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Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein lead to persistent lung bacterial infections, mainly due to Pseudomonas aeruginosa, causing loss of respiratory function and finally death of people affected by CF. Unfortunately, even in the era of CFTR modulation therapies, management of pulmonary infections in CF remains highly challenging especially for patients with advanced stages of lung disease. Recently, we identified antimicrobial peptides (AMPs), namely Esc peptides, with potent antipseudomonal activity. In this study, by means of electrophysiological techniques and computational studies we discovered their ability to increase the CFTR-controlled ion currents, by direct interaction with the F508del-CFTR mutant. Remarkably, this property was not explored previously with any AMPs or peptides in general. More interestingly, in contrast with clinically used CFTR modulators, Esc peptides would give particular benefit to CF patients by combining their capability to eradicate lung infections and to act as promoters of airway wound repair with their ability to ameliorate the activity of the channel with conductance defects. Overall, our findings not only highlighted Esc peptides as the first characterized AMPs with a novel property, that is the potentiator activity of CFTR, but also paved the avenue to investigate the functions of AMPs and/or other peptide molecules, for a new up-and-coming pharmacological approach to address CF lung disease. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-04030-2.

Bacterial biofilms are a serious threat for human health, and the Gram-positive bacterium Staphylococcus aureus is one of the microorganisms that can easily switch from a planktonic to a sessile lifestyle, providing protection from a large variety of adverse environmental conditions. Dormant non-dividing cells with low metabolic activity, named persisters, are tolerant to antibiotic treatment and are the principal cause of recalcitrant and resistant infections, including skin infections. Antimicrobial peptides (AMPs) hold promise as new anti-infective agents to treat such infections. Here for the first time, we investigated the activity of the frog-skin AMP temporin G (TG) against preformed S. aureus biofilm including persisters, as well as its efficacy in combination with tobramycin, in inhibiting S. aureus growth. TG was found to provoke ~50 to 100% reduction of biofilm viability in the concentration range from 12.5 to 100 &micro, M vs ATCC and clinical isolates and to be active against persister cells (about 70&ndash, 80% killing at 50&ndash, 100 &micro, M). Notably, sub-inhibitory concentrations of TG in combination with tobramycin were able to significantly reduce S. aureus growth, potentiating the antibiotic power. No critical cytotoxicity was detected when TG was tested in vitro up to 100 &micro, M against human keratinocytes, confirming its safety profile for the development of a new potential anti-infective drug, especially for treatment of bacterial skin infections.

The activity of many antibiotics depends on the initial density of cells used in bacteria growth inhibition assays. This phenomenon, termed the inoculum effect, can have important consequences for the therapeutic efficacy of the drugs, since bacterial loads vary by several orders of magnitude in clinically relevant infections. Antimicrobial peptides are a promising class of molecules to fight drug-resistant bacteria, since they act mainly by perturbing the cell membranes rather than by inhibiting intracellular targets. Here we report the first systematic characterization of the inoculum effect for this class of antibacterial compounds. Thirteen peptides (including all-D enantiomers) and peptidomimetics were analyzed by measuring minimum inhibitory concentration values, covering more than 7 orders of magnitude in inoculated cell density. In all cases, we observed a significant inoculum effect for cell densities above 5 × 104cells/mL, while the active concentrations remained constant (within the micromolar range) for lower densities. In the case of membrane-active peptides, these data can be rationalized by considering a simple model, taking into account peptide-cell association and hypothesizing that a threshold number of cell-bound peptide molecules is required in order to cause a killing effect. The observed effects question the clinical utility of activity and selectivity determinations performed at a fixed, standardized cell density. A routine evaluation of the inoculum dependence of the activity of antimicrobial peptides and peptidomimetics should be considered.Significance statementBacterial drug resistance is a crucial threat to global health and antimicrobials with novel mechanisms of action are severely needed. Antimicrobial peptides are natural molecules that kill bacteria mostly by perturbing their membranes and represent promising compounds to fight resistant microbes. Their activity is normally tested under standardized conditions of bacterial density. However, the bacterial load in clinically relevant infections varies by many orders of magnitude. Here we showed that the minimum peptide concentration needed for bacterial killing can vary by more than 100 times with an increase in the density of cells in the initial inoculum of the assay (inoculum effect) These findings question utility of the presently used activity screening assays and our current understanding of antimicrobial peptides.

Antibiotic resistance is now considered a worldwide problem that puts public health at risk. The onset of bacterial strains resistant to conventional antibiotics and the scarcity of new drugs have prompted scientific research to re-evaluate natural products as molecules with high biological and chemical potential. A class of natural compounds of significant importance is represented by alkaloids derived from higher plants. In this review, we have collected data obtained from various research groups on the antimicrobial activities of these alkaloids against conventional antibiotic-resistant strains. In addition, the structure–function relationship was described and commented on, highlighting the high potential of alkaloids as antimicrobials.

