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Antimicrobial peptides (AMPs) have the potential to treat multidrug-resistant bacterial infections. However, the clinical application of AMPs is prevented by their toxicity and poor proteolytic stability. Here, a site-specific approach is used to generate new AMPs to improve their efficacy against bacterial pathogens while reducing their toxicity. We modified and generated a new series of antimicrobial peptides from the leucine- and lysine-rich antimicrobial peptide Amp1L (LKLLKKLLKKLLKLL) by the site-specific incorporation of an isopeptide bond while retaining the peptide's size, sequence, charge, and molecular weight. This single bond switch provides the peptides with a weak helical conformation, strong antimicrobial activity, resistance to proteolytic degradation, low toxicity, and lower hemolytic activity. This new site-specific approach offers a powerful tool for developing potent and nontoxic antimicrobial drugs.

Previously, we reported that intratumor or systemic inoculation of a cationic 15-mer, innate immunity-like lytic peptide composed of d- and l-amino acids ([D]-K6L9) caused growth arrest of 22RV1 prostate carcinoma xenografts in a mouse model. However, despite its therapeutic potential, this peptide has significant systemic toxicity at concentrations slightly higher than the therapeutic one. Here, we used the acidic environment created by solid tumors as a trigger to activate anticancer lytic peptides by making them cationic only at low pH levels. We achieved this selectivity by substituting lysines (pKa, ∼10.5) for histidines (pKa, ∼6.1) in the parental peptide [D]-K6L9. Histidine is protonated below pH 7. For that purpose, we replaced either three or all six lysines in the parental peptide with histidines to obtain the peptides [D]-K3H3L9 and [D]-H6L9. Interestingly, in vitro experiments showed pH-dependent activity only with [D]-H6L9 mainly toward cancer cell lines. However, both peptides showed reduced systemic toxicity compared with the parental peptide. Intratumor and systemic inoculation of these peptides resulted in a significant decrease in the 22RV1 prostate cancer tumor volume and systemic secretion of prostate-specific antigen in a xenograft mice model. Moreover, histologic modifications revealed a significant reduction in new blood vessels selectively in tumor tissues after treatment with the peptides compared with the untreated tumors. The lytic mode of action of these new peptides, which makes it difficult for the cancer cells to develop resistance, and their selective and pH-dependent activity make them potential candidates for treatment of solid cancer tumors. [Cancer Res 2009;69(8):3458–63]

Supplementary Figures 1-3 from Suppression of Human Solid Tumor Growth in Mice by Intratumor and Systemic Inoculation of Histidine-Rich and pH-Dependent Host Defense–like Lytic Peptides

The human immunodeficiency virus enters its host cells by membrane fusion, initiated by the gp41 subunit of its envelope protein. gp41 has also been shown to bind T-cell receptor (TCR) complex components, interfering with TCR signaling leading to reduced T-cell activation. This immunoinhibitory activity is suggested to occur during the membrane fusion process and is attributed to various membranotropic regions of the gp41 ectodomain and to the transmembrane domain. Although extensively studied, the cytosolic region of gp41, termed the cytoplasmic tail (CT), has not been examined in the context of immune suppression. Here we investigated whether the CT inhibits T-cell activation in different T-cell models by utilizing gp41-derived peptides and expressed full gp41 proteins. We found that a conserved region of the CT, termed lentiviral lytic peptide 2 (LLP2), specifically inhibits the activation of mouse, Jurkat, and human primary T-cells. This inhibition resulted in reduced T-cell proliferation, gene expression, cytokine secretion, and cell surface expression of CD69. Differential activation of the TCR signaling cascade revealed that CT-based immune suppression occurs downstream of the TCR complex. Moreover, LLP2 peptide treatment of Jurkat and primary human T-cells impaired Akt but not NFκB and ERK1/2 activation, suggesting that immune suppression occurs through the Akt pathway. These findings identify a novel gp41 T-cell suppressive element with a unique inhibitory mechanism that can take place post-membrane fusion.

Antimicrobial peptides (AMPs), which can be modified to kill a broad spectrum of microoganisms or a specific microorganism, are considered as promising alternatives to combat the rapidly widespread, resistant bacterial infections. However, there are still several obstacles to overcome. These include toxicity, stability, and the ability to interfere with the immune response and bacterial resistance. To overcome these challenges, we herein replaced the regular peptide bonds with isopeptide bonds to produce new AMPs based on the well-known synthetic peptides Amp1L and MSI-78 (pexiganan). Two new peptides Amp1EP and MSIEP were generated while retaining properties such as size, sequence, charge, and molecular weight. These new peptides have reduced toxicity toward murine macrophage (RAW 264.7) cells, human monocytic (THP-1) cells, and human red blood cells (hRBCs) and enhanced the stability toward proteolytic degradation. Importantly, the new peptides do not repress the pro-inflammatory cytokine and hence should not modulate the immune response. Structurally, the new peptides, Amp1EP and MSIEP, have a structure of random coils in contrast to the helical structures of the parental peptides as revealed by circular dichroism (CD) analysis. Their mode of action, assessed by flow cytometry, includes permeabilization of the bacterial membrane. Overall, we present here a new approach to modulate AMPs to develop antimicrobial peptides for future therapeutic purposes.

