram-positive bacteria Bacillus subtilis. This selectivity derives from the fact that PR-AMPs act internally, and require a specific cytoplasmic membrane protein transport system to translocate to the cytoplasm, which is present in some Gram-negative bacterial species (e.g., E. coli and A. baumannii), but not in others (e.g., P. aeruginosa) or Gram-positive ones [60,61]. It is worth taking into account that a peculiar, lytic and not yet fully characterized mode of action has been suggested for PR-AMPs against P. aeruginosa, instead of the canonical non-lytic mechanism [62]. ...
The application of high-throughput sequencing technologies to non-model organisms has brought new opportunities for the identification of bioactive peptides from genomes and transcriptomes. From this point of view, marine invertebrates represent a potentially rich, yet largely unexplored resource for de novo discovery due to their adaptation to diverse challenging habitats. Bioinformatics analyses of available genomic and transcriptomic data allowed us to identify myticalins, a novel family of antimicrobial peptides (AMPs) from the mussel Mytilus galloprovincialis, and a similar family of AMPs from Modiolus spp., named modiocalins. Their coding sequence encompasses two conserved N-terminal (signal peptide) and C-terminal (propeptide) regions and a hypervariable central cationic region corresponding to the mature peptide. Myticalins are taxonomically restricted to Mytiloida and they can be classified into four subfamilies. These AMPs are subject to considerable interindividual sequence variability and possibly to presence/absence variation. Functional assays performed on selected members of this family indicate a remarkable tissue-specific expression (in gills) and broad spectrum of activity against both Gram-positive and Gram-negative bacteria. Overall, we present the first linear AMPs ever described in marine mussels and confirm the great potential of bioinformatics tools for the de novo discovery of bioactive peptides in non-model organisms.
... Therefore, the recovered peptide amounts in kidneys of Api137 treated animals (2.4 µg/g) were slightly higher than in Api88 treated animals (1.8 µg/g). Notably, PrAMPs usually do not penetrate mammalian cells, but immune cells and HeLa cancer cells can internalize apidaecins and Bac7(1-35) after long incubation times (Tavano et al., 2011;Hansen et al., 2012;Pelillo et al., 2014;Bluhm et al., 2016). However, it is unlikely that PrAMPs internalized in kidney cells within 10 min (first time point of the pharmacokinetic study). ...
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.
With the growing crisis of antimicrobial resistance, it is critical to continue to seek out new sources of novel antibiotics. This need has led to renewed interest in natural product antimicrobials, specifically antimicrobial peptides. Nonlytic antimicrobial peptides are highly promising due to their unique mechanisms of action. One such peptide is apidaecin (Api), which inhibits translation termination through stabilization of the quaternary complex of the ribosome-apidaecin-tRNA-release factor. Synthetic derivatives of apidaecin have been developed, but structure-guided modifications have yet to be considered. In this work, we have focused on modifying key residues in the Api sequence that are responsible for the interactions that stabilize the quaternary complex. We present one of the first examples of a highly modified Api peptide that maintains its antimicrobial activity and interaction with the translation complex. These findings establish a starting point for further structure-guided optimization of Api peptides.
The rapid emergence of multi-drug resistant microorganisms has become one of the most critical threats to public health. A decrease in the effectiveness of available antibiotics has led to the failure of infection control, resulting in a high risk of death. Among several alternatives, antimicrobial peptides serve as potential alternatives to antibiotics to resolve the emergence and spread of multidrug-resistant pathogens. These small proteins exhibit potent antimicrobial activity and are also an essential component of the immune system. Though several AMPs have been reported and characterized, studies associated with their potential medical applications are limited. This review highlights the novel sources of AMPs with high antimicrobial activities, including the entomopathogenic nematode/bacterium (EPN/EPB) symbiotic complex. Additionally, the AMPs derived from insects, nematodes, marine organisms and the design of peptidomimetic antimicrobial agents that can complement the defects of therapeutic peptides have been used as a template.
Proline‐rich antimicrobial peptides expressed in insects are primarily active against Enterobacteriaceae. Mechanistically, they target the bacterial (70S) ribosome after partially transporter‐based cellular uptake, as revealed for Api137 and Onc112 on Escherichia coli. Following molecular modeling indicating that the Onc112 contact site is conserved among the ribosomes of high‐priority pathogens, the ribosome binding of Api137 and Onc112 was studied. The dissociation constants (Kd) of Onc112 were ∼75 nmol/L for Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, 36 nmol/L for Pseudomonas aeruginosa, and 102 nmol/L for Staphylococcus aureus, thus indicating a very promising lead structure for developing broad‐spectrum antibiotics. Api137 bound weaker with Kd values ranging from 155 nmol/L to 13 μmol/L. For most bacteria, the antibacterial activities were lower than predicted from the Kd values, which was only partially explained by their ability to enter bacterial cells. Other factors limiting the activity expected from the ribosome binding might be off‐target binding.
