Biophysical Insights into the Antitumoral Activity of Crotalicidin against Breast Cancer Model Membranes.

Bioactive peptides have emerged as promising therapeutic agents with antimicrobial, antifungal, antiparasitic, and, recently, antitumoral properties with a mechanism of action based on membrane destabilization and cell death, often involving a conformational change in the peptide. This biophysical study aims to provide preliminary insights into the membrane-level antitumoral mode of action of crotalicidin, a cationic host defense peptide from rattlesnake venom, toward breast cancer cell lines. The lipid composition of breast cancer cell lines was obtained after lipid extraction and quantification to prepare representative cell membrane models. Membrane-peptide interaction studies were performed using differential scanning calorimetry and Fourier-transform infrared spectroscopy. The outcome evidences the potential antitumoral activity and selectivity of crotalicidin toward breast cancer cell lines and suggests a mechanism initiated by the electrostatic interaction of the peptide with the lipid bilayer surface and posterior conformation change with membrane intercalation between the acyl chains in negatively charged lipid systems. This research provides valuable information that clears up the antitumoral mode of action of crotalicidin.

ΔM4: Membrane-Active Peptide with Antitumoral Potential against Human Skin Cancer Cells.

Peptides have become attractive potential agents due to their affinity to cancer cells. In this work, the biological activity of the peptide ΔM4 against melanoma cancer cell line A375, epidermoid carcinoma cell line A431, and non-tumoral HaCaT cells was evaluated. The cytotoxic MTT assay demonstrates that ΔM4 show five times more activity against cancer than non-cancer cells. The potential membrane effect of ΔM4 was evaluated through lactate dehydrogenase release and Sytox uptake experiments. The results show a higher membrane activity of ΔM4 against A431 in comparison with the A375 cell line at a level of 12.5 µM. The Sytox experiments show that ΔM4 has a direct effect on the permeability of cancer cells in comparison with control cells. Infrared spectroscopy was used to study the affinity of the peptide to membranes resembling the composition of tumoral and non-tumoral cells. The results show that ΔM4 induces a fluidization effect on the tumoral lipid system over 5% molar concentration. Finally, to determine the appearance of phosphatidylserine on the surface of the cell, flow cytometry analyses were performed employing an annexin V-PE conjugate. The results suggest that 12.5 µM of ΔM4 induces phosphatidylserine translocation in A375 and A431 cancer cells. The findings of this study support the potential of ΔM4 as a selective agent for targeting cancer cells. Its mechanism of action demonstrated selectivity, membrane-disrupting effects, and induction of phosphatidylserine translocation.

Simulation Study of the Effect of Antimicrobial Peptide Associations on the Mechanism of Action with Bacterial and Eukaryotic Membranes.

Antimicrobial peptides (AMPs) can be directed to specific membranes based on differences in lipid composition. In this study, we performed atomistic and coarse-grained simulations of different numbers of the designed AMP adepantin-1 with a eukaryotic membrane, cytoplasmic Gram-positive and Gram-negative membranes, and an outer Gram-negative membrane. At the core of adepantin-1's behavior is its amphipathic α-helical structure, which was implemented in its design. The amphipathic structure promotes rapid self-association of peptide in water or upon binding to bacterial membranes. Aggregates initially make contact with the membrane via positively charged residues, but with insertion, the hydrophobic residues are exposed to the membrane's hydrophobic core. This adaptation alters the aggregate's stability, causing the peptides to diffuse in the polar region of the membrane, mostly remaining as a single peptide or pairing up to form an antiparallel dimer. Thus, the aggregate's proposed role is to aid in positioning the peptide into a favorable conformation for insertion. Simulations revealed the molecular basics of adepantin-1 binding to various membranes, and highlighted peptide aggregation as an important factor. These findings contribute to the development of novel anti-infective agents to combat the rapidly growing problem of bacterial resistance to antibiotics.

Biophysical Characterization of LTX-315 Anticancer Peptide Interactions with Model Membrane Platforms: Effect of Membrane Surface Charge.

