Designing New Hybrid Antibiotics: Proline-Rich Antimicrobial Peptides Conjugated to the Aminoglycoside Tobramycin.

Resistance to aminoglycoside antibiotics is a serious problem, typically arising from inactivating enzymes, reduced uptake, or increased efflux in the important pathogens for which they are used as treatment. Conjugating aminoglycosides to proline-rich antimicrobial peptides (PrAMPs), which also target ribosomes and have a distinct bacterial uptake mechanism, might mutually benefit their individual activities. To this aim we have developed a strategy for noninvasively modifying tobramycin to link it to a Cys residue and through this covalently link it to a Cys-modified PrAMP by formation of a disulfide bond. Reduction of this bridge in the bacterial cytosol should release the individual antimicrobial moieties. We found that the conjugation of tobramycin to the well-characterized N-terminal PrAMP fragment Bac7(1-35) resulted in a potent antimicrobial capable of inactivating not only tobramycin-resistant bacterial strains but also those less susceptible to the PrAMP. To a certain extent, this activity also extends to the shorter and otherwise poorly active fragment Bac7(1-15). Although the mechanism that allows the conjugate to act when its individual components do not is as yet unclear, results are very promising and suggest this may be a way of resensitizing pathogens that have developed resistance to the antibiotic.

Natural and Synthetic Halogenated Amino Acids-Structural and Bioactive Features in Antimicrobial Peptides and Peptidomimetics.

The 3D structure and surface characteristics of proteins and peptides are crucial for interactions with receptors or ligands and can be modified to some extent to modulate their biological roles and pharmacological activities. The introduction of halogen atoms on the side-chains of amino acids is a powerful tool for effecting this type of tuning, influencing both the physico-chemical and structural properties of the modified polypeptides, helping to first dissect and then rationally modify features that affect their mode of action. This review provides examples of the influence of different types of halogenation in amino acids that replace native residues in proteins and peptides. Examples of synthetic strategies for obtaining halogenated amino acids are also provided, focusing on some representative compounds and their biological effects. The role of halogenation in native and designed antimicrobial peptides (AMPs) and their mimetics is then discussed. These are in the spotlight for the development of new antimicrobial drugs to counter the rise of antibiotic-resistant pathogens. AMPs represent an interesting model to study the role that natural halogenation has on their mode of action and also to understand how artificially halogenated residues can be used to rationally modify and optimize AMPs for pharmaceutical purposes.

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.

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.

Antimicrobial and host cell-directed activities of Gly/Ser-rich peptides from salmonid cathelicidins.

Cathelicidins, a major family of vertebrate antimicrobial peptides (AMPs), have a recognized role in the first line of defense against infections. They have been identified in several salmonid species, where the putative mature peptides are unusually long and rich in serine and glycine residues, often arranged in short multiple repeats (RLGGGS/RPGGGS) intercalated by hydrophobic motifs. Fragments of 24-40 residues, spanning specific motifs and conserved sequences in grayling or brown, rainbow and brook trout, were chemically synthesized and examined for antimicrobial activity against relevant Gram-positive and Gram-negative salmonid pathogens, as well as laboratory reference strains. They were not active in complete medium, but showed varying potency and activity spectra in diluted media. Bacterial membrane permeabilization also occurred only under these conditions and was indicated by rapid propidium iodide uptake in peptide-treated bacteria. However, circular dichroism analyses indicated that they did not significantly adopt ordered conformations in membrane-like environments. The peptides were not hemolytic or cytotoxic to trout cells, including freshly purified head kidney leukocytes (HKL) and the fibroblastic RTG-2 cell line. Notably, when exposed to them, HKL showed increased metabolic activity, while a growth-promoting effect was observed on RTG-2 cells, suggesting a functional interaction of salmonid cathelicidins with host cells similar to that shown by mammalian ones. The three most active peptides produced a dose-dependent increase in phagocytic uptake by HKL simultaneously stimulated with bacterial particles. The peptide STF(1-37), selected for further analyses, also enhanced phagocytic uptake in the presence of autologous serum, and increased intracellular killing of live E. coli. Furthermore, when tested on HKL in combination with the immunostimulant ß-glucan, it synergistically potentiated both phagocytic uptake and the respiratory burst response, activities that play a key role in fish immunity. Collectively, these data point to a role of salmonid cathelicidins as modulators of fish microbicidal mechanisms beyond a salt-sensitive antimicrobial activity, and encourage further studies also in view of potential applications in aquaculture.