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 (MoskowitzS. M.;Antimicrob. Agents Chemother.2012, 56, 1019−103022106224; ChengH. Y.;J. Biomed. Sci.2010, 17, 6020653976). 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.

Background: The concept of a multimodality therapy in the treatment of soft tissue sarcomas (STS) has been discussed with controversy. Surgical resection with clear margins and radiation therapy remain gold standard in STS therapy. It is still questionable whether a systemic therapy with chemotherapeutics has a positive impact on the overall survival rate especially in early stages of disease, because the therapeutic effect in the treatment of STS is limited by its toxicities and its low responding rates. Treatment options are rare. As a result the search for combination therapies by using low dose approaches is of high importance. Recent studies showed the therapeutic efficiency of a designer host defense-like lytic D,L- amino acid peptide [D]-K 3 H 3 L 9 . Therefore we tested a combination of this peptide with Doxorubicin on two different sarcoma cell lines in vitro and also in a syngeneic immunocompetent murine fibrosarcoma mouse model. Methods: In vitro the human synovial sarcoma cell line SW 982 and the murine fibrosarcoma cell line BFS-1 were exposed to the oncolytic peptide [D]-K 3 H 3 L 9 , to the Anthracycline Doxorubicin and to both agents simultaneously. In vivo the murine fibrosarcoma cell line BFS-1 was injected subcutaneously into the syngeneic mice. When the tumors engrafted the oncolytic designer peptide [D]-K 3 H 3 L 9 , Doxorubicin or a combination of both was administered thrice a week for a three weeks’ follow-up. Results: The combination treatment approach using an oncolytic designer host defense peptide and Doxorubicin inhibited the in vitro sarcoma cell proliferation significantly. The single therapies, either with local intratumoral application of [D]-K 3 H 3 L 9 or with intraperitoneal application of Doxorubicin in the syngeneic mouse model, inhibited at least the tumor progression. The combination of both substances revealed a significant inhibition of tumor growth and weight. Conclusion: The in vivo low dose combination schedule inhibited the tumor growth significantly. Histological analyses of the tumor sections revealed an antiproliferative and antiangiogenic effect. So, these results demonstrate the effectiveness of combined low-dose application forms with designer host defense-like lytic peptides and chemotherapeutics.

The HIV gp160 envelope fusion protein is situated in the viral membrane and mediates virus entry into its host cell. Increasing evidence suggests that virtually all parts of the HIV envelope are structurally and functionally dependent on membranes. Protein-lipid interactions and membrane properties influence the dynamics of a manifold of gp160 biological activities such as membrane fusion, immune suppression and gp160 incorporation into virions during HIV budding and assembly. In the following we will summarize our current understanding of this interdependence between membrane interaction, structural conformation and functionality of the different gp160 domains. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

Bacterial resistance to antibiotics is a major concern worldwide, leading to an extensive search for alternative drugs. Promising candidates are antimicrobial peptides (AMPs), innate immunity molecules, shown to be highly efficient against multidrug resistant bacteria. Therefore, it is essential to study bacterial resistance mechanisms against them. For that purpose, we used experimental evolution, and isolated a Salmonella enterica serovar typhimurium-resistant line to the AMP 4DK5L7. This AMP displayed promising features including widespread activity against Gram-negative bacteria and protection from proteolytic degradation. However, the resistance that evolved in the isolated strain was particularly high. Whole genome sequencing revealed that five spontaneous mutations had evolved. Of these, three are novel in the context of acquired AMP resistance. Two mutations are related to the AcrAB-TolC multidrug efflux pump. One occurred in AcrB, the substrate-binding domain of the system, and the second in RamR, a transcriptional regulator of the system. Together, the mutations increased the minimal inhibitory concentration (MIC) by twofold toward this AMP. Moreover, the mutation in AcrB induced hypersusceptibility toward ampicillin and colistin. The last mutation occurred in Skp, a periplasmic chaperone that participates in the biogenesis of outer membrane proteins (OMPs). This mutation increased the MIC by twofold to 4DK5L7 and by fourfold to another AMP, seg5D. Proteomic analysis revealed that the mutation abolished Skp expression, reduced OMP abundance, and increased DegP levels. DegP, a protease that was reported to have an additional chaperone activity, escorts OMPs through the periplasm along with Skp, but is also associated with AMP resistance. In conclusion, our data demonstrate that both loss of Skp and manipulation of the AcrAB-TolC system are alternative strategies of AMP acquired resistance in Salmonella typhimurium and might represent a common mechanism in other Gram-negative bacteria.