LTX-315 is a clinical-stage, anticancer peptide therapeutic that disrupts cancer cell membranes. Existing mechanistic knowledge about LTX-315 has been obtained from cell-based biological assays, and there is an outstanding need to directly characterize the corresponding membrane-peptide interactions from a biophysical perspective. Herein, we investigated the membrane-disruptive properties of the LTX-315 peptide using three cell-membrane-mimicking membrane platforms on solid supports, namely the supported lipid bilayer, intact vesicle adlayer, and tethered lipid bilayer, in combination with quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) measurements. The results showed that the cationic LTX-315 peptide selectively disrupted negatively charged phospholipid membranes to a greater extent than zwitterionic or positively charged phospholipid membranes, whereby electrostatic interactions were the main factor to influence peptide attachment and membrane curvature was a secondary factor. Of note, the EIS measurements showed that the LTX-315 peptide extensively and irreversibly permeabilized negatively charged, tethered lipid bilayers that contained high phosphatidylserine lipid levels representative of the outer leaflet of cancer cell membranes, while circular dichroism (CD) spectroscopy experiments indicated that the LTX-315 peptide was structureless and the corresponding membrane-disruptive interactions did not involve peptide conformational changes. Dynamic light scattering (DLS) measurements further verified that the LTX-315 peptide selectively caused irreversible disruption of negatively charged lipid vesicles. Together, our findings demonstrate that the LTX-315 peptide preferentially disrupts negatively charged phospholipid membranes in an irreversible manner, which reinforces its potential as an emerging cancer immunotherapy and offers a biophysical framework to guide future peptide engineering efforts.

Integrating In Vitro and In Silico Analysis of a Cationic Antimicrobial Peptide Interaction with Model Membranes of Colistin-Resistant Pseudomonas aeruginosa Strains.

Bacterial antibiotic resistance is a serious global public health concern. Infections caused by colistin-resistant Pseudomonas aeruginosa (CRPa) strains represent a serious threat due to their considerable morbidity and mortality rates, since most of the current empirical antibiotic therapies are ineffective against these strains. Accordingly, cationic antimicrobial peptides (CAMPs) have emerged as promising alternatives to control resistant bacteria. In this study, the interaction of a CAMP derived from cecropin D-like (∆M2) with model membranes mimicking bacterial biomembranes of wild-type (WTPa) strains of P. aeruginosa and CRPa was evaluated through in vitro and in silico approaches. In vitro interaction was determined by infrared spectroscopy, whereas in silico molecular dynamics was performed to predict specific interactions between amino acids of ∆M2 and lipids of model membrane systems. Experimental analysis showed this peptide interacted with the lipids of bacterial-like model membranes of WTPa and CRPa. In both cases, an increase in the concentration of peptides induced an increase in the phase transition temperature of the lipid systems. On the other hand, the peptides in solution underwent a transition from a random to a helical secondary structure after interacting with the membranes mostly favored in the CRPa system. The α-helix structure percentage for ΔM2 interacting with WTPa and CRPa lipid systems was 6.4 and 33.2%, respectively. Finally, molecular dynamics showed ∆M2 to have the most affinities toward the phospholipids palmitoyl-oleyl-phosphatidylglycerol (POPG) and palmitoyl-oleoyl-phosphatidylethanolamine (POPE) that mimic membranes of WTPa and CRPa, respectively. This work provides clues for elucidating the membrane-associated mechanism of action of ∆M2 against colistin-susceptible and -resistant strains of Pseudomonas aeruginosa.

Membrane Activity of LL-37 Derived Antimicrobial Peptides against Enterococcus hirae: Superiority of SAAP-148 over OP-145.