In vitro and in vivo evaluation of BMAP-derived peptides for the treatment of cystic fibrosis-related pulmonary infections.

Patients with cystic fibrosis require pharmacological treatment against chronic lung infections. The alpha-helical antimicrobial peptides BMAP-27 and BMAP-28 have shown to be highly active in vitro against planktonic and sessile forms of multidrug-resistant Pseudomonas aeruginosa, Staphylococcus aureus, and Stenotrophomonas maltophilia cystic fibrosis strains. To develop small antibacterial peptides for therapeutic use, we tested shortened/modified BMAP fragments, and selected the one with the highest in vitro antibacterial activity and lowest in vivo acute pulmonary toxicity. All the new peptides have shown to roughly maintain their antibacterial activity in vitro. The 1-18 N-terminal fragment of BMAP-27, showing MIC90 of 16 µg/ml against P. aeruginosa isolates and strain-dependent anti-biofilm effects, showed the lowest pulmonary toxicity in mice. However, when tested in a murine model of acute lung infection by P. aeruginosa, BMAP-27(1-18) did not show any curative effect. If exposed to murine broncho-alveolar lavage fluid BMAP-27(1-18) was degraded within 10 min, suggesting it is not stable in pulmonary environment, probably due to murine proteases. Our results indicate that shortened BMAP peptides could represent a starting point for antibacterial drugs, but they also indicate that they need a further optimization for effective in vivo use.

The human cathelicidin LL-37--A pore-forming antibacterial peptide and host-cell modulator.

The human cathelicidin hCAP18/LL-37 has become a paradigm for the pleiotropic roles of peptides in host defence. It has a remarkably wide functional repertoire that includes direct antimicrobial activities against various types of microorganisms, the role of 'alarmin' that helps to orchestrate the immune response to infection, the capacity to locally modulate inflammation both enhancing it to aid in combating infection and limiting it to prevent damage to infected tissues, the promotion of angiogenesis and wound healing, and possibly also the elimination of abnormal cells. LL-37 manages to carry out all its reported activities with a small and simple, amphipathic, helical structure. In this review we consider how different aspects of its primary and secondary structures, as well as its marked tendency to form oligomers under physiological solution conditions and then bind to molecular surfaces as such, explain some of its cytotoxic and immunomodulatory effects. We consider its modes of interaction with bacterial membranes and capacity to act as a pore-forming toxin directed by our organism against bacterial cells, contrasting this with the mode of action of related peptides from other species. We also consider its different membrane-dependent effects on our own cells, which underlie many of its other activities in host defence. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

Helical peptide-polyamine and -polyether conjugates as synthetic ionophores.

Two new synthetic ionophores in which the hydrophobic portion is represented by a short helical Aib-peptide (Aib=α-amino-isobutyric acid) and the hydrophilic one is a poly-amino (1a) or a polyether (1b) chain have been prepared. The two conjugates show a high ionophoric activity in phospholipid membranes being able to efficiently dissipate a pH gradient and, in the case of 1b, to transport Na(+) across the membrane. Bioactivity evaluation of the two conjugates shows that 1a has a moderate antimicrobial activity against a broad spectrum of microorganisms and it is able to permeabilize the inner and the outer membrane of Escherichia coli cells.

Lipopolysaccharide Phosphorylation by the WaaY Kinase Affects the Susceptibility of Escherichia coli to the Human Antimicrobial Peptide LL-37.

