An Overview of Frog Skin-Derived Esc Peptides: Promising Multifunctional Weapons against Pseudomonas aeruginosa-Induced Pulmonary and Ocular Surface Infections.

Antimicrobial resistance is a silent pandemic harming human health, and Pseudomonas aeruginosa is the most common bacterium responsible for chronic pulmonary and eye infections. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics. In this review, the in vitro/in vivo activities of the frog skin-derived AMP Esc(1-21) are shown. Esc(1-21) rapidly kills both the planktonic and sessile forms of P. aeruginosa and stimulates migration of epithelial cells, likely favoring repair of damaged tissue. However, to undertake preclinical studies, some drawbacks of AMPs (cytotoxicity, poor biostability, and limited delivery to the target site) must be overcome. For this purpose, the stereochemistry of two amino acids of Esc(1-21) was changed to obtain the diastereomer Esc(1-21)-1c, which is more stable, less cytotoxic, and more efficient in treating P. aeruginosa-induced lung and cornea infections in mouse models. Incorporation of these peptides (Esc peptides) into nanoparticles or immobilization to a medical device (contact lens) was revealed to be an effective strategy to ameliorate and/or to prolong the peptides' antimicrobial efficacy. Overall, these data make Esc peptides encouraging candidates for novel multifunctional drugs to treat lung pathology especially in patients with cystic fibrosis and eye dysfunctions, characterized by both tissue injury and bacterial infection.

The antimicrobial potential of adarotene derivatives against Staphylococcus aureus strains.

Multidrug-resistant (MDR) pathogens are severely impacting our ability to successfully treat common infections. Here we report the synthesis of a panel of adarotene-related retinoids showing potent antimicrobial activity on Staphylococcus aureus strains (including multidrug-resistant ones). Fluorescence and molecular dynamic studies confirmed that the adarotene analogues were able to induce conformational changes and disfunctions to the cell membrane, perturbing the permeability of the phospholipid bilayer. Since the major obstacle for developing retinoids is their potential cytotoxicity, a selected candidate was further investigated to evaluate its activity on a panel of human cell lines. The compound was found to be well tolerated, with IC50 5-15-fold higher than the MIC on S. aureus strains. Furthermore, the adarotene analogue had a good pharmacokinetic profile, reaching a plasma concentration of about 6 µM after 0.5 h after administration (150 mg/kg), at least twice the MIC observed against various bacterial strains. Moreover, it was demonstrated that the compound potentiated the growth-inhibitory effect of the poorly bioavailable rifaximin, when used in combination. Overall, the collected data pave the way for the development of synthetic retinoids as potential therapeutics for hard-to-treat infectious diseases caused by antibiotic-resistant Gram-positive pathogens.

Antimicrobial peptides for novel antiviral strategies in the current post-COVID-19 pandemic.

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted how urgent and necessary the discovery of new antiviral compounds is for novel therapeutic approaches. Among the various classes of molecules with antiviral activity, antimicrobial peptides (AMPs) of innate immunity are among the most promising ones, mainly due to their different mechanisms of action against viruses and additional biological properties. In this review, the main physicochemical characteristics of AMPs are described, with particular interest toward peptides derived from amphibian skin. Living in aquatic and terrestrial environments, amphibians are one of the richest sources of AMPs with different primary and secondary structures. Besides describing the various antiviral activities of these peptides and the underlying mechanism, this review aims at emphasizing the high potential of these small molecules for the development of new antiviral agents that likely reduce the selection of resistant strains.

The antimicrobial peptide Esc(1-21)-1c increases susceptibility of Pseudomonas aeruginosa to conventional antibiotics by decreasing the expression of the MexAB-OprM efflux pump.