The development of antimicrobial agents against multidrug-resistant bacteria is an important medical challenge. Antimicrobial peptides (AMPs), human cathelicidin LL-37 and its derivative OP-145, possess a potent antimicrobial activity and were under consideration for clinical trials. In order to overcome some of the challenges to their therapeutic potential, a very promising AMP, SAAP-148 was designed. Here, we studied the mode of action of highly cationic SAAP-148 in comparison with OP-145 on membranes of Enterococcus hirae at both cellular and molecular levels using model membranes composed of major constituents of enterococcal membranes, that is, anionic phosphatidylglycerol (PG) and cardiolipin (CL). In all assays used, SAAP-148 was consistently more efficient than OP-145, but both peptides displayed pronounced time and concentration dependences in killing bacteria and performing at the membrane. At cellular level, Nile Red-staining of enterococcal membranes showed abnormalities and cell shrinkage, which is also reflected in depolarization and permeabilization of E. hirae membranes. At the molecular level, both peptides abolished the thermotropic phase transition and induced disruption of PG/CL. Interestingly, the membrane was disrupted before the peptides neutralized the negative surface charge of PG/CL. Our results demonstrate that SAAP-148, which kills bacteria at a significantly lower concentration than OP-145, shows stronger effects on membranes at the cellular and molecular levels.

Preparation and pharmacokinetics of glycyrrhetinic acid and cell transmembrane peptides modified with liposomes for liver targeted-delivery.

The article presents a hepatocellular carcinoma cell surface-specific ligand glycyrrhetinic acid (GA) and cell-penetrating peptide (TAT) with good cell membrane penetration to modify the anti-tumor drug pingyangmycin (PYM) liver delivery system, which achieve targeted delivery of drugs and improve anti-tumor efficiency. In this study, we synthesized the pingyangmycin liposome modified by glycyrrhetinic acid and cell penetrating peptide(GA-TAT-PYM-L) and evaluated the anti-tumor effect of GA-TAT-PYM-Lin vitro. Using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylte-trazolium bromidecell proliferation method, GA-TAT-PYM-L had a stronger inhibitory effect on HepG2 cells than the free drug PYM at the same concentration. Acridine orange-ethidium bromide staining assays showed that GA-TAT-PYM-L had stronger apoptosis promotion effects on HepG2 cells in comparison to PYM. Pharmacokinetic studies indicated that, compared with PYM, GA-TAT-PYM-L enhanced mean residence time (MRT0-∞) and area under curve (AUC0-∞) by about 2.79-fold and 2.45-fold. TheT1/2was prolonged to 140.23 ± 14.13 min. Tissue distribution results showed that the PYM concentrations in livers from the GA-TAT-PYM-L group were always higher than other tissues at each monitoring period after 5 min, indicating that GA-TAT-PYM-L can achieve liver targeting.

Antiviral peptide engineering for targeting membrane-enveloped viruses: Recent progress and future directions.

Membrane-enveloped viruses are a major cause of global health challenges, including recent epidemics and pandemics. This mini-review covers the latest efforts to develop membrane-targeting antiviral peptides that inhibit enveloped viruses by 1) preventing virus-cell fusion or 2) disrupting the viral membrane envelope. The corresponding mechanisms of antiviral activity are discussed along with peptide engineering strategies to modulate membrane-peptide interactions in terms of potency and selectivity. Application examples are presented demonstrating how membrane-targeting antiviral peptides are useful therapeutics and prophylactics in animal models, while a stronger emphasis on biophysical concepts is proposed to refine mechanistic understanding and support potential clinical translation.

iRGD Peptide-Mediated Liposomal Nanoparticles with Photoacoustic/Ultrasound Dual-Modality Imaging for Precision Theranostics Against Hepatocellular Carcinoma.