Recruited blood monocytes contribute to the establishment, perpetuation, and resolution of tissue inflammation. Specifically, in the inflamed intestine, monocyte ablation was shown to ameliorate colitis scores in preclinical animal models. However, the majority of intestinal macrophages that seed the healthy gut are also monocyte derived. Monocyte ablation aimed to curb inflammation would therefore likely interfere with intestinal homeostasis. In this study, we used a TLR2 trans-membrane peptide that blocks TLR2 dimerization that is critical for TLR2/1 and TLR2/6 heterodimer signaling to blunt inflammation in a murine colitis model. We show that although the TLR2 peptide treatment ameliorated colitis, it allowed recruited monocytes to give rise to macrophages that lack the detrimental proinflammatory gene signature and reduced potentially damaging neutrophil infiltrates. Finally, we demonstrate TLR blocking activity of the peptide on in vitro cultured human monocyte-derived macrophages. Collectively, we provide a significantly improved anti-inflammatory TLR2 peptide and critical insights in its mechanism of action toward future potential use in the clinic.

Hantavirus assembly and budding are governed by the surface glycoproteins Gn and Gc. In this study, we investigated the glycoproteins of Puumala, the most abundant Hantavirus species in Europe, using fluorescently labeled wild-type constructs and cytoplasmic tail (CT) mutants. We analyzed their intracellular distribution, co-localization and oligomerization, applying comprehensive live, single-cell fluorescence techniques, including confocal microscopy, imaging flow cytometry, anisotropy imaging and Number&Brightness analysis. We demonstrate that Gc is significantly enriched in the Golgi apparatus in absence of other viral components, while Gn is mainly restricted to the endoplasmic reticulum (ER). Importantly, upon co-expression both glycoproteins were found in the Golgi apparatus. Furthermore, we show that an intact CT of Gc is necessary for efficient Golgi localization, while the CT of Gn influences protein stability. Finally, we found that Gn assembles into higher-order homo-oligomers, mainly dimers and tetramers, in the ER while Gc was present as mixture of monomers and dimers within the Golgi apparatus. Our findings suggest that PUUV Gc is the driving factor of the targeting of Gc and Gn to the Golgi region, while Gn possesses a significantly stronger self-association potential., Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe, 648