Introduction: The increase in bacterial strains resistant to conventional antibiotics is an alarming problem for human health and could lead to pandemics in the future. Among bacterial pathogens responsible for a large variety of severe infections there is Pseudomonas aeruginosa. Therefore, there is an urgent need for new molecules with antimicrobial activity or that can act as adjuvants of antibiotics already in use. In this scenario, antimicrobial peptides (AMPs) hold great promise. Recently, we characterized a frog-skin AMP derived from esculentin-1a, namely Esc(1-21)-1c, endowed with antipseudomonal activity without being cytotoxic to human cells. Methods: The combinatorial effect of the peptide and antibiotics was investigated through the checkerboard assay, differential proteomic and transcriptional analysis. Results: Here, we found that Esc(1-21)-1c can synergistically inhibit the growth of P. aeruginosa cells with three different antibiotics, including tetracycline. We therefore investigated the underlying mechanism implemented by the peptide using a differential proteomic approach. The data revealed a significant decrease in the production of three proteins belonging to the MexAB-OprM efflux pump upon treatment with sub-inhibitory concentration of Esc(1-21)-1c. Down-regulation of these proteins was confirmed by transcriptional analysis and direct measurement of their relative levels in bacterial cells by tandem mass spectrometry analysis in multiple reaction monitoring scan mode. Conclusion: These evidences suggest that treatment with Esc(1-21)-1c in combination with antibiotics would increase the intracellular drug content making bacteria more susceptible to the antibiotic. Overall, these results highlight the importance of characterizing new molecules able to synergize with conventional antibiotics, paving the way for the development of alternative therapeutic strategies based on AMP/antibiotic formulations to counteract the emergence of resistant bacterial strains and increase the use of "old" antibiotics in medical practice.

Novel Peptides with Dual Properties for Treating Pseudomonas aeruginosa Keratitis: Antibacterial and Corneal Wound Healing.

The corneal epithelium is a layer in the anterior part of eye that contributes to light refraction onto the retina and to the ocular immune defense. Although an intact corneal epithelium is an excellent barrier against microbial pathogens and injuries, corneal abrasions can lead to devastating eye infections. Among them, Pseudomonas aeruginosa-associated keratitis often results in severe deterioration of the corneal tissue and even blindness. Hence, the discovery of new drugs able not only to eradicate ocular infections, which are often resistant to antibiotics, but also to elicit corneal wound repair is highly demanded. Recently, we demonstrated the potent antipseudomonal activity of two peptides, Esc(1-21) and its diastereomer Esc(1-21)-1c. In this study, by means of a mouse model of P. aeruginosa keratitis and an in vivo corneal debridement wound, we discovered the efficacy of these peptides, particularly Esc(1-21)-1c, to cure keratitis and to promote corneal wound healing. This latter property was also supported by in vitro cell scratch and ELISA assays. Overall, the current study highlights Esc peptides as novel ophthalmic agents for treating corneal infection and injury, being able to display a dual function, antimicrobial and wound healing, rarely identified in a single peptide at the same micromolar concentration range.

Rapid Assessment of Susceptibility of Bacteria and Erythrocytes to Antimicrobial Peptides by Single-Cell Impedance Cytometry.

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight antibiotic-resistant infections. In most cases, they kill bacteria by making their membrane permeable and therefore exhibit low propensity to induce bacterial resistance. In addition, they are often selective, killing bacteria at concentrations lower than those at which they are toxic to the host. However, clinical applications of AMPs are hindered by a limited understanding of their interactions with bacteria and human cells. Standard susceptibility testing methods are based on the analysis of the growth of a bacterial population and therefore require several hours. Moreover, different assays are required to assess the toxicity to host cells. In this work, we propose the use of microfluidic impedance cytometry to explore the action of AMPs on both bacteria and host cells in a rapid manner and with single-cell resolution. Impedance measurements are particularly well-suited to detect the effects of AMPs on bacteria, due to the fact that the mechanism of action involves perturbation of the permeability of cell membranes. We show that the electrical signatures of Bacillus megaterium cells and human red blood cells (RBCs) reflect the action of a representative antimicrobial peptide, DNS-PMAP23. In particular, the impedance phase at high frequency (e.g., 11 or 20 MHz) is a reliable label-free metric for monitoring DNS-PMAP23 bactericidal activity and toxicity to RBCs. The impedance-based characterization is validated by comparison with standard antibacterial activity assays and absorbance-based hemolytic activity assays. Furthermore, we demonstrate the applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the way to study AMP selectivity for bacterial versus eukaryotic cells in the presence of both cell types.

