Bioactive compounds: a goldmine for defining new strategies against pathogenic bacterial biofilms?

Most human infectious diseases are caused by microorganisms growing as biofilms. These three-dimensional self-organized communities are embedded in a dense matrix allowing microorganisms to persistently inhabit abiotic and biotic surfaces due to increased resistance to both antibiotics and effectors of the immune system. Consequently, there is an urgent need for novel strategies to control biofilm-associated infections. Natural products offer a vast array of chemical structures and possess a wide variety of biological properties; therefore, they have been and continue to be exploited in the search for potential biofilm inhibitors with a specific or multi-locus mechanism of action. This review provides an updated discussion of the major bioactive compounds isolated from several natural sources - such as plants, lichens, algae, microorganisms, animals, and humans - with the potential to inhibit biofilm formation and/or to disperse established biofilms by bacterial pathogens. Despite the very large number of bioactive products, their exact mechanism of action often remains to be clarified and, in some cases, the identity of the active molecule is still unknown. This knowledge gap should be filled thus allowing development of these products not only as novel drugs to combat bacterial biofilms, but also as antibiotic adjuvants to restore the therapeutic efficacy of current antibiotics.

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.

Effects of Lipidation on a Proline-Rich Antibacterial Peptide.

The emergence of multidrug-resistant bacteria is a worldwide health problem. Antimicrobial peptides have been recognized as potential alternatives to conventional antibiotics, but still require optimization. The proline-rich antimicrobial peptide Bac7(1-16) is active against only a limited number of Gram-negative bacteria. It kills bacteria by inhibiting protein synthesis after its internalization, which is mainly supported by the bacterial transporter SbmA. In this study, we tested two different lipidated forms of Bac7(1-16) with the aim of extending its activity against those bacterial species that lack SbmA. We linked a C12-alkyl chain or an ultrashort cationic lipopeptide Lp-I to the C-terminus of Bac7(1-16). Both the lipidated Bac-C12 and Bac-Lp-I forms acquired activity at low micromolar MIC values against several Gram-positive and Gram-negative bacteria. Moreover, unlike Bac7(1-16), Bac-C12, and Bac-Lp-I did not select resistant mutants in E. coli after 14 times of exposure to sub-MIC concentrations of the respective peptide. We demonstrated that the extended spectrum of activity and absence of de novo resistance are likely related to the acquired capability of the peptides to permeabilize cell membranes. These results indicate that C-terminal lipidation of a short proline-rich peptide profoundly alters its function and mode of action and provides useful insights into the design of novel broad-spectrum antibacterial agents.

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.

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

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.

Fragments of the Nonlytic Proline-Rich Antimicrobial Peptide Bac5 Kill Escherichia coli Cells by Inhibiting Protein Synthesis.

Unlike most antimicrobial peptides (AMPs), the main mode of action of the subclass of proline-rich antimicrobial peptides (PrAMPs) is not based on disruption of the bacterial membrane. Instead, PrAMPs exploit the inner membrane transporters SbmA and YjiL/MdtM to pass through the bacterial membrane and enter the cytosol of specific Gram-negative bacteria, where they exert an inhibitory effect on protein synthesis. Despite sharing a high proline and arginine content with other characterized PrAMPs, the PrAMP Bac5 has a low sequence identity with them. Here we investigated the mode of action of three N-terminal Bac5 fragments, Bac5(1-15), Bac5(1-25), and Bac5(1-31). We show that Bac5(1-25) and Bac5(1-31) retained excellent antimicrobial activity toward Escherichia coli and low toxicity toward eukaryotic cells, whereas Bac5(1-15) was inactive. Bac5(1-25) and Bac5(1-31) inhibited bacterial protein synthesis in vitro and in vivo Competition assays suggested that the binding site of Bac5 is within the ribosomal tunnel, where it prevents the transition from the initiation to the elongation phase of translation, as reported for other PrAMPs, such as the bovine PrAMP Bac7. Surprisingly, unlike Bac7, Bac5(1-25) exhibited species-specific inhibition, being an excellent inhibitor of protein synthesis on E. coli ribosomes but a poor inhibitor on Thermus thermophilus ribosomes. This indicates that while Bac5 most likely has an overlapping binding site with Bac7, the mode of interaction is distinct, suggesting that Bac5 fragments may be interesting alternative lead compounds for the development of new antimicrobial agents.

Induced expression of cathelicidins in trout (Oncorhynchus mykiss) challenged with four different bacterial pathogens.