The human cathelicidin LL-37 is a multifunctional host defense peptide with immunomodulatory and antimicrobial roles. It kills bacteria primarily by altering membrane barrier properties, although the exact sequence of events leading to cell lysis has not yet been completely elucidated. Random insertion mutagenesis allowed isolation of Escherichia coli mutants with altered susceptibility to LL-37, pointing to factors potentially relevant to its activity. Among these, inactivation of the waaY gene, encoding a kinase responsible for heptose II phosphorylation in the LPS inner core, leads to a phenotype with decreased susceptibility to LL-37, stemming from a reduced amount of peptide binding to the surface of the cells, and a diminished capacity to lyse membranes. This points to a specific role of the LPS inner core in guiding LL-37 to the surface of Gram-negative bacteria. Although electrostatic interactions are clearly relevant, the susceptibility of the waaY mutant to other cationic helical cathelicidins was unaffected, indicating that particular structural features or LL-37 play a role in this interaction.

PEGylation of the peptide Bac7(1-35) reduces renal clearance while retaining antibacterial activity and bacterial cell penetration capacity.

The proline-rich antibacterial peptide Bac7(1-35) protects mice against Salmonella typhimurium infection, despite its rapid clearance. To overcome this problem the peptide was linked to a polyethylene glycol (PEG) molecule either via a cleavable ester bond or via a non-hydrolysable amide bond. Both the PEGylated conjugates retained most of the in vitro activity against S. typhimurium. In addition, the ester bond was cleaved in human serum or plasma, releasing a carboxymethyl derivative of Bac7(1-35) which accounts for a higher activity of this peptide with relative to the other, non-hydrolysable form. Both PEGylated peptides maintained the capacity of the unconjugated form to kill bacteria without permeabilizing the bacterial membranes, by penetrating into cells. They exploited the same transporter as unmodified Bac7(1-35), suggesting it has the capacity to internalize quite sizeable cargo if this is linked to Bac7 fragment. PEGylation allows the peptide to have a wide distribution in mice, and a slow renal clearance, indicating that this strategy would improve the bioavailability of Bac7, and in principle of other antimicrobial peptides. This can be an equally important issue to reducing cytotoxicity for therapeutic use of these antibacterials.

Effect of size and N-terminal residue characteristics on bacterial cell penetration and antibacterial activity of the proline-rich peptide Bac7.

Bac7 is a proline-rich antimicrobial peptide, selective for Gram-negative bacteria, which acts intracellularly after membrane translocation. Progressively shortened fragments of Bac7 allowed determining the minimal sequence required for entry and antimicrobial activity as a 16-residue, N-terminal fragment, while further shortening led to a marked decrease in both functions. Furthermore, two N-terminal arginine residues were required for efficient translocation and activity. Analogues in which these residues were omitted, or where the side chain steric or physicochemical characteristics were systematically altered, were tested on different Escherichia coli strains, including a mutant with a destabilized outer membrane and one lacking the relevant SbmA membrane transport protein. H-bonding capacity, stereochemistry, and charge, in that order, played a determining role for efficient transit through both the outer and cytoplasmic membranes. Our studies allowed building a more detailed model for the mode-of-action of Bac7, and confirming its potential as an anti-infective agent, also suggesting it may be a vehicle for internalization of other antibiotic cargo.

The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 µM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis.

Proteolytic activity of Escherichia coli oligopeptidase B against proline-rich antimicrobial peptides.

Oligopeptidase B (OpdB) is a serine peptidase widespread among bacteria and protozoa that has emerged as a virulence factor despite its function has not yet been precisely established. By using an OpdB-overexpressing Escherichia coli strain, we found that the overexpressed peptidase makes the bacterial cells specifically less susceptible to several proline-rich antimicrobial peptides known to penetrate into the bacterial cytosol, and that its level of activity directly correlates with the degree of resistance. We established that E. coli OpdB can efficiently hydrolyze in vitro cationic antimicrobial peptides up to 30 residues in length, even though they contained several prolines, shortening them to inactive fragments. Two consecutive basic residues are a preferred cleavage site for the peptidase. In the case of a single basic residue, there is no cleavage if proline residues are present in the P1 and P2 positions. These results also indicate that cytosolic peptidases may cause resistance to antimicrobial peptides that have an intracellular mechanism of action, such as the proline-rich peptides, and may contribute to define the substrate specificity of the E. coli OpdB.

