Peptide Translocation Research Articles

Charged Antimicrobial Peptides Can Translocate across Membranes without Forming Channel-like Pores

How can highly charged, cationic antimicrobial peptides (AMPs) translocate across hydrophobic lipid bilayers despite the prohibitive energetic penalty to do so? A common explanation has been the formation of peptide-lined channels. However, for most AMPs, no structures of membrane pores have been found despite clear evidence of membrane leakage and antimicrobial activity. The study here suggests an alternative and simple reason: for the AMP PGLa from Xenopus laevis (charge +5), such pores are not needed to explain both leakage and peptide translocation. Elevated-temperature multimicrosecond equilibrium simulations at all-atomistic level reveal that peptides spontaneously translocate across the membrane individually on a timescale of tens of microseconds, without forming pores. Both surface-bound peptides and lipids assist in the one-by-one translocation of the charged side chains. Single peptides can remain in a transmembrane orientation for many microseconds, snorkeling some charged residues to one interface and some to the opposite, but without inducing a water channel. Instead of stable pores, short-lived water bridges occur when two or three peptides connect at their termini, allowing both ion translocation and lipid flip-flop via a brushlike mechanism usually involving the C terminus of one peptide. The results here suggest that for some specific antimicrobial and other membrane active peptides, pore formation may not have to be invoked at all to explain peptide translocation and membrane permeabilization, which may explain why no channel structures for them have been determined experimentally.

Peptide translocation across MOMP, the major outer membrane channel from Campylobacter jejuni

Here we report on translocation of short poly-arginines across the MOMP porin, the major outer membrane protein in the cell wall of Campylobacter jejuni. MOMP was purified to homogeneity from a pathogenic strain of C. jejuni. Its reconstitution in lipid membranes and measuring the ion-current revealed two main distinct populations of protein channels which we interpreted as mono and trimers. Addition of poly-arginines causes concentration and voltage dependent ion-current fluctuations. Increasing the transmembrane potential decreases the residence time of the peptide inside the channel indicating successful translocation. We conclude that poly-arginines can cross the outer membrane of Campylobacter through the MOMP channel.

Translocation of cell-penetrating peptides into Candida fungal pathogens.

Cell-penetrating peptides (CPPs) are small peptides capable of crossing cellular membranes while carrying molecular cargo. Although they have been widely studied for their ability to translocate nucleic acids, small molecules, and proteins into mammalian cells, studies of their interaction with fungal cells are limited. In this work, we evaluated the translocation of eleven fluorescently labeled peptides into the important human fungal pathogens Candida albicans and C. glabrata and explored the mechanisms of translocation. Seven of these peptides (cecropin B, penetratin, pVEC, MAP, SynB, (KFF)3 K, and MPG) exhibited substantial translocation (>80% of cells) into both species in a concentration-dependent manner, and an additional peptide (TP-10) exhibiting strong translocation into only C. glabrata. Vacuoles were involved in translocation and intracellular trafficking of the peptides in the fungal cells and, for some peptides, escape from the vacuoles and localization in the cytosol were correlated to toxicity toward the fungal cells. Endocytosis was involved in the translocation of cecropin B, MAP, SynB, MPG, (KFF)3 K, and TP-10, and cecropin B, penetratin, pVEC, and MAP caused membrane permeabilization during translocation. These results indicate the involvement of multiple translocation mechanisms for some CPPs. Although high levels of translocation were typically associated with toxicity of the peptides toward the fungal cells, SynB was translocated efficiently into Candida cells at concentrations that led to minimal toxicity. Our work highlights the potential of CPPs in delivering antifungal molecules and other bioactive cargo to Candida pathogens.