BackgroundThe outcome of a given antibiotic treatment on the growth capacity of bacteria is largely dependent on the initial population size (the Inoculum Effect, IE). For some specific classical antibiotic drugs this phenomenon is well established in both in-vitro and in-vivo studies, and its precise mechanisms, its clinical implications and its mathematical modelling are at the forefront of current research. Traditional view of the IE is that it is mainly attributed to β-lactam antibiotics in relation to β-lactamase producing bacteria, and that some antibiotics do not induce an IE at all. The study of antimicrobial peptides had emerged in the past two decades as a possible additional strategy for combatting infections, and their mechanism of operation and clinical implications are extensively studied. Yet, no previous studies addressed the possible induction of IE under the action of classical cationic antimicrobial peptides (CAMPs).Based on mathematical reasoning regarding bacteria-neutrophils interaction, we hypothesized that CAMPs also induce an IE in bacterial growth, and questioned what are the similarities and differences between the IE induced by CAMPs and that induced by classical antibiotics. To this aim we also needed to better understand the characteristics of the IE induced by classical antibiotics.Principal FindingsWe characterized and built a model of in-vitro IE in E. coli cultures using a large variety of antimicrobials, including 6 conventional antibiotics, and for the first time, 4 cationic antimicrobial peptides (CAMPs). Each combination of bacterial initial load and antimicrobial concentration experiment was done in duplicate, with 48 such combinations in each experiment. Each experiment was repeated 4-6 times, sometimes with some adjustments in the tested concentrations to get better resolution of the IE. Each growth curve was processed independently, to correctly reflect the initial exponential growth that might lead to large deviations even between duplicates. By using Optical Density (OD) to monitor the bacterial density, we were able to gather growth curves from this extensive data set and from these curves extract, by data processing, the corresponding growth functions. We show that this process allows us to clearly differentiate between simple one-dimensional deterministic bacterial growth dynamics and more complex behaviour.In all agents we tested, including all cationic antimicrobial peptides and all conventional antibiotics, independently of their biochemical mechanism of action, an “inoculum effect” was found. At a certain range of concentrations, which is specific for every drug and experimental setting, the system exhibits a bistable behaviour in which large loads survive and small loads are inhibited. Moreover, we characterized three distinct classes of drug-induced bi-stable growth dynamics and demonstrated that in rich medium, CAMPs correspond to the simplest class, bacteriostatic antibiotics to the second class and all other traditional antibiotics to the third, more complex class. In particular, for the first two classes, of cationic antimicrobial peptides and of the commercial bacteriostatic antibiotics, the bacterial growth can be explained by a very simple deterministic one-dimensional mathematical model. These findings provide a unifying universal framework to describe the dynamics of the inoculum effect induced by antimicrobials with inherently different killing mechanisms.Limitations of the results: The IE we detect is in-vitro, in rich medium, and the simple deterministic one dimensional models apply to this setting for the CAMPs and the bacteriostatic antibiotics only. While these findings can be used as a building block to more complex settings, with in-vivo being the most complex of all, it is clear that additional studies are needed in order to address these complexities. Another limitation is the OD methodology which does not clearly differentiate between live, dormant and dead cells and also does not detect small bacterial loads that are below the reader detection level. Nonetheless, since only live bacteria grow, the growth functions that we find experimentally are independent of the dead and dormant bacteria, and the bacterial density axis may be at most shifted by small amount due to this effect. The behaviour at small loads, below the OD detection level, is also irrelevant for the current study as we are concerned with the IE at high inoculum. Finally, this study is conducted at the population level only, with the point of view that IE is induced by deterministic non-linear interactions between the bacteria and the anti-microbial agent, without delving into the details of the particular molecular mechanisms that lead to this particular interaction. Such detailed nonlinear molecular mechanisms that induce IE are known to exist for some of the agents we use. Future studies are needed to better understand the detailed molecular mechanisms in the other cases.Conclusions & SignificanceThe vast increase in bacterial resistance, highlights the need for new approaches to eradicate bacterial infections, by either the development of new antimicrobial agents, or new strategies of treatment. Developing treatment strategies requires a better understanding of the Inoculum Effect (IE). We demonstrate that IE is abundant in the application of both classical antimicrobial peptides and classical antibiotics to bacteria. Furthermore, we show that IE falls into three universality classes of bi-stable behaviours and that classical antimicrobial peptides form a class of their own – the simplest and most predictable class. These findings propose a new exciting viewpoint on the universality features of IE that may serve as building blocks for the design of better treatment strategies for infection.We stress that the detection of IE in CAMPs may have important implications for their mode of operation, and this finding may lead to further explorations of this phenomenon both in terms of mechanistic models and in terms of clinical and biological implications.While bacterial IE was identified in previous studies of particular conventional antibiotic agents and bacteria, previous explanations of its appearance included genetic and/or phenotypic population heterogeneity and additional time-dependent factors. These were modelled, for example, by deterministic multi-dimensional equations of classical reaction kinetics. Here we show that for some cases (the bacteriostatic antibiotics) a one dimensional model can explain the resulting growth curves by density dependant mechanisms alone. By Ockham’s razor principle, we assert that such models are adequate for describing the IE in bacteriostatic antibiotics. On the other hand, we also show that for all other cases (growth with all other classical antibiotics and growth in poor medium) simple one dimensional deterministic models cannot describe the dynamics, and thus multi-dimensional models may be needed to describe IE in these cases. Additionally, contrary to some other studies, we show that IE appears in every antibiotic we tested (in particular antibiotics that are not β-lactams), so additional molecular mechanisms for creating the non-linear bacterial-drug interaction need to be identified.Finally, density dependent phenomena are abundant in biology and may appear in other pathogenesis systems, where densities matter. Here we demonstrated that such phenomena can sometimes be described by very simple growth dynamics. Such simple models may serve as building blocks to more complex models such as in-vivo ones and may also inspire detailed studies aimed at deciphering the specific dominant molecular mechanisms of the detected IE. We propose that the principles and methodologies developed here for studying IE by observing the population level dynamics may be applicable to diverse biological situations.Authors SummaryThe vast increase in bacterial resistance highlights the need for new approaches to eradicate bacterial infections, by either the development of new antimicrobial agents, or new strategies of treatment. Since the outcome of a given antibiotic treatment on the growth capacity of bacteria is largely dependent on the initial population size (Inoculum Effect, IE), developing treatment strategies requires a better understanding of this effect. We characterized and built a model of this effect in E. coli cultures using a large variety of antimicrobials, including conventional antibiotics, and for the first time, cationic antimicrobial peptides (CAMPs). Our results show that all classes of antimicrobial drugs induce an inoculum effect. Moreover, we characterized three distinct classes of drug-induced bi-stable growth dynamics and demonstrated that in rich medium, CAMPs correspond to the simplest class, bacteriostatic antibiotics to the second class and all other traditional antibiotics to the third, more complex class. These findings provide a unifying universal framework to describe the dynamics of the inoculum effect induced by antimicrobials with inherently different killing mechanisms. These findings propose a new exciting viewpoint on the universality features of IE that may serve as building blocks for the design of better treatment strategies for infection.