Cellular internalization and cytotoxicity of the antimicrobial proline-rich peptide Bac7(1-35) in monocytes/macrophages, and its activity against phagocytosed Salmonella typhimurium.

Bac7(1-35) is an active fragment of the bovine cathelicidin antimicrobial peptide Bac7, which selectively inactivates Gram-negative bacteria both in vitro and in mice infected with Salmonella typhimurium. It has a non-lytic mechanism of action, is rapidly internalized by susceptible bacteria and mammalian cells and likely acts by binding to internal targets. In this study we show that Bac7(1-35) accumulates selectively within primed macrophages with respect to resting monocytes. Confocal microscopy analysis showed that the peptide mainly distributes in the cytoplasm and perinuclear region of macrophages within 3 hours of incubation, without affecting cell viability. Cytotoxicity studies showed that the peptide does not induce necrotic or apoptotic damage up to concentrations 50-100-fold higher than minimal inhibitory concentrations (MIC). Moreover, Bac7(1-35) did not affect the ability of macrophages to engulf S. typhimurium, a species that may proliferate within this cell type. Conversely, when added to macrophages after phagocytosis, Bac7(1-35) caused a significant reduction in the number of recovered bacteria, indicating that it can kill the engulfed microorganisms directly and/or indirectly, via activation of the defense response of the cells.

Functional characterization of SbmA, a bacterial inner membrane transporter required for importing the antimicrobial peptide Bac7(1-35).

SbmA is an inner membrane protein of Gram-negative bacteria that is involved in the internalization of glycopeptides and prokaryotic and eukaryotic antimicrobial peptides, as well as of peptide nucleic acid (PNA) oligomers. The SbmA homolog BacA is required for the development of Sinorhizobium meliloti bacteroids within plant cells and favors chronic infections with Brucella abortus and Mycobacterium tuberculosis in mice. Here, we investigated functional features of SbmA/BacA using the proline-rich antimicrobial peptide Bac7(1-35) as a substrate. Circular dichroism and affinity chromatography studies were used to investigate the ability of SbmA to bind the peptide, and a whole-cell transport assay with fluorescently labeled peptide allowed the determination of transport kinetic parameters with a calculated Km value of 6.95 ± 0.89 µM peptide and a Vmax of 53.91 ± 3.17 nmol/min/mg SbmA. Use of a bacterial two-hybrid system coupled to SEC-MALLS (size exclusion chromatography coupled with multiangle laser light scattering) analyses established that SbmA is a homodimer in the membrane, and treatment of the cells with arsenate or ionophores indicated that the peptide transport mediated by SbmA is driven by the electrochemical gradient. Overall, these results shed light on the SbmA-mediated internalization of peptide substrates and suggest that the transport of an unknown substrate(s) represents the function of this protein.

Potential novel therapeutic strategies in cystic fibrosis: antimicrobial and anti-biofilm activity of natural and designed α-helical peptides against Staphylococcus aureus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia.

BACKGROUND: Treatment of cystic fibrosis-associated lung infections is hampered by the presence of multi-drug resistant pathogens, many of which are also strong biofilm producers. Antimicrobial peptides, essential components of innate immunity in humans and animals, exhibit relevant in vitro antimicrobial activity although they tend not to select for resistant strains. RESULTS: Three α-helical antimicrobial peptides, BMAP-27 and BMAP-28 of bovine origin, and the artificial P19(9/B) peptide were tested, comparatively to Tobramycin, for their in vitro antibacterial and anti-biofilm activity against 15 Staphylococcus aureus, 25 Pseudomonas aeruginosa, and 27 Stenotrophomonas maltophilia strains from cystic fibrosis patients. All assays were carried out in physical-chemical experimental conditions simulating a cystic fibrosis lung. All peptides showed a potent and rapid bactericidal activity against most P. aeruginosa, S. maltophilia and S. aureus strains tested, at levels generally higher than those exhibited by Tobramycin and significantly reduced biofilm formation of all the bacterial species tested, although less effectively than Tobramycin did. On the contrary, the viability-reducing activity of antimicrobial peptides against preformed P. aeruginosa biofilms was comparable to and, in some cases, higher than that showed by Tobramycin. CONCLUSIONS: The activity shown by α-helical peptides against planktonic and biofilm cells makes them promising "lead compounds" for future development of novel drugs for therapeutic treatment of cystic fibrosis lung disease.