Direct and indirect activation of eukaryotic elongation factor 2 kinase by AMP-activated protein kinase

BackgroundEukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a key regulator of protein synthesis in mammalian cells. It phosphorylates and inhibits eEF2, the translation factor necessary for peptide translocation during the elongation phase of protein synthesis. When cellular energy demand outweighs energy supply, AMP-activated protein kinase (AMPK) and eEF2K become activated, leading to eEF2 phosphorylation, which reduces the rate of protein synthesis, a process that consumes a large proportion of cellular energy under optimal conditions. AimThe goal of the present study was to elucidate the mechanisms by which AMPK activation leads to increased eEF2 phosphorylation to decrease protein synthesis. MethodsUsing genetically modified mouse embryo fibroblasts (MEFs), effects of treatments with commonly used AMPK activators to increase eEF2 phosphorylation were compared with that of the novel compound 991. Bacterially expressed recombinant eEF2K was phosphorylated in vitro by recombinant activated AMPK for phosphorylation site-identification by mass spectrometry followed by site-directed mutagenesis of the identified sites to alanine residues to study effects on the kinetic properties of eEF2K. Wild-type eEF2K and a Ser491/Ser492 mutant were retrovirally re-introduced in eEF2K-deficient MEFs and effects of 991 treatment on eEF2 phosphorylation and protein synthesis rates were studied in these cells. Results & conclusionsAMPK activation leads to increased eEF2 phosphorylation in MEFs mainly by direct activation of eEF2K and partly by inhibition of mammalian target of rapamycin complex 1 (mTORC1) signaling. Treatment of MEFs with AMPK activators can also lead to eEF2K activation independently of AMPK probably via a rise in intracellular Ca2+. AMPK activates eEF2K by multi-site phosphorylation and the newly identified Ser491/Ser492 is important for activation, leading to mTOR-independent inhibition of protein synthesis. Our study provides new insights into the control of eEF2K by AMPK, with implications for linking metabolic stress to decreased protein synthesis to conserve energy reserves, a pathway that is of major importance in cancer cell survival.

Glycosaminoglycan Binding and Non-Endocytic Membrane Translocation of Cell-Permeable Octaarginine Monitored by Real-Time In-Cell NMR Spectroscopy.

Glycosaminoglycans (GAGs), which are covalently-linked membrane proteins at the cell surface have recently been suggested to involve in not only endocytic cellular uptake but also non-endocytic direct cell membrane translocation of arginine-rich cell-penetrating peptides (CPPs). However, in-situ comprehensive observation and the quantitative analysis of the direct membrane translocation processes are challenging, and the mechanism therefore remains still unresolved. In this work, real-time in-cell NMR spectroscopy was applied to investigate the direct membrane translocation of octaarginine (R8) into living cells. By introducing 4-trifluoromethyl-l-phenylalanine to the N terminus of R8, the non-endocytic membrane translocation of 19F-labeled R8 (19F-R8) into a human myeloid leukemia cell line was observed at 4 °C with a time resolution in the order of minutes. 19F NMR successfully detected real-time R8 translocation: the binding to anionic GAGs at the cell surface, followed by the penetration into the cell membrane, and the entry into cytosol across the membrane. The NMR concentration analysis enabled quantification of how much of R8 was staying in the respective translocation processes with time in situ. Taken together, our in-cell NMR results provide the physicochemical rationale for spontaneous penetration of CPPs in cell membranes.

Regulation of Protein Synthesis by Post‐translational Modification of Eukaryotic Translation Elongation Factor 1A

Protein synthesis is an intricate multi‐step process accomplished through a series of concerted actions of several different factors. Post initiation, translation elongation continues in a cyclic manner with the delivery of cognate aa‐tRNA to ribosomal A site, peptide bond formation and translocation to P‐site. The broad mechanism of action of elongation factors is known, however the regulation of translation elongation remains poorly understood. To safeguard timely and accurate gene expression, the protein synthesis machinery must be strictly regulated. Indeed the eukaryotic Elongation Factor 1A (eEF1A) shows extensive post‐translational modifications, both phosphorylation and methylation. In Saccharomyces cerevisiae, global proteomic analyses have reported at least 15 residues in eEF1A that are phosphorylated under different growth conditions. We have created a set of corresponding alanine (non‐phosphorylatable) and aspartate (phosphomimetic) mutations in these residues to study their phenotypic as well as enzymatic effects. Our in vivo and in vitro analyses in budding yeast show that S53D, Y416D and T430D mutant alleles are unable to support cell growth as the only form of eEF1A, while S18D, T259D and Y416A show slow growth and an initiation defect manifested by Gcn2 activation. Further experiments to test the translation fidelity, in vitro enzymatic activity and their ability to interact with cofactors are ongoing. In addition to translation elongation, eEF1A has been implicated in a wide variety of cellular processes, for example cytoskeleton organization through actin bundling, protein quality control, viral invasion and replication, etc. This work is aimed at identifying the events and factors that modulate eEF1A activity ensuring careful control of gene expression and appropriate cell function.Support or Funding InformationNIH grant RO1 GM057483

Crystal structure of eukaryotic ribosome and its complexes with inhibitors.