Effects of antimicrobial peptides on membrane dynamics: A comparison of fluorescence and NMR experiments.

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight resistant infections. They are commonly thought to kill bacteria by perturbing the permeability of their cell membranes. However, bacterial killing requires a high coverage of the cell surface by bound peptides, at least in the case of cationic and amphipathic AMPs. Therefore, it is conceivable that peptide accumulation on the bacterial membranes might interfere with vital cellular functions also by perturbing bilayer dynamics, a hypothesis that has been termed "sand in the gearbox". Here we performed a systematic study of such possible effects, for two representative peptides (the cationic cathelicidin PMAP-23 and the peptaibol alamethicin), employing fluorescence and NMR spectroscopies. These approaches are commonly applied to characterize lipid order and dynamics, but sample different time-scales and could thus report on different membrane properties. In our case, fluorescence anisotropy measurements on liposomes labelled with probes localized at different depths in the bilayer showed that both peptides perturb membrane fluidity and order. Pyrene excimer-formation experiments showed a peptide-induced reduction in lipid lateral mobility. Finally, laurdan fluorescence indicated that peptide binding reduces water penetration below the headgroups region. Comparable effects were observed also in fluorescence experiments performed directly on live bacterial cells. By contrast, the fatty acyl chain order parameters detected by deuterium NMR spectroscopy remained virtually unaffected by addition of the peptides. The apparent discrepancy between the two techniques confirms previous sporadic observations and is discussed in terms of the different characteristic times of the two approaches. The perturbation of membrane dynamics in the ns timescale, indicated by the multiple fluorescence approaches reported here, could contribute to the antimicrobial activity of AMPs, by affecting the function of membrane proteins, which is strongly dependent on the physicochemical properties of the bilayer.

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.

KDEON WK-11: A short antipseudomonal peptide with promising potential.

The plight of antimicrobial resistance continues to limit the availability of antibiotic treatment effective in combating resistant bacterial infections. Despite efforts made to rectify this issue and minimise its effects on both patients and the wider community, progress in this area remains minimal. Here, we de-novo designed a peptide named KDEON WK-11, building on previous work establishing effective residues and structures active in distinguished antimicrobial peptides such as lactoferrin. We assessed its antimicrobial activity against an array of bacterial strains and identified its most potent effect, against Pseudomonas aeruginosa with an MIC value of 3.12 µM, lower than its counterparts developed with similar residues and chain lengths. We then determined its anti-biofilm properties, potential mechanism of action and in vitro cytotoxicity. We identified that KDEON WK-11 had a broad range of antimicrobial activity and specific capabilities to fight Pseudomonas aeruginosa with low in vitro cytotoxicity and promising potential to express anti-lipopolysaccharide qualities, which could be exploited to expand its properties into an anti-sepsis agent.

Pulmonary Safety Profile of Esc Peptides and Esc-Peptide-Loaded Poly(lactide-co-glycolide) Nanoparticles: A Promising Therapeutic Approach for Local Treatment of Lung Infectious Diseases.

In recent years, we have discovered Esc(1-21) and its diastereomer (Esc peptides) as valuable candidates for the treatment of Pseudomonas lung infection, especially in patients with cystic fibrosis (CF). Furthermore, engineered poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) were revealed to be a promising pulmonary delivery system of antimicrobial peptides. However, the "ad hoc" development of novel therapeutics requires consideration of their stability, tolerability, and safety. Hence, by means of electrophysiology experiments and preclinical studies on healthy mice, we demonstrated that neither Esc peptides or Esc-peptide-loaded PLGA NPs significantly affect the integrity of the lung epithelium, nor change the global gene expression profile of lungs of treated animals compared to those of vehicle-treated animals. Noteworthy, the Esc diastereomer endowed with the highest antimicrobial activity did not provoke any pulmonary pro-inflammatory response, even at a concentration 15-fold higher than the efficacy dosage 24 h after administration in the free or encapsulated form. The therapeutic index was ≥70, and the peptide was found to remain available in the bronchoalveolar lavage of mice, after two days of incubation. Overall, these studies should open an avenue for a new up-and-coming pharmacological approach, likely based on inhalable peptide-loaded NPs, to address CF lung disease.