Pseudomonas aeruginosa is the major microorganism colonizing the respiratory epithelium in cystic fibrosis (CF) sufferers. The widespread use of available antibiotics has drastically reduced their efficacy, and antimicrobial peptides (AMPs) are a promising alternative. Among them, the frog skin-derived AMPs, i.e., Esc(1-21) and its diastereomer, Esc(1-21)-1c, have recently shown potent activity against free-living and sessile forms of P. aeruginosa . Importantly, this pathogen also escapes antibiotics treatment by invading airway epithelial cells. Here, we demonstrate that both AMPs kill Pseudomonas once internalized into bronchial cells which express either the functional or the ΔF508 mutant of the CF transmembrane conductance regulator. A higher efficacy is displayed by Esc(1-21)-1c (90% killing at 15 μM in 1 h). We also show the peptides' ability to stimulate migration of these cells and restore the induction of cell migration that is inhibited by Pseudomonas lipopolysaccharide when used at concentrations mimicking lung infection. This property of AMPs was not investigated before. Our findings suggest new therapeutics that not only eliminate bacteria but also can promote reepithelialization of the injured infected tissue. Confocal microscopy indicated that both peptides are intracellularly localized with a different distribution. Biochemical analyses highlighted that Esc(1-21)-1c is significantly more resistant than the all- l peptide to bacterial and human elastase, which is abundant in CF lungs. Besides proposing a plausible mechanism underlying the properties of the two AMPs, we discuss the data with regard to differences between them and suggest Esc(1-21)-1c as a candidate for the development of a new multifunctional drug against Pseudomonas respiratory infections.

Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll‐like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2‐p) ameliorated DSS‐induced colitis by interfering specifically with the activation of Ly6C + monocytes without affecting their recruitment to the colon. We report that TLR2‐p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2–TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal‐regulated kinases (ERK) signaling and reduced secretion of pro‐inflammatory cytokines, such as interleukin (IL)‐6, IL‐23, IL‐12, and IL‐1β. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro‐inflammatory Ly6C hi monocytes and suggests that inhibition of this aggregation by TLR2‐p might have therapeutic potential in the treatment of acute gut inflammation.

The ability of the Lentivirus HIV-1 to inhibit T-cell activation by its gp41 fusion protein is well documented, yet limited data exists regarding other viral fusion proteins. HIV-1 utilizes membrane binding region of gp41 to inhibit T-cell receptor (TCR) complex activation. Here we examined whether this T-cell suppression strategy is unique to the HIV-1 gp41. We focused on T-cell modulation by the gp21 fusion peptide (FP) of the Human T-lymphotropic Virus 1 (HTLV-1), a Deltaretrovirus that like HIV infects CD4+ T-cells. Using mouse and human in-vitro T-cell models together with in-vivo T-cell hyper activation mouse model, we reveal that HTLV-1’s FP inhibits T-cell activation and unlike the HIV FP, bypasses the TCR complex. HTLV FP inhibition induces a decrease in Th1 and an elevation in Th2 responses observed in mRNA, cytokine and transcription factor profiles. Administration of the HTLV FP in a T-cell hyper activation mouse model of multiple sclerosis alleviated symptoms and delayed disease onset. We further pinpointed the modulatory region within HTLV-1’s FP to the same region previously identified as the HIV-1 FP active region, suggesting that through convergent evolution both viruses have obtained the ability to modulate T-cells using the same region of their fusion protein. Overall, our findings suggest that fusion protein based T-cell modulation may be a common viral trait., Author summary In order to successfully infect and persist in their hosts, viruses utilize multiple strategies to evade the immune system. HIV utilizes membrane interacting regions of its envelope protein, primarily used to fuse with its target cells, to inhibit T-cell activation. Yet, it is unknown whether this ability is shared with other viruses. In this study we examined the T-cell inhibitory activity of the envelope protein of the Human T-lymphotropic virus 1 (HTLV-1), which infects T-cells. We focused on a functionally conserved region of HTLV’s and HIV’s fusion proteins, the fusion peptide (FP). Here, we reveal that HTLV’s FP inhibits the activity of T-cells in-vitro and in a T-cell hyper activation model in mice. This inhibition is characterized by downregulation of the T-cell Th1/type 1 response, leading to an elevated T-cell Th2/type 2 response observed by transition in the profiles of mRNA, cytokines and regulatory proteins. Furthermore, we demonstrate that the HTLV and HIV FPs inhibit T-cell activation at different levels of the signaling cascade. Although the HTLV FP’s mechanism of T-cell inhibition differs from the HIV’s FP, our findings suggest that FP mediated immune evasion might be a trait shared between different viruses.