PURPOSE: Prepare a multifunctional ultrasound molecular probe, cell-penetrating peptide-modified 10-hydroxycamptothecin-loaded phase-transformation lipid nanoparticles (iRGD-ICG-10-HCPT-PFP-NPs), and to combine iRGD-ICG-10-HCPT-PFP -NPs with low-intensity focused ultrasound (LIFU) for precision theranostics against hepatocellular carcinoma (HCC). MATERIALS AND METHODS: The morphology of nanoparticles (NPs) and iRGD-ICG-10-HCPT-PFP-NPs was detected. In vitro, we examined targeting ability by flow cytometry and confocal laser scanning microscopy (CLSM), assessed penetration ability into hepatoma cells, and assessed killing ability. In vivo, we examined the targeting ability of the NPs with a photoacoustic (PA) imager and fluorometer (FL), while LIFU irradiation was used to trigger the release of chemotherapeutic drugs, which had a therapeutic effect on tumors. RESULTS: The particle size of iRGD-ICG-10-HCPT-PFP-NPs was 298.4 ± 10.42 nm. In vitro, iRGD-ICG-10-HCPT-PFP-NPs bound more to SK-Hep1 cells than ICG-10-HCPT-PFP-NPs. iRGD-ICG-10-HCPT-PFP-NPs could achieve PA/ultrasound imaging. The percentage of antiproliferative and apoptotic cells in the iRGD-ICG-10-HCPT-PFP-NPs+LIFU group was significantly higher. In vivo, iRGD-ICG-10-HCPT-PFP-NPs can target tumor sites and achieve PA/ultrasound imaging. The tumor volume in the iRGD-ICG-10-HCPT-PFP-NPs+LIFU group was significantly smaller, and the antiproliferative and proapoptotic effects were higher. CONCLUSION: We successfully prepared a novel molecular probe that has good targeting, can perform ultrasound/PA dual-modality imaging, and can penetrate deep into tumors to achieve better therapeutic tumor effects, providing a new idea and method for theranostics of HCC.

Membrane interactions of Ocellatins. Where do antimicrobial gaps stem from?

The antimicrobial peptides Ocellatin-LB1, -LB2 and -F1, isolated from frogs, are identical from residue 1 to 22, which correspond to the -LB1 sequence, whereas -LB2 carries an extra N and -F1 additional NKL residues at their C-termini. Despite the similar sequences, previous investigations showed different spectra of activities and biophysical investigations indicated a direct correlation between both membrane-disruptive properties and activities, i.e., ocellatin-F1 > ocellatin-LB1 > ocellatin-LB2. This study presents experimental evidence as well as results from theoretical studies that contribute to a deeper understanding on how these peptides exert their antimicrobial activities and how small differences in the amino acid composition and their secondary structure can be correlated to these activity gaps. Solid-state NMR experiments allied to the simulation of anisotropic NMR parameters allowed the determination of the membrane topologies of these ocellatins. Interestingly, the extra Asn residue at the Ocellatin-LB2 C-terminus results in increased topological flexibility, which is mainly related to wobbling of the helix main axis as noticed by molecular dynamics simulations. Binding kinetics and thermodynamics of the interactions have also been assessed by Surface Plasmon Resonance and Isothermal Titration Calorimetry. Therefore, these investigations allowed to understand in atomic detail the relationships between peptide structure and membrane topology, which are in tune within the series -F1 > > -LB1 ≥ -LB2, as well as how peptide dynamics can affect membrane topology, insertion and binding.

PepBiotics, novel cathelicidin-inspired antimicrobials to fight pulmonary bacterial infections.

BACKGROUND: Antimicrobial peptides are considered potential alternatives to antibiotics. Here we describe the antibacterial properties of a family of novel cathelicidin-related (CR-) peptides, which we named PepBiotics, against bacteria typically present in cystic fibrosis (CF) patients. METHODS: Broth dilution assays were used to determine antibacterial activity of PepBiotics under physiological conditions, as well as development of bacterial resistance against these peptides. Toxicity was tested in mice and cell cultures while molecular interactions of PepBiotics with bacterial membrane components was determined using CD, ITC and LPS/LTA induced macrophage studies. RESULTS: A relatively small number of PepBiotics remained highly antibacterial against CF-related respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus, at high ionic strength and low pH. Interestingly, these PepBiotics also prevented LPS/LTA induced activation of macrophages and was shown to be non-toxic to primary human nasal epithelial cells. Furthermore, both P. aeruginosa and S. aureus were unable to induce resistance against CR-163 and CR-172, two PepBiotics selected for their excellent antimicrobial and immunomodulatory properties. Toxicity studies in mice indicated that intratracheal administration of CR-163 was well tolerated in vivo. Finally, interaction of CR-163 with bacterial-type anionic membranes but not with mammalian-type (zwitterionic lipid) membranes was confirmed using ITC and 31P solid state NMR. CONCLUSIONS: PepBiotics are a promising novel class of highly active antimicrobial peptides, of which CR-163 showed the most potential for treatment of clinically relevant (CF-) pathogens in physiological conditions. GENERAL SIGNIFICANCE: These observations emphasize the therapeutic potential of PepBiotics against CF-related bacterial respiratory infections.