Use of unnatural amino acids to probe structure-activity relationships and mode-of-action of antimicrobial peptides.

Endogenous antimicrobial peptides (AMPs) can have multimodal mechanisms of bacterial inactivation, such as membrane lysis, interference with cell wall biosynthesis or membrane-based protein machineries, or translocation through the membrane to intracellular targets. The controlled variation of side-chain characteristics in their amino acid residues can provide much useful information on structure-activity relationships and mode-of-action, and also lead to improved activities. The small size and relatively low complexity of AMPs make them amenable to solid-phase peptide synthesis, facilitating the use of nonproteinogenic amino acids and vastly increasing the accessible molecular diversity of side chains. Here, we describe how such residues can be used to modulate such key parameters as cationicity, hydrophobicity, steric factors conformational stability, and H-bonding.

Activity of Cathelicidin Peptides against Simkania negevensis.

The in vitro activity of six cathelicidin peptides against the reference strain Z of Simkania negevensis was investigated. Five peptides-PG-1, Bac7, SMAP-29, BMAP-27, and BMAP-28-proved to be active at very low concentrations (1 to 0.1 µg/mL), while LL-37 cathelicidin was ineffective even at a concentration of 100 µg/mL. In comparison to chlamydiae, S. negevensis proved to be more susceptible to the antimicrobial peptides tested.

Proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action.

Proline-rich antimicrobial peptides are a group of cationic host defense peptides of vertebrates and invertebrates characterized by a high content of proline residues, often associated with arginine residues in repeated motifs. Those isolated from some mammalian and insect species, although not evolutionarily related, use a similar mechanism to selectively kill Gram-negative bacteria, with a low toxicity to animals. Unlike other types of antimicrobial peptides, their mode of action does not involve the lysis of bacterial membranes but entails penetration into susceptible cells, where they then act intracellularly. Some aspects of the transport system and cytoplasmic targets have been elucidated. These features make them attractive both as anti-infective lead compounds and as a new class of potential cell-penetrating peptides capable of internalising membrane-impermeant drugs into both bacterial and eukaryotic cells.

Antifungal activity of amphotericin B conjugated to carbon nanotubes.

Amphotericin B (AMB) has long been considered the most effective drug in the treatment of serious invasive fungal infections. There are, however, major limitations to its use, due to several adverse effects, including acute infusional reactions and, most relevant, a dose-dependent nephrotoxicity. At least some of these effects are attributed to the aggregation of AMB as a result of its poor water solubility. To overcome this problem, reformulated versions of the drug have been developed, including a micellar dispersion of AMB with sodium deoxycholate (AMBD), its encapsulation into liposomes, or its incorporation into lipidic complexes. The development of nanobiotechnologies provides novel potential drug delivery systems that make use of nanomaterials such as functionalized carbon nanotubes (f-CNTs), which are emerging as an innovative and efficient tool for the transport and cellular translocation of therapeutic molecules. In this study, we prepared two conjugates between f-CNTs and AMB. The antifungal activity of these conjugates was tested against a collection of reference and clinical fungal strains, in comparison to that of AMB alone or AMBD. Measured minimum inhibition concentration (MIC) values for f-CNT-AMB conjugates were either comparable to or better than those displayed by AMB and AMBD. Furthermore, AMBD-resistant Candida strains were found to be susceptible to f-CNT-AMB 1. Additional studies, aimed at understanding the mechanism of action of the conjugates, suggest a nonlytic mechanism, since the compounds show a major permeabilizing effect on the tested fungal strains only after extended incubation. Interestingly, the f-CNT-AMB 1 does not show any significant toxic effect on Jurkat cells at antifungal concentrations.