Multidrug resistant bacteria possess various mechanisms that can sense environmental stresses such as antibiotics and antimicrobial peptides and rapidly respond to defend themselves. Two known defense strategies are biofilm formation and lipopolysaccharide (LPS) modification. Though LPS modifications are observed in biofilm-embedded bacteria, their effect on biofilm formation is unknown. Using biochemical and biophysical methods coupled with confocal microscopy, atomic force microscopy, and transmission electron microscopy, we show that biofilm formation is promoted in a Pseudomonas aeruginosa PAO1 strain with a loss of function mutation in the arnB gene. This loss of function prevents the addition of the positively charged sugar 4-amino-4-deoxy-l-arabinose to lipid A of LPS under restrictive magnesium conditions. The data reveal that the arnB mutant, which is susceptible to antimicrobial peptides, forms a biofilm that is more robust than that of the wild type. This is in line with the observations that the arnB mutant exhibits outer surface properties such as hydrophobicity and net negative charge that promote the formation of biofilms. Moreover, when grown under Mg2+ limitation, both the wild type and the arnB mutant exhibited a reduction in the level of membrane-bound polysaccharides. The data suggest that the loss of polysaccharides exposes the membrane and alters its biophysical properties, which in turn leads to more biofilm formation. In summary, we show for the first time that blocking a specific lipid A modification promotes biofilm formation, suggesting a trade-off between LPS remodeling and resistance mechanisms of biofilm formation.

Many bacteria live as biofilms to cope with unfavourable surroundings. Biofilms start from (i) a planktonic stage, (ii) initial adhesion to surfaces and (iii) formation of sessile micro-colonies that secrete extracellular polymeric substance (EPS), leading to bacterial resistance to antibiotics. Antimicrobial peptides (AMPs) are extensively studied with regard to planktonic bacteria but much less so with regard to biofilm formation. In the present study, we investigated how the above three steps are affected by the properties of the AMPs using a series of peptides composed of six lysines and nine leucines, which differ in their sequences and hence their biophysical properties. Treatment with bactericidal peptides at non-inhibitory concentrations resulted in reduced biofilm growth, for some starting from 25 nM which is 0.2 and 0.4% of their minimum inhibitory concentration (MIC 6.3 and 12.5 μM, respectively), continuing in a dose-dependent manner. We suggest that reduced bacterial adhesion to surfaces and decreased biofilm growth are due to the peptide's ability to coat either the biomaterial surface or the bacterium itself. Degradation of established biofilms by bactericidal and non-bactericidal peptides, within 1 h of incubation, occurs by either killing of embedded bacteria or detachment of live ones. In addition to shedding light on the mechanism of biofilm inhibition and degradation, these data may assist in the design of anti-biofilm AMPs.

Cell surfaces are densely populated with various proteins. Aggregation of these proteins to nanoscale clusters can be critical for various cellular functions such as signaling, motility and division. Quantitative characterization of corresponding structures and their changes might be useful to understand these basic cell processes and serve as an early marker of cellular stress or diseases. Atomic force microscopy (AFM) allows high-resolution imaging of cell surface structures, resolving fine details of these structures. Moreover, AFM enables simultaneous imaging of cell surface morphology and mapping of its' mechanical characteristics. This review focuses on applying the fractal dimension measure as a sensitive method to quantify single cell surface structures and their changes from AFM images.

Recently, a single study revealed a new complex composed of Toll-like receptor 4 (TLR4), TLR6, and CD36 induced by fibrillary Aβ peptides, the hallmark of Alzheimer's disease. Unlike TLRs located on the plasma membrane that dimerize on the membrane after ligand binding to their extracellular domain, the TLR4-TLR6-CD36 complex assembly has been suggested to be induced by intracellular signals from CD36, similar to integrin inside-out signaling. However, the assembly site of TLR4-TLR6-CD36 and the domains participating in Aβ-induced signaling is still unknown. By interfering with TLR4-TLR6 dimerization using a TLR4-derived peptide, we show that receptor assembly is abrogated within the plasma membrane. Furthermore, we reveal that the transmembrane domains of TLR4 and TLR6 have an essential role in receptor dimerization and activation. Inhibition of TLR4-TLR6 assembly was associated with reduced secretion of proinflammatory mediators from microglia cells, ultimately rescuing neurons from death. Our findings support TLR4-TLR6 dimerization induced by Aβ. Moreover, we shed new light on TLR4-TLR6 assembly and localization and show the potential of inhibiting TLR4-TLR6 dimerization as a treatment of Alzheimer's disease.

Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus–cell and cell–cell contact sites. Overall, the findings of the present study may suggest lipid–protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes., We show that sphinganine lipidated peptides affect membrane fusion, modulate the membrane and disrupt protein assembly. In addition the findings may aid in deciphering the function of DHSM in biological membranes.

The mammalian innate immune response is responsible for the early stages of defense against invading pathogens. One of the major receptor families facilitating innate immune activation is the Toll-like receptor (TLR) family. These receptors are type 1 membrane proteins spanning the membrane with a single transmembrane domain (TMD). All TLRs form homo- and hetero-dimers within membranes and new data suggest that the single transmembrane domain of some of these receptors is involved in their dimerization and function. Newly identified TLR dimers are continuously reported but only little is known about the importance of the TMDs for their dimer assembly and signaling regulation. Uncontrolled or untimely activation of TLRs is related to a large number of pathologies ranging from cystic fibrosis to sepsis and cancer. In this review we will focus on the contribution of the TMDs of innate immune receptors – specifically TLR2–to their regulation and function. In addition, we will address the current issues remaining to be solved regarding the mechanistic insights of this regulation. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

An immunosuppressive motif was recently found within the HIV-1 gp41 fusion protein (termed immunosuppressive loop-associated determinant core motif (ISLAD CM)). Peptides containing the motif interact with the T-cell receptor (TCR) complex; however, the mechanism by which the motif exerts its immunosuppressive activity is yet to be determined. Recent studies showed that interactions between protein domains in the membrane milieu are not always sterically controlled. Therefore, we utilized the unique membrane leniency toward association between d- and l-stereoisomers to investigate the detailed mechanism by which ISLAD CM inhibits T-cell activation. We show that a d-enantiomer of ISLAD CM (termed ISLAD d-CM) inhibited the proliferation of murine myelin oligodendrocyte glycoprotein (MOG)-(35–55)-specific line T-cells to the same extent as the l-motif form. Moreover, the d- and l-forms preferentially bound spleen-derived T-cells over B-cells by 13-fold. Furthermore, both forms of ISLAD CM co-localized with the TCR on activated T-cells and interacted with the transmembrane domain of the TCR. FRET experiments revealed the importance of basic residues for the interaction between ISLAD CM forms and the TCR transmembrane domain. Ex vivo studies demonstrated that ISLAD d-CM administration inhibited the proliferation (72%) and proinflammatory cytokine secretion of pathogenic MOG(35–55)-specific T-cells. This study provides insights into the immunosuppressive mechanism of gp41 and demonstrates that chirality-independent interactions in the membrane can take place in diverse biological systems. Apart from HIV pathogenesis, the d-peptide reported herein may serve as a potential tool for treating T-cell-mediated pathologies.

Background: HIV-1 infectivity is decreased by specific mutations that alter the hydrophobicity level in the HIV-1 gp41 loop core. Results: Antibody recognition, disulfide-bond formation, and lipid mixing of loop domain peptides are strongly affected by these mutations. Conclusion: The hydrophobic core maintains proper function and structure of the loop region. Significance: A better understanding of the membrane fusion mechanism of HIV and similar viruses is provided. The gp41 disulfide loop region switches from a soluble state to a membrane-bound state during the human immunodeficiency virus type 1 (HIV-1) envelope-mediated membrane fusion process. The loop possesses a hydrophobic core at the center of the region with an unusual basic residue (Lys-601). Furthermore, two loop core mutations, K601A and L602A, are found to inhibit HIV-1 infectivity while keeping wild type-like levels of the envelope, implying that they exert an inhibitory effect on gp41 during the membrane fusion event. Here, we investigated the mode of action of these mutations on the loop region. We show that the K601A mutation, but not the L602A mutation, abolished the binding of a loop-specific monoclonal antibody to a loop domain peptide. Additionally, the K601A, but not the L602A, impaired disulfide bond formation in the peptides. This was correlated with changes in the circular dichroism spectrum imposed by the K601A mutation. In the membrane, however, the L602A, but not the K601A, reduced the lipid mixing ability of the loop peptides, which was correlated with decreased α-helical content of the L602A mutant. The results suggest that the Lys-601 residue provides a moderate hydrophobicity level within the gp41 loop core that contributes to the proper structure and function of the loop inside and outside the membrane. Because basic residues are found between the loop Cys residues of several lentiviral fusion proteins, the findings may contribute to understanding the fusion mechanism of other viruses as well.