A high-resolution structure of the eukaryotic ribosome has been determined and has led to increased interest in studying protein biosynthesis and regulation of biosynthesis in cells. The functional complexes of the ribosome crystals obtained from bacteria and yeast have permitted researchers to identify the precise residue positions in different states of ribosome function. This knowledge, together with electron microscopy studies, enhances our understanding of how basic ribosome processes, including mRNA decoding, peptide bond formation, mRNA, and tRNA translocation and cotranslational transport of the nascent peptide, are regulated. In this review, we discuss the crystal structure of the entire 80S ribosome from yeast, which reveals its eukaryotic-specific features, and application of X-ray crystallography of the 80S ribosome for investigation of the binding mode for distinct compounds known to inhibit or modulate the protein-translation function of the ribosome. We also refer to a challenging aspect of the structural study of ribosomes, from higher eukaryotes, where the structures of major distinctive features of higher eukaryote ribosome-the high-eukaryote-specific long ribosomal RNA segments (about 1MDa)-remain unresolved. Presently, the structures of the major part of these high-eukaryotic expansion ribosomal RNA segments still remain unresolved.This article is part of the themed issue 'Perspectives on the ribosome'.

Revealing the sequence of interactions of PuroA peptide with Candida albicans cells by live-cell imaging

To determine the mechanism(s) of action of antimicrobial peptides (AMPs) it is desirable to provide details of their interaction kinetics with cellular, sub-cellular and molecular targets. The synthetic peptide, PuroA, displays potent antimicrobial activities which have been attributed to peptide-induced membrane destabilization, or intracellular mechanisms of action (DNA-binding) or both. We used time-lapse fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM) to directly monitor the localization and interaction kinetics of a FITC- PuroA peptide on single Candida albicans cells in real time. Our results reveal the sequence of events leading to cell death. Within 1 minute, FITC-PuroA was observed to interact with SYTO-labelled nucleic acids, resulting in a noticeable quenching in the fluorescence lifetime of the peptide label at the nucleus of yeast cells, and cell-cycle arrest. A propidium iodide (PI) influx assay confirmed that peptide translocation itself did not disrupt the cell membrane integrity; however, PI entry occurred 25–45 minutes later, which correlated with an increase in fractional fluorescence of pores and an overall loss of cell size. Our results clarify that membrane disruption appears to be the mechanism by which the C. albicans cells are killed and this occurs after FITC-PuroA translocation and binding to intracellular targets.

Structural Analysis of a Temperature-Induced Transition in a Viral Capsid Probed by HDX-MS

Icosahedral viral capsids are made of a large number of symmetrically organized protein subunits whose local movements can be essential for infection. In the capsid of the minute virus of mice, events required for infection that involve translocation of peptides through capsid pores are associated with a subtle conformational change. In vitro, this change can be reversibly induced by overcoming the energy barrier through mild heating of the capsid, but little is known about the capsid regions involved in the process. Here, we use hydrogen-deuterium exchange coupled to mass spectrometry to analyze the dynamics of the minute virus of mice capsid at increasing temperatures. Our results indicate that the transition associated with peptide translocation involves the structural rearrangement of regions distant from the capsid pores. These alterations are reflected in an increased dynamics of some secondary-structure elements in the capsid shell from which spikes protrude, and a decreased dynamics in the long intertwined loops that form the large capsid spikes. Thus, the translocation events through capsid pores involve a global conformational rearrangement of the capsid and a complex alteration of its equilibrium dynamics. This study additionally demonstrates the potential of hydrogen-deuterium exchange coupled to mass spectrometry to explore in detail temperature-dependent structural dynamics in large macromolecular protein assemblies. Most importantly, it paves the way for undertaking novel studies of the relationship between structure, dynamics, and biological function in virus particles and other large protein cages.

Effects of Mechanical Properties of Lipid Bilayers on the Entry of Cell-Penetrating Peptides into Single Vesicles.