Synthetic Amphipathic ß-Sheet Temporin-Derived Peptide with Dual Antibacterial and Anti-Inflammatory Activities.

Temporin family is one of the largest among antimicrobial peptides (AMPs), which act mainly by penetrating and disrupting the bacterial membranes. To further understand the relationship between the physical-chemical properties and their antimicrobial activity and selectivity, an analogue of Temporin L, [Nle1, dLeu9, dLys10]TL (Nle-Phe-Val-Pro-Trp-Phe-Lys-Phe-dLeu-dLys-Arg-Ile-Leu-CONH2) has been developed in the present work. The design strategy consisted of the addition of a norleucine residue at the N-terminus of the lead peptide sequence, [dLeu9, dLys10]TL, previously developed by our group. This modification promoted an increase of peptide hydrophobicity and, interestingly, more efficient activity against both Gram-positive and Gram-negative strains, without affecting human keratinocytes and red blood cells survival compared to the lead peptide. Thus, this novel compound was subjected to biophysical studies, which showed that the peptide [Nle1, dLeu9, dLys10]TL is unstructured in water, while it adopts ß-type conformation in liposomes mimicking bacterial membranes, in contrast to its lead peptide forming α-helical aggregates. After its aggregation in the bacterial membrane, [Nle1, dLeu9, dLys10]TL induced membrane destabilization and deformation. In addition, the increase of peptide hydrophobicity did not cause a loss of anti-inflammatory activity of the peptide [Nle1, dLeu9, dLys10]TL in comparison with its lead peptide. In this study, our results demonstrated that positive net charge, optimum hydrophobic-hydrophilic balance, and chain length remain the most important parameters to be addressed while designing small cationic AMPs.

The Inhibition of DNA Viruses by the Amphibian Antimicrobial Peptide Temporin G: A Virological Study Addressing HSV-1 and JPCyV.

Herpes simplex virus type-1 (HSV-1) and John Cunningham polyomavirus (JCPyV) are widely distributed DNA viruses causing mainly asymptomatic infection, but also mild to very severe diseases, especially when these viruses reach the brain. Some drugs have been developed to inhibit HSV-1 replication in host cells, but their prolonged use may induce resistance phenomena. In contrast, to date, there is no cure for JCPyV. The search for alternative drugs that can reduce viral infections without undermining the host cell is moving toward antimicrobial peptides (AMPs) of natural occurrence. These include amphibian AMPs belonging to the temporin family. Herein, we focus on temporin G (TG), showing that it strongly affects HSV-1 replication by acting either during the earliest stages of its life cycle or directly on the virion. Computational studies have revealed the ability of TG to interact with HSV-1 glycoprotein B. We also found that TG reduced JCPyV infection, probably affecting both the earliest phases of its life cycle and the viral particle, likely through an interaction with the viral capsid protein VP1. Overall, our results are promising for the development of short naturally occurring peptides as antiviral agents used to counteract diseases related to HSV-1 and JCPyV.

Antifungal Activity of the Frog Skin Peptide Temporin G and Its Effect on Candida albicans Virulence Factors.

The increasing resistance to conventional antifungal drugs is a widespread concern, and a search for new compounds, active against different species of fungi, is demanded. Antimicrobial peptides (AMPs) hold promises in this context. Here we investigated the activity of the frog skin AMP Temporin G (TG) against a panel of fungal strains, by following the Clinical and Laboratory Standards Institute protocols. TG resulted to be active against (i) Candida species and Cryptococcus neoformans, with MIC50 between 4 µM and 64 µM after 24 h of incubation; (ii) dermatophytes with MIC80 ranging from 4 to 32 µM, and (iii) Aspergillus strains with MIC80 of 128 µM. In addition, our tests revealed that TG reduced the metabolic activity of Candida albicans cells, with moderate membrane perturbation, as proven by XTT and Sytox Green assays, respectively. Furthermore, TG was found to be effective against some C. albicans virulence factors; indeed, at 64 µM it was able to inhibit ~90% of yeast-mycelial switching, strongly prevented biofilm formation, and led to a 50% reduction of metabolic activity in mature biofilm cells, and ~30-35% eradication of mature biofilm biomass. Even though further studies are needed to deepen our knowledge of the mechanisms of TG antifungal activity, our results suggest this AMP as an attractive lead compound for treatment of fungal diseases.