Membrane Fractionation of Protein Hydrolysates from By-Products: Recovery of Valuable Compounds from Spent Yeasts.

Spent brewer's yeast (Saccharomyces sp.), the second most generated by-product from the brewing industry, contains bioactive and nutritional compounds with high added value such as proteins (40-50%), polysaccharides, fibers and vitamins. Molecules of interest from agro-industrial by-products need to be extracted, separated, concentrated, and/or purified so that a minimum purity level is achieved, allowing its application. Enzymatic hydrolysis has been successfully used in the production of peptides and protein hydrolysates. The obtained hydrolysates require efficient downstream processes such as membrane technology, which is an important tool for the recovery of thermolabile and sensitive compounds from complex mixtures, with low energy consumption and high specificity. The integration of membrane techniques that promote the separation through sieving and charge-based mechanisms is of great interest to improve the purity of the recovered fractions. This review is specifically addressed to the application of membrane technologies for the recovery of peptides from yeast protein hydrolysates. Fundamental concepts and practical aspects relative to the ultrafiltration of agro-industrial protein hydrolysates will be described. Challenges and perspectives involving the recovery of peptides from yeast protein hydrolysates will be presented and thoroughly discussed.

Molecular dynamics study of membrane permeabilization by wild-type and mutant lytic peptides from the non-enveloped Flock House virus.

Flock House virus (FHV) serves as a model system for understanding infection mechanisms utilized by non-enveloped viruses to transport across cellular membranes. During the infection cycle of FHV, a fundamental stage involves disruption of the endosomal membrane by membrane active peptides, following externalization of the peptides from the capsid interior. The FHV lytic agents are the 44 C-terminal amino acids residues of the capsid protein, which are auto-catalytically cleaved during the capsid maturation process. The cleaved peptides are termed γ peptides. In this study, we perform multi-scale molecular dynamics simulations including 40 µs all-atom molecular dynamics simulations to study the behavior of pre-inserted transmembrane lytic peptides at a high concentration in a neutral membrane. We study the dynamical organization among peptides to form oligomeric bundles in four systems including the wild-type γ peptide and three mutant forms; namely, a truncation mutant in which the 23 C-terminal residues are deleted (γ1), a construct where the 8 C-terminal residues of γ are fused to γ1 (Δ385-399 γ) and a single-point mutant (F402A γ), all of which have been experimentally shown to drastically affect infectivity and lytic activity compared to the wild-type γ. Our results shed light on the actions of varied forms of the FHV lytic peptide including membrane insertion, trans-membrane stability, peptide oligomerization, water permeation activity and dynamic pore formation. Findings from this study provide detailed structural information and rationale for the differences in lytic activity among variants of FHV γ.

Effect of saturation in phospholipid/fatty acid monolayers on interaction with amyloid ß peptide.