The translocation of cell-penetrating peptides (CPPs) through plasma membranes of living cells is an important physiological phenomenon in biomembranes. To reveal the mechanism underlying the translocation of a CPP, transportan 10 (TP10), through lipid bilayers, we examined the effects of the mechanical properties of lipid bilayers on the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) into a giant unilamellar vesicle (GUV) using the single GUV method. First, we examined the effect of lateral tension in membranes on the entry of CF-TP10 into single GUVs comprising a mixture of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (2/8). CF-TP10 entered the GUV lumen before the membrane permeation of Alexa Fluor 647 hydrazide (AF647) from the GUV and thus before pore formation in the membrane. The fraction of entry of CF-TP10 before pore formation and the rate of membrane rupture increased with tension. The CF-TP10-induced fractional change in the membrane area increased continuously with time until membrane rupture, but it increased more slowly than did the CF-TP10 concentration in the GUV membrane. A high mole fraction of cholesterol inhibited the entry of CF-TP10 into single GUVs by suppressing the translocation of CF-TP10 from the external to the internal monolayer, although higher concentrations of CF-TP10 induced the formation of pores through which CF-TP10 rapidly translocated. Suppression of the translocation of CF-TP10 by cholesterol can be reasonably explained by the large line tension of a prepore. We discussed the role of mechanical properties in membranes on the entry of CF-TP10 into single GUVs and proposed a hypothesis of the mechanism that CF-TP10 translocates across a bilayer through transient hydrophilic prepores in the membrane.

Dynamic Phenylalanine Clamp Interactions Define Single-Channel Polypeptide Translocation through the Anthrax Toxin Protective Antigen Channel

Anthrax toxin is an intracellularly acting toxin where sufficient detail is known about the structure of its channel, allowing for molecular investigations of translocation. The toxin is composed of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). The toxin's translocon, PA, translocates the large enzymes, LF and EF, across the endosomal membrane into the host cell's cytosol. Polypeptide clamps located throughout the PA channel catalyze the translocation of LF and EF. Here, we show that the central peptide clamp, the ϕ clamp, is a dynamic site that governs the overall peptide translocation pathway. Single-channel translocations of a 10-residue, guest–host peptide revealed that there were four states when peptide interacted with the channel. Two of the states had intermediate conductances of 10% and 50% of full conductance. With aromatic guest–host peptides, the 50% conducting intermediate oscillated with the fully blocked state. A Trp guest–host peptide was studied by manipulating its stereochemistry and prenucleating helix formation with a covalent linkage in the place of a hydrogen bond or hydrogen-bond surrogate (HBS). The Trp peptide synthesized with ʟ-amino acids translocated more efficiently than peptides synthesized with D- or alternating D,ʟ-amino acids. HBS stapled Trp peptide exhibited signs of steric hindrance and difficulty translocating. However, when mutant ϕ clamp (F427A) channels were tested, the HBS peptide translocated normally. Overall, peptide translocation is defined by dynamic interactions between the peptide and ϕ clamp. These dynamics require conformational flexibility, such that the peptide productively forms both extended-chain and helical states during translocation.

Depicting Binding-Mediated Translocation of HIV-1 Tat Peptides in Living Cells with Nanoscale Pens of Tat-Conjugated Quantum Dots.

Cell-penetrating peptides (CPPs) can translocate across cell membranes, and thus have great potential for the cellular delivery of macromolecular cargoes. However, the mechanism of this cellular uptake process is not yet fully understood. In this study, a time-lapse single-particle light-sheet microscopy technique was implemented to obtain a parallel visualization of the translocating process of individual human immunodeficiency virus 1 (HIV-1) transactivator of transcription (Tat) peptide conjugated quantum dots (TatP-QDs) in complex cellular terrains. Here, TatP-QDs served as nanoscale dynamic pens, which depict remarkable trajectory aggregates of TatP-QDs on the cell surface. Spectral-embedding analysis of the trajectory aggregates revealed a manifold formed by isotropic diffusion and a fraction of directed movement, possibly caused by interaction between the Tat peptides and heparan sulfate groups on the plasma membrane. Further analysis indicated that the membrane deformation induced by Tat-peptide attachment increased with the disruption of the actin framework in cytochalasin D (cyto D)-treated cells, yielding higher interactions on the TatP-QDs. In native cells, the Tat peptides can remodel the actin framework to reduce their interaction with the local membrane environment. Characteristic hot spots for interaction were detected on the membrane, suggesting that a funnel passage may have formed for the Tat-coated particles. This finding offers valuable insight into the cellular delivery of nanoscale cargo, suggesting an avenue for direct therapeutic delivery.