Derivatives of Esculentin-1 Peptides as Promising Candidates for Fighting Infections from Escherichia coli O157:H7.

New strategies are needed to fight the emergence of multidrug-resistant bacteria caused by an overuse of antibiotics in medical and veterinary fields. Due to the importance of biofilms in clinical infections, antibiofilm peptides have a great potential to treat infections. In recent years, an increased interest has emerged in antimicrobial peptides (AMPs). One of the richest sources of AMPs is represented by amphibian skin. In the present work, we investigated the effects of two peptides derived from the frog skin AMP esculentin-1, namely, Esc(1-21) and Esc(1-18), on the growth, biofilm formation, and gene expression of the non-pathogenic Escherichia coli strain K12 and of enterohemorrhagic E. coli O157:H7. Both peptides showed minimal bactericidal concentrations ranging from 4 to 8 µM for Esc(1-21) and from 32 to 64 µM for Esc(1-18). They also, at sub-MIC doses, reduced the formation of biofilm, as supported by both microbiological assays and scanning electron microscopy, while they displayed no marked activity against the planktonic form of the bacteria. Transcriptional analysis in E. coli O157:H7 showed that both AMPs induced the expression of several genes involved in the regulation of formation and dispersal of biofilm, as well as in the stress response. In conclusion, we demonstrated that these AMPs affect E. coli O157:H7 growth and biofilm formation, thus suggesting a great potential to be developed as novel therapeutics against infections caused by bacterial biofilms.

The Antimicrobial Peptide Esc(1-21) Synergizes with Colistin in Inhibiting the Growth and in Killing Multidrug Resistant Acinetobacter baumannii Strains.

Multidrug-resistant microbial infections and the scarce availability of new antibiotics capable of eradicating them are posing a serious problem to global health security. Among the microorganisms that easily acquire resistance to antibiotics and that are the etiological cause of severe infections, there is Acinetobacter baumannii. Carbapenems are the principal agents used to treat A. baumannii infections. However, when strains develop resistance to this class of antibiotics, colistin is considered one of the last-resort drugs. However, the appearance of resistance to colistin also makes treatment of the Acinetobacter infections very difficult. Antimicrobial peptides (AMP) from the innate immunity hold promise as new alternative antibiotics due to their multiple biological properties. In this study, we characterized the activity and the membrane-perturbing mechanism of bactericidal action of a derivative of a frog-skin AMP, namely Esc(1-21), when used alone or in combination with colistin against multidrug-resistant A. baumannii clinical isolates. We found that the mixture of the two compounds had a synergistic effect in inhibiting the growth and killing of all of the tested strains. When combined at dosages below the minimal inhibitory concentration, the two drugs were also able to slow down the microbial growth and to potentiate the membrane-perturbing effect. To the best of our knowledge, this is the first report showing a synergistic effect between AMPs, i.e., Esc(1-21), and colistin against colistin-resistant A. baumannii clinical isolates, highlighting the potential clinical application of such combinational therapy.

Exposure to b-LED Light While Exerting Antimicrobial Activity on Gram-Negative and -Positive Bacteria Promotes Transient EMT-like Changes and Growth Arrest in Keratinocytes.