The effect of the saturation of fatty acid (FA) in 1,2-dimyristoyl-sn-glycero-3-phosphocoline (DMPC)/FA membrane on the interaction between lipid membrane and amyloid ß monomer was investigated by using the Langmuir monolayer technique. The surface pressure (Π)-mean molecular area (A) isotherms and fluorescent measurements reveal that DMPC and octadecanoic acid (stearic acid, SA) molecules were somewhat miscible in the mixed membrane, which was maintained to homogeneous gel phase by enhance of the intermolecular hydrophobic interactions because of the all trans acyl chains. On the other hand, DMPC and 9Z,12Z-octadecadienoic acid (linoleic acid, LA) molecules were considered to be well miscible in the mixed membrane, where the membrane partially transferred from gel phase to liquid-crystalline phase. The Π-A isotherms of the monolayers on amyloid ß-peptide (Aß) solution indicated that Aß monomers tend to be inserted into the saturated acyl chain region of monolayers at low surface pressure and that the Aß monomers were then extruded from the monolayer at higher surface pressure. It was observed that behaviors of Aß monomers at higher surface pressure depended on membrane microstructures. In the DMPC/SA monolayers, Aß aggregated and then was extruded from monolayers at about 20 mN m-1 of surface pressure irrespective of the SA proportion. On the other hand, in the DMPC/LA monolayers, Aß, which favors to interact with DMPC, is dispersed in the monolayer even at high surface pressure because DMPC and LA molecules were well miscible in the monolayer.

Endogenous and Exogenous KdpF Peptide Increases Susceptibility of Mycobacterium bovis BCG to Nitrosative Stress and Reduces Intramacrophage Replication.

Emerging antibiotic resistance in pathogenic bacteria like Mycobacterium sp., poses a threat to human health and therefore calls for the development of novel antibacterial strategies. We have recently discovered that bacterial membrane peptides, such as KdpF, possess anti-virulence properties when overproduced in pathogenic bacterial species. Overproduction of the KdpF peptide in Mycobacterium bovis BCG decreased bacterial replication within macrophages, without presenting antibacterial activity. We propose that KdpF functions as a regulatory molecule and interferes with bacterial virulence, potentially through interaction with the PDIM transporter MmpL7. We demonstrate here that KdpF overproduction in M. bovis BCG, increased bacterial susceptibility to nitrosative stress and thereby was responsible for lower replication rate within macrophages. Moreover, in a bacterial two-hybrid system, KdpF was able to interact not only with MmpL7 but also with two membrane proteins involved in nitrosative stress detoxification (NarI and NarK2), and a membrane protein of unknown function that is highly induced upon nitrosative stress (Rv2617c). Interestingly, we showed that the exogenous addition of KdpF synthetic peptide could affect the stability of proteins that interact with this peptide. Finally, the exogenous KdpF peptide presented similar biological effects as the endogenously expressed peptide including nitrosative stress susceptibility and reduced intramacrophage replication rate for M. bovis BCG. Taken together, our results establish a link between high levels of KdpF and nitrosative stress susceptibility to further highlight KdpF as a potent molecule with anti-virulence properties.

Use of the Salmonella MgtR peptide as an antagonist of the Mycobacterium MgtC virulence factor.

BACKGROUND: The MgtC virulence factor has been proposed as an attractive target for antivirulence strategies because it is shared by several important bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis (Mtb). AIM: A natural antagonistic peptide, MgtR, which interacts with MgtC and modulates its stability, has been identified in Salmonella, and we investigated its efficiency to target MgtC in another pathogen. MATERIALS & METHODS: We evaluated the interaction between Salmonella MgtR peptide and the Mtb MgtC protein using an in vivo bacterial two-hybrid system and we addressed the effect of exogenously added synthetic MgtR and endogenously expressed peptide. RESULTS: MgtR peptide strongly interacted with Mtb MgtC protein and exogenously added synthetic MgtR peptide-reduced Mtb MgtC level and interfered with the dimerization of Mtb MgtC. Importantly, heterologous expression of MgtR in Mycobacterium bovis BCG resulted in increased phagocytosis and reduced intramacrophage survival. CONCLUSION: MgtR peptide can target Mtb MgtC protein and reduce mycobacterial macrophage resistance, thus providing a promising new scaffold for the development of antivirulence compounds.