Structural effects and translocation of spontaneous membrane-translocating peptides with POPC bilayer

Martini coarse-grained force field simulations have been carried out to estimate the free energy profiles of the spontaneous membrane-translocating peptide TP2 and one negative control peptide ONEG with POPC as the model bilayer. The results show that the free energy minimum of TP2 is [Formula: see text]20[Formula: see text]kJ/mol lower than that of ONEG. In addition, the minimum of TP2 shifts slightly to the bilayer center compared with ONEG. The translocation barrier height for TP2 and ONEG are 119.0[Formula: see text]kJ/mol and 155.7[Formula: see text]kJ/mol, respectively. The lower central energy barrier of TP2 facilitates the transition between two leaflets of POPC. Both translocating peptides induce the formation of funnel-shaped structures at the bilayer center, but TP2 has a more compact structure and brings less perturbation compared with ONEG. Subsequently all atom molecular simulations testify the findings. It is indicated that compared with its negative control ONEG, TP2 binds better with lipid and penetrates deeper into bilayer with less perturbation to the bilayer structure. Our findings may shed light on the design and virtual screening of spontaneous membrane-translocating peptides.

Channel of viral DNA packaging motor for real time kinetic analysis of peptide oxidation states.

Nanopore technology has become a powerful tool in single molecule sensing, and protein nanopores appear to be more advantageous than synthetic counterparts with regards to channel amenability, structure homogeneity, and production reproducibility. However, the diameter of most of the well-studied protein nanopores is too small to allow the passage of protein or peptides that are typically in multiple nanometers scale. The portal channel from bacteriophage SPP1 has a large channel size that allows the translocation of peptides with higher ordered structures. Utilizing single channel conductance assay and optical single molecule imaging, we observed translocation of peptides and quantitatively analyzed the dynamics of peptide oligomeric states in real-time at single molecule level. The oxidative and the reduced states of peptides were clearly differentiated based on their characteristic electronic signatures. A similar Gibbs free energy (ΔG0) was obtained when different concentrations of substrates were applied, suggesting that the use of SPP1 nanopore for real-time quantification of peptide oligomeric states is feasible. With the intrinsic nature of size and conjugation amenability, the SPP1 nanopore has the potential for development into a tool for the quantification of peptide and protein structures in real time.

Label-free discrimination of membrane-translocating peptides on porous silicon microfluidic biosensors.

A label-free optical sensor was constructed by integrating electrochemically etched porous silicon (pSi) and supported phospholipid bilayers in a microfluidic chip. The translocation of peptides through the phospholipid bilayers could induce a red shift in effective optical thickness of the pSi layer, which could be monitored by reflective interferometric Fourier transform spectroscopy. By measuring the kinetic data of membrane translocating on the phospholipid bilayers/pSi chip, the relationship between the behavior of membrane-translocating peptides (MTPs) and translocating mechanism was established. With these optical data, MTPs with different action modes on the cell membrane can be correctly discriminated. The bio-functionalized microfluidic sensor will provide a reliable and cost-effective platform to study the transmembrane behavior of peptides, which is of great importance in the MTP screening and peptide function study.

Charge Distribution Fine-Tunes the Translocation of α-Helical Amphipathic Peptides across Membranes

Hundreds of cationic antimicrobial and cell-penetrating peptides (CPPs) form amphipathic α-helices when bound to lipid membranes. Here, we test two hypotheses for the differences in the ability of these peptides to translocate across membranes. The first, which we now call the hydrophobicity hypothesis, is that peptide translocation is determined by the Gibbs energy of insertion into the bilayer from the membrane interface. The second, which we call the charge-distribution hypothesis, is that translocation is determined by whether the distribution of cationic residues in the peptide can transiently stabilize a high-energy inserted intermediate by forming salt bridges to the phosphates of lipid headgroups. To test these hypotheses, we measured translocation of two series of peptide variants. The first series was based on TP10W, a peptide derived from the amphipathic CPP transportan 10; the second was based on DL1a, a synthetic peptide derived from staphylococcal δ-lysin. The peptides in those two series had small sequence changes relative to TP10W and DL1a: either single-residue substitutions or two-residue switches, which were designed to increase or decrease translocation differently according to the two hypotheses. We found that with regard to the changes introduced in the sequences, five out of six peptide variants translocated in agreement with the charge-distribution hypothesis, whereas none showed agreement with the hydrophobicity hypothesis. We conclude that large effects on translocation are probably determined by hydrophobicity, but the fine tuning appears to arise from the distribution of cationic residues along the peptide sequence.