While blue LED (b-LED) light is increasingly being studied for its cytotoxic activity towards bacteria in therapy of skin-related infections, its effects on eukaryotic cells plasticity are less well characterized. Moreover, since different protocols are often used, comparing the effect of b-LED towards both microorganisms and epithelial surfaces may be difficult. The aim of this study was to analyze, in the same experimental setting, both the bactericidal activity and the effects on human keratinocytes. Exposure to b-LED induced an intense cytocidal activity against Gram-positive (i.e, Staphylococcus aureus) and Gram-negative (i.e., Pseudomonas aeruginosa) bacteria associated with catheter-related infections. Treatment with b-LED of a human keratinocyte cell line induced a transient cell cycle arrest. At the molecular level, exposure to b-LED induced a transient downregulation of Cyclin D1 and an upregulation of p21, but not signs of apoptosis. Interestingly, a transient induction of phosphor-histone γ-H2Ax, which is associated with genotoxic damages, was observed. At the same time, keratinocytes underwent a transient epithelial to mesenchymal transition (EMT)-like phenotype, characterized by E-cadherin downregulation and SNAIL/SLUG induction. As a functional readout of EMT induction, a scratch assay was performed. Surprisingly, b-LED treatment provoked a delay in the scratch closure. In conclusion, we demonstrated that b-LED microbicidal activity is associated with complex responses in keratinocytes that certainly deserve further analysis.

The Triprenylated Anthranoid Ferruginin A, a Promising Scaffold for the Development of Novel Antibiotics against Gram-Positive Bacteria.

In today's post-antibiotic era, the search for new antimicrobial compounds is of major importance and nature represents one of the primary sources of bioactive molecules. In this work, through a cheminformatics approach, we clustered an in-house library of natural products and their derivatives based on a combination of fingerprints and substructure search. We identified the prenylated emodine-type anthranoid ferruginin A as a novel antimicrobial compound. We tested its ability to inhibit and kill a panel of Gram-positive and Gram-negative bacteria, and compared its activity with that of two analogues, vismione B and ferruanthrone. Furthermore, the capability of these three anthranoids to disrupt staphylococcal biofilm was investigated, as well as their effect on the viability of human keratinocytes. Ferruginin A showed a potent activity against both the planktonic and biofilm forms of Gram-positive bacteria (i.e., Staphylococcus aureus and S. epidermidis) and had the best therapeutic index compared to vismione B and ferruanthrone. In conclusion, ferruginin A represents a promising scaffold for the further development of valuable antimicrobial agents.

Esc peptides as novel potentiators of defective cystic fibrosis transmembrane conductance regulator: an unprecedented property of antimicrobial peptides.

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.

Opposing Effects of PhoPQ and PmrAB on the Properties of Salmonella enterica serovar Typhimurium: Implications on Resistance to Antimicrobial Peptides.

The increasing number of resistant bacteria is a major threat worldwide, leading to the search for new antibiotic agents. One of the leading strategies is the use of antimicrobial peptides (AMPs), cationic and hydrophobic innate immune defense peptides. A major target of AMPs is the bacterial membrane. Notably, accumulating data suggest that AMPs can activate the two-component systems (TCSs) of Gram-negative bacteria. These include PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB), responsible for remodeling of the bacterial cell surface. To better understand this mechanism, we utilized bacteria deficient either in one system alone or in both and biophysical tools including fluorescence spectroscopy, single-cell atomic force microscopy, electron microscopy, and mass spectrometry (Moskowitz, S. M.; Antimicrob. Agents Chemother. 2012, 56, 1019-1030; Cheng, H. Y.; J. Biomed. Sci. 2010, 17, 60). Our data suggested that the two systems have opposing effects on the properties of Salmonella enterica. The knockout of PhoPQ made the bacteria more susceptible to AMPs by making the surface less rigid, more polarized, and permeable with a slightly more negatively charged cell wall. In addition, the periplasmic space is thinner. In contrast, the knockout of PmrAB did not affect its susceptibility, while it made the bacterial outer layer very rigid, less polarized, and less permeable than the other two mutants, with a negatively charged cell wall similar to the WT. Overall, the data suggest that the coexistence of systems with opposing effects on the biophysical properties of the bacteria contribute to their membrane flexibility, which, on the one hand, is important to accommodate changing environments and, on the other hand, may inhibit the development of meaningful resistance to AMPs.