Determining the Secondary Structure of Membrane Proteins and Peptides Via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

Revealing detailed structural and dynamic information of membrane embedded or associated proteins is challenging due to their hydrophobic nature which makes NMR and X-ray crystallographic studies challenging or impossible. Electron paramagnetic resonance (EPR) has emerged as a powerful technique to provide essential structural and dynamic information for membrane proteins with no size limitations in membrane systems which mimic their natural lipid bilayer environment. Therefore, tremendous efforts have been devoted toward the development and application of EPR spectroscopic techniques to study the structure of biological systems such as membrane proteins and peptides. This chapter introduces a novel approach established and developed in the Lorigan lab to investigate membrane protein and peptide local secondary structures utilizing the pulsed EPR technique electron spin echo envelope modulation (ESEEM) spectroscopy. Detailed sample preparation strategies in model membrane protein systems and the experimental setup are described. Also, the ability of this approach to identify local secondary structure of membrane proteins and peptides with unprecedented efficiency is demonstrated in model systems. Finally, applications and further developments of this ESEEM approach for probing larger size membrane proteins produced by overexpression systems are discussed.

Kinetics of peptide folding in lipid membranes.

Despite our extensive understanding of water-soluble protein folding kinetics, much less is known about the folding dynamics and mechanisms of membrane proteins. However, recent studies have shown that for relatively simple systems, such as peptides that form a transmembrane α-helix, helical dimer, or helix-turn-helix, it is possible to assess the kinetics of several important steps, including peptide binding to the membrane from aqueous solution, peptide folding on the membrane surface, helix insertion into the membrane, and helix-helix association inside the membrane. Herein, we provide a brief review of these studies and also suggest new initiation and probing methods that could lead to improved temporal and structural resolution in future experiments.

Overexpression of the Salmonella KdpF membrane peptide modulates expression of kdp genes and intramacrophage growth.

Membrane peptides appear as an emerging class of regulatory molecules in bacteria, which can interact with membrane proteins, including transporters and sensor kinases. The KdpF peptide, which is cotranscribed with kdpABC genes and regulated by the KdpDE two-component system, is supposed to stabilize the KdpABC potassium transporter complex but may also exhibit unsuspected regulatory function(s). The mycobacterial KdpF can interact with the KdpD histidine kinase, and kdpF overexpression has been shown to reduce intramacrophage replication of Mycobacterium bovis BCG. In this study, we investigated whether KdpF displays similar behavior in another intracellular pathogen, Salmonella enterica serovar Typhimurium. We show that Salmonella KdpF can interact with KdpD in a bacterial two-hybrid assay. We have constructed a Salmonella strain overexpressing kdpF, and we have investigated expression of the kdp regulon, as well as intramacrophage survival. We show that kdpF overexpression reduces expression of kdpA and kdpD genes under potassium limitation. Moreover, kdpF overexpression increases intramacrophage multiplication of S. Typhimurium. Hence, our results indicate that KdpF can play a regulatory role in S. Typhimurium, modulating kdp gene expression and intramacrophage survival, but in a way that differs from the one reported for M. bovis BCG.

The influenza fusion peptide promotes lipid polar head intrusion through hydrogen bonding with phosphates and N-terminal membrane insertion depth.

Influenza infection requires fusion between the virus envelope and a host cell endosomal membrane. The influenza hemagglutinin fusion peptide (FP) is essential to viral membrane fusion. It was recently proposed that FPs would fuse membranes by increasing lipid tail protrusion, a membrane fusion transition state. The details of how FPs induce lipid tail protrusion, however, remain to be elucidated. To decipher the molecular mechanism by which FPs promote lipid tail protrusion, we performed molecular dynamics simulations of the wild-type (WT) FP, fusogenic mutant F9A, and nonfusogenic mutant W14A in model bilayers. This article presents the peptide-lipid interaction responsible for lipid tail protrusion and a related lipid perturbation, polar head intrusion, where polar heads are sunk under the membrane surface. The backbone amides from the four N-terminal peptide residues, deeply inserted in the membrane, promoted both perturbations through H bonding with lipid phosphates. Polar head intrusion correlated with peptides N-terminal insertion depth and activity: the N-termini of WT and F9A were inserted deeper into the membrane than nonfusogenic W14A. Based on these results, we propose that FP-induced polar head intrusion would complement lipid tail protrusion in catalyzing membrane fusion by reducing repulsions between juxtaposed membranes headgroups. The presented model provides a framework for further research on membrane fusion and influenza antivirals.