Oriented single directional insertion of nanochannel of bacteriophage SPP1 DNA packaging motor into lipid bilayer via polar hydrophobicity.

Insertion of biological nanopore into artificial membrane is of fundamental importance in nanotechnology. Many applications require control and knowledge of channel orientation. In this work, the insertion orientation of the bacteriophage SPP1 and phi29 DNA packaging motors into lipid membranes was investigated. Single molecule electrophysiological assays and Ni-NTA-nanogold binding assays revealed that both SPP1 and phi29 motor channels exhibited a one-way traffic property for TAT peptide translocation from N- to C-termini of the protein channels. SPP1 motor channels preferentially inserts into liposomes with their C-terminal wider region facing inward. Changing the hydrophobicity of the N- or C-termini of phi29 connector alters the insertion orientation, suggesting that the hydrophobicity and hydrophilicity of the termini of the protein channel governs the orientation of the insertion into lipid membrane. It is proposed that the specificity in motor channel orientation is a result of the hydrophilic/hydrophobic interaction at the air/water interface when the protein channels are incorporating into liposome membranes.

Kinetics of Spontaneous and EF-G-Accelerated Rotation of Ribosomal Subunits

Ribosome dynamics play an important role in translation. The rotation of the ribosomal subunits relative to one another is essential for tRNA-mRNA translocation. An important unresolved question is whether subunit rotation limits the rate of translocation. Here,we monitor subunit rotation relative to peptide bond formation and translocation using ensemble kineticsand single-molecule FRET. We observe that spontaneous forward subunit rotation occurs at a rate of 40s(-1), independent of the rate of preceding peptidebond formation. Elongation factor G (EF-G) accelerates forward subunit rotation to 200 s(-1). tRNA-mRNA movement is much slower (10-40 s(-1)), suggesting that forward subunit rotation does not limit the rate of translocation. The transition back to the non-rotated state of the ribosome kinetically coincides with tRNA-mRNA movement. Thus, large-scale movements of the ribosome are intrinsically rapid and gated by its ligands such as EF-G and tRNA.

Fingerprinting of Peptides with a Large Channel of Bacteriophage Phi29 DNA Packaging Motor.

Nanopore technology has become a highly sensitive and powerful tool for single molecule sensing of chemicals and biopolymers. Protein pores have the advantages of size amenability, channel homogeneity, and fabrication reproducibility. But most well-studied protein pores for sensing are too small for passage of peptide analytes that are typically a few nanometers in dimension. The funnel-shaped channel of bacteriophage phi29 DNA packaging motor has previously been inserted into a lipid membrane to serve as a larger pore with a narrowest N-terminal constriction of 3.6 nm and a wider C-terminal end of 6 nm. Here, the utility of phi29 motor channel for fingerprinting of various peptides using single molecule electrophysiological assays is reported. The translocation of peptides is proved unequivocally by single molecule fluorescence imaging. Current blockage percentage and distinctive current signatures are used to distinguish peptides with high confidence. Each peptide generated one or two distinct current blockage peaks, serving as typical fingerprint for each peptide. The oligomeric states of peptides can also be studied in real time at single molecule level. The results demonstrate the potential for further development of phi29 motor channel for detection of disease-associated peptide biomarkers.

Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides.

Studies attempting to characterize the membrane translocation of antimicrobial and cell-penetrating peptides are frequently limited by the resolution of conventional light microscopy. This study shows that spheroplasts provide a valuable approach to overcome these limits. Spheroplasts produce less ambiguous images and allow for more systematic analyses of localization. Data collected with spheroplasts are consistent with studies using normal bacterial cells and imply that a particular peptide may not always follow the same mechanism of action.