Cathelicidins are an important family of antimicrobial peptide effectors of innate immunity in vertebrates. Two members of this group, CATH-1 and CATH-2, have been identified and characterized in teleosts (ray-finned fish). In this study, we investigated the expression of these genes in different tissues of rainbow trout challenged with 4 different inactivated pathogens. By using qPCR, we detected a strong induction of both cath-1 and cath-2 genes within 24 hours after intraperitoneal inoculation with Lactococcus garvieae, Yersinia ruckeri, Aeromonas salmonicida, or Flavobacterium psychrophilum cells. Up to 700-fold induction of cath-2 was observed in the spleen of animals challenged with Y. ruckeri. Moreover, we found differences in the intensity and timing of gene up-regulation in the analyzed tissues. The overall results highlight the importance of cathelicidins in the immune response mechanisms of salmonids.

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.

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.

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.

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.

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.

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.

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.

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.

Enteric YaiW is a surface-exposed outer membrane lipoprotein that affects sensitivity to an antimicrobial peptide.

yaiW is a previously uncharacterized gene found in enteric bacteria that is of particular interest because it is located adjacent to the sbmA gene, whose bacA ortholog is required for Sinorhizobium meliloti symbiosis and Brucella abortus pathogenesis. We show that yaiW is cotranscribed with sbmA in Escherichia coli and Salmonella enterica serovar Typhi and Typhimurium strains. We present evidence that the YaiW is a palmitate-modified surface exposed outer membrane lipoprotein. Since BacA function affects the very-long-chain fatty acid (VLCFA) modification of S. meliloti and B. abortus lipid A, we tested whether SbmA function might affect either the fatty acid modification of the YaiW lipoprotein or the fatty acid modification of enteric lipid A but found that it did not. Interestingly, we did observe that E. coli SbmA suppresses deficiencies in the VLCFA modification of the lipopolysaccharide of an S. meliloti bacA mutant despite the absence of VLCFA in E. coli. Finally, we found that both YaiW and SbmA positively affect the uptake of proline-rich Bac7 peptides, suggesting a possible connection between their cellular functions.

Paenilamicins from the honey bee pathogen Paenibacillus larvae are context-specific translocation inhibitors of protein synthesis.

The paenilamicins are a group of hybrid non-ribosomal peptide-polyketide compounds produced by the honey bee pathogen Paenibacillus larvae that display activity against Gram-positive pathogens, such as Staphylococcus aureus. While paenilamicins have been shown to inhibit protein synthesis, their mechanism of action has remained unclear. Here, we have determined structures of the paenilamicin PamB2 stalled ribosomes, revealing a unique binding site on the small 30S subunit located between the A- and P-site tRNAs. In addition to providing a precise description of interactions of PamB2 with the ribosome, the structures also rationalize the resistance mechanisms utilized by P. larvae. We could further demonstrate that PamB2 interferes with the translocation of mRNA and tRNAs through the ribosome during translation elongation, and that this inhibitory activity is influenced by the presence of modifications at position 37 of the A-site tRNA. Collectively, our study defines the paenilamicins as a new class of context-specific translocation inhibitors.

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.

Functional and structural study of the dimeric inner membrane protein SbmA.

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability.

Structural and biological characterization of shortened derivatives of the cathelicidin PMAP-36.

Cathelicidins, a family of host defence peptides in vertebrates, play an important role in the innate immune response, exhibiting antimicrobial activity against many bacteria, as well as viruses and fungi. This work describes the design and synthesis of shortened analogues of porcine cathelicidin PMAP-36, which contain structural changes to improve the pharmacokinetic properties. In particular, 20-mers based on PMAP-36 (residues 12-31) and 13-mers (residues 12-24) with modification of amino acid residues at critical positions and introduction of lipid moieties of different lengths were studied to identify the physical parameters, including hydrophobicity, charge, and helical structure, required to optimise their antibacterial activity. Extensive conformational analysis, performed by CD and NMR, revealed that the substitution of Pro25-Pro26 with Ala25-Lys26 increased the α-helix content of the 20-mer peptides, resulting in broad-spectrum antibacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus epidermidis strains. Interestingly, shortening to just 13 residues resulted in only a slight decrease in antibacterial activity. Furthermore, two sequences, a 13-mer and a 20-mer, did not show cytotoxicity against HaCat cells up to 64 µM, indicating that both derivatives are not only effective but also selective antimicrobial peptides. In the short peptide, the introduction of the helicogenic α-aminoisobutyric acid forced the helix toward a prevailing 310 structure, allowing the antimicrobial activity to be maintained. Preliminary tests of resistance to Ser protease chymotrypsin indicated that this modification resulted in a peptide with an increased in vivo lifespan. Thus, some of the PMAP-36 derivatives studied in this work show a good balance between chain length, antibacterial activity, and selectivity, so they represent a good starting point for the development of even more effective and proteolysis-resistant active peptides.