BackgroundColistin is a last-resort treatment option for many MDR Gram-negative bacteria. The covalent addition of l-aminoarabinose to the lipid A moiety of LPS is the main colistin resistance mechanism in the human pathogen Pseudomonas aeruginosa.ObjectivesIdentification (by in silico screening of a chemical library) of potential inhibitors of ArnT, which catalyses the last committed step of lipid A aminoarabinosylation, and their validation in vitro as colistin adjuvants.MethodsThe available ArnT crystal structure was used for a docking-based virtual screening of an in-house library of natural products. The resulting putative ArnT inhibitors were tested in growth inhibition assays using a reference colistin-resistant P. aeruginosa strain. The most promising compound was further characterized for its range of activity, specificity and cytotoxicity. Additionally, the effect of the compound on lipid A aminoarabinosylation was verified by MS analyses of lipid A.ResultsA putative ArnT inhibitor (BBN149) was discovered by molecular docking and demonstrated to specifically potentiate colistin activity in colistin-resistant P. aeruginosa isolates, without relevant effect on colistin-susceptible strains. BBN149 also showed adjuvant activity against colistin-resistant Klebsiella pneumoniae and low toxicity to bronchial epithelial cells. Lipid A aminoarabinosylation was reduced in BBN149-treated cells, although only partially.ConclusionsThis study demonstrates that in silico screening targeting ArnT can successfully identify inhibitors of colistin resistance and provides a promising lead compound for the development of colistin adjuvants for the treatment of MDR bacterial infections.

Antimicrobial peptides (AMPs) selectively kill bacteria by disrupting their cell membranes, and are promising compounds to fight drug-resistant microbes. Biophysical studies on model membranes have characterized AMP/membrane interactions and the mechanism of bilayer perturbation, showing that accumulation of cationic peptide molecules in the external leaflet leads to the formation of pores ("carpet" mechanism). However, similar quantitative studies on real cells are extremely limited. Here, we investigated the interaction of the dansylated PMAP23 peptide (DNS-PMAP23) with a Gram-positive bacterium, showing that 107 bound peptide molecules per cell are needed to kill it. This result is consistent with our previous finding for Gram-negative strains, where a similar high threshold for killing was determined, demonstrating the general relevance of the carpet model for real bacteria. However, in the case of the Gram-positive strain, this number of molecules even exceeds the total surface available on the bacterial membrane. The high affinity of DNS-PMAP23 for the anionic teichoic acids of the Gram-positive cell wall, but not for the lipopolysaccharides of Gram-negative bacteria, provides a rationale for this finding. To better define the role of anionic lipids in peptide/cell association, we studied DNS-PMAP23 interaction with E. coli mutant strains lacking phosphatidylglycerol and/or cardiolipin. Surprisingly, these strains showed a peptide affinity similar to that of the wild type. This finding was rationalized by observing that these bacteria have an increased content of other anionic lipids, thus maintaining the total membrane charge essentially constant. Finally, studies of DNS-PMAP23 association to dead bacteria showed an affinity an order of magnitude higher compared to that of live cells, suggesting strong peptide binding to intracellular components that become accessible after membrane perturbation. This effect could play a role in population resistance to AMP action, since dead bacteria could protect the surviving cells by sequestering significant amounts of peptide molecules. Overall, our data indicate that quantitative studies of peptide association to bacteria can lead to a better understanding of the mechanism of action of AMPs.

Antimicrobial peptides (AMPs) represent new alternatives to cope with the increasing number of multi-drug resistant microbial infections. Recently, a derivative of the frog-skin AMP esculentin-1a, Esc(1-21), was found to rapidly kill both the planktonic and biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action. Lately, its diastereomer Esc(1-21)-1c containing two d-amino acids i.e. DLeu14 and DSer17 revealed to be less cytotoxic, more stable to proteolytic degradation and more efficient in eradicating Pseudomonas biofilm. When tested in vitro against the free-living form of this pathogen, it displayed potent bactericidal activity, but this was weaker than that of the all-l peptide. To investigate the reason accounting for this difference, mechanistic studies were performed on Pseudomonas spheroplasts and anionic or zwitterionic membranes, mimicking the composition of microbial and mammalian membranes, respectively. Furthermore, structural studies by means of optical and nuclear magnetic resonance spectroscopies were carried out. Our results suggest that the different extent in the bactericidal activity between the two isomers is principally due to differences in their interaction with the bacterial cell wall components. Indeed, the lower ability in binding and perturbing anionic phospholipid bilayers for Esc(1-21)-1c contributes only in a small part to this difference, while the final effect of membrane thinning once the peptide is inserted into the membrane is identical to that provoked by Esc(1-21). In addition, the presence of two d-amino acids is sufficient to reduce the α-helical content of the peptide, in parallel with its lower cytotoxicity.

Staphylococcus aureus is a major human pathogen causing a wide range of nosocomial infections including pulmonary, urinary, and skin infections. Notably, the emergence of bacterial strains resistant to conventional antibiotics has prompted researchers to find new compounds capable of killing these pathogens. Nature is undoubtedly an invaluable source of bioactive molecules characterized by an ample chemical diversity. They can act as unique platform providing new scaffolds for further chemical modifications in order to obtain compounds with optimized biological activity. A class of natural compounds with a variety of biological activities is represented by alkaloids, important secondary metabolites produced by a large number of organisms including bacteria, fungi, plants, and animals. In this work, starting from the screening of 39 alkaloids retrieved from a unique in-house library, we identified a heterodimer -carboline alkaloid, nigritanine, with a potent anti-Staphylococcus action. Nigritanine, isolated from Strychnos nigritana, was characterized for its antimicrobial activity against a reference and three clinical isolates of S. aureus. Its potential cytotoxicity was also evaluated at short and long term against mammalian red blood cells and human keratinocytes, respectively. Nigritanine showed a remarkable antimicrobial activity (minimum inhibitory concentration of 128 &micro, M) without being toxic in vitro to both tested cells. The analysis of the antibacterial activity related to the nigritanine scaffold furnished new insights in the structure&ndash, activity relationships (SARs) of -carboline, confirming that dimerization improves its antibacterial activity. Taking into account these interesting results, nigritanine can be considered as a promising candidate for the development of new antimicrobial molecules for the treatment of S. aureus-induced infections.

Due to their excellent in vitro activity against multidrug resistant bacteria, antimicrobial peptides (AMPs) hold promise for treatment of Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) sufferers. In this work, poly(lactide- co-glycolide) (PLGA) nanoparticles for lung delivery of AMPs deriving from the frog-skin esculentin-1a, namely, Esc(1-21) and Esc(1-21)-1c (Esc peptides), were successfully developed. Improved peptide transport through artificial CF mucus and simulated bacterial extracellular matrix was achieved in vitro. The formulations were effectively delivered through a liquid jet nebulizer already available to patients. Notably, Esc peptide-loaded nanoparticles displayed an improved efficacy in inhibiting P. aeruginosa growth in vitro and in vivo in the long term. A single intratracheal administration of Esc peptide-loaded nanoparticles in a mouse model of P. aeruginosa lung infection resulted in a 3-log reduction of pulmonary bacterial burden up to 36 h. Overall, results unravel the potential of PLGA nanoparticles as a reliable delivery system of AMPs to lungs.

Pseudomonas aeruginosa is a pathogenic bacterium known to cause serious human infections, especially in immune-compromised patients. This is due to its unique ability to transform from a drug-tolerant planktonic to a more dangerous and treatment-resistant sessile life form, called biofilm. Recently, two derivatives of the frog skin antimicrobial peptide esculentin-1a, i.e. Esc(1-21) and its D-amino acids containing diastereomer Esc(1-21)-1c, were characterized for their powerful anti-Pseudomonal activity against both forms. Prevention of biofilm formation already in its early stages could be even more advantageous for counteracting infections induced by this bacterium. In this work, we studied how the diastereomer Esc(1-21)-1c can inhibit Pseudomonas biofilm formation in comparison to the parent peptide and two clinically-used conventional antibiotics, i.e. colistin and aztreonam, when applied at dosages below the minimal growth inhibitory concentration. Biofilm prevention was correlated to the peptides' ability to inhibit Pseudomonas motility and to reduce the production of virulent metabolites, for example, pyoverdine and rhamnolipids. Furthermore, the molecular mechanism underlying these activities was evaluated by studying the peptides' effect on the expression of key genes involved in the virulence and motility of bacteria, as well as by monitoring the peptides' binding to the bacterial signaling nucleotide ppGpp. Our results demonstrate that the presence of only two D-amino acids in Esc(1-21)-1c is sufficient to downregulate ppGpp-mediated expression of biofilm-associated genes, presumably as a result of higher peptide stability and therefore prolonged interaction with the nucleotide. Overall, these studies should assist efficient design and optimization of new anti-infective agents with multiple pharmacologically beneficial properties.

In this study we designed and synthesized a new library of antimicrobial peptides correlated to [Pro

Antimicrobial peptides (AMPs) represent new alternatives to cope with the increasing number of multi-drug resistant microbial infections. Recently, a derivative of the frog-skin AMP esculentin-1a, Esc(1-21), was found to rapidly kill both the planktonic and biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action. Lately, its diastereomer Esc(1-21)-1c containing two d-amino acids i.e.

In this study we designed and synthesized a new library of antimicrobial peptides correlated to [Pro3,DLeu9]TL 1, a temporin L derivative devoid of cytolytic effects in vitro, and investigated the correlation between the α-helical content of the compounds and their antibacterial, cytotoxic and hemolytic activities. We systematically replaced Gly in position 10 of reference peptide with several amino acids. Structure-activity relationship studies of these analogues were performed by means of antimicrobial and cytotoxicity assays along with CD spectroscopy analyses. NMR analysis was also accomplished for compound 10. As well, the most promising peptides were additionally evaluated for their activity against some clinical strains isolated from human skin and for their mechanism of action by studying the kinetics of membrane perturbation of some representative microbial strains. We identified novel analogues with interesting properties that make them attractive lead compounds for potential topical applications.