Fusion of the HIV membrane with that of a target T cell is an essential first step in the viral infection process. Here we describe single-particle tracking (SPT) studies of a 16-amino-acid peptide derived from the HIV fusion protein (FP16), as it interacts with a supported lipid bilayer. FP16 was found to spontaneously insert into and move within the bilayer with two different modes of diffusion, a fast mode with a diffusion coefficient typical of protein motion in membranes and a much slower one. We observed transitions between the two modes: slow peptides were found to speed up, and fast peptides could slow down. Hidden Markov model analysis was employed as a method for the identification of the two modes in single-molecule trajectories and analysis of their interconversion rates. Surprisingly, the diffusion coefficients of the two modes were found to depend differently on solution viscosity. Thus, whereas the fast diffusive mode behaved as predicted by the Saffman-Delbrück theory, the slow mode behaved according to the Stokes-Einstein relation. To further characterize the two diffusive modes, FP16 molecules were studied in bilayers cooled through their liquid crystalline-to-gel phase transition. Our analysis suggested that the slow diffusive mode might originate from the formation of large objects, such as lipid domains or local protrusions, which are induced by the peptides and move together with them.

The T-cell receptor-CD3 complex (TCR-CD3) serves a critical role in protecting organisms from infectious agents. The TCR is a heterodimer composed of α- and β-chains, which are responsible for antigen recognition. Within the transmembrane domain of the α-subunit, a region has been identified to be crucial for the assembly and function of the TCR. This region, termed core peptide (CP), consists of nine amino acids (GLRILLLKV), two of which are charged (lysine and arginine) and are crucial for the interaction with CD3. Earlier studies have shown that a synthetic peptide corresponding to the CP sequence can suppress the immune response in animal models of T-cell-mediated inflammation, by disrupting proper assembly of the TCR. As a step towards the understanding of the source of the CP activity, we focused on CP in egg phosphatidylcholine/cholesterol (9:1, mol/mol) model membranes and determined its secondary structure, oligomerization state, and orientation with respect to the membrane. To achieve this goal, 15-residue segments of TCRα, containing the CP, were synthesized and spin-labeled at different locations with a nitroxide derivative. Electron spin-echo envelope modulation spectroscopy was used to probe the position and orientation of the peptides within the membrane, and double electron-electron resonance measurements were used to probe its conformation and oligomerization state. We found that the peptide is predominantly helical in a membrane environment and tends to form oligomers (mostly dimers) that are parallel to the membrane plane.

TLR2, together with TLR1 and TLR6, is essential for detecting lipopeptides and bacterial cell wall components such as lipoteichoic acid from Gram-positive bacteria. In this study, we report that transmembrane domain (TMD)–derived peptides from TLR2 and TLR6 specifically inhibit TLR2 activation. Secretion of the cytokines TNF-α and IL-6 by cultured macrophages (RAW264.7 cell line) was inhibited by these peptides in response to TLR2 activation by lipoteichoic acid (TLR2/6 activator) or palmitoyl (3)-Cys-Ser-Lys(4)-OH (TLR2/1 activator) but not by LPS (TLR4 activator). Extensive biophysical and biochemical assays, combined with GALLEX experiments, show that these peptides heterodimerize with their complementary TMDs on their reciprocal protein. These results suggest that TLR2/6/1 TMD assembly is essential for activating this complex. Importantly, when administered to mice inflicted by TLR2, but not TLR4-driven lethal inflammation, a selected peptide rescued 60% of these septic mice, showing potent in vivo inhibition of TNF-α and IL-6 secretion. Furthermore, this peptide also showed high protection in a whole bacteria model. Owing to the importance of TLR2 regulation under a variety of pathological conditions, compounds that can fine-tune this activity are of great importance.

The organization and orientation of membrane−inserted helices is important for better understanding the mode of action of membrane−active peptides and of protein−membrane interactions. Here we report on the application of ESEEM (electron spin−echo envelope modulation) and DEER (double electron−electron resonance) techniques to probe the orientation and oligomeric state of an α−helical trans−membrane model peptide, WALP23, under conditions of negative mismatch between the hydrophobic cores of the model membrane and the peptide. Using ESEEM, we measured weak dipolar interactions between spin−labeled WALP23 and (2)H nuclei of either the solvent (D(2)O) or of lipids specifically deuterated at the choline group. The ESEEM data obtained from the deuterated lipids were fitted using a model that provided the spin label average distance from a layer of (2)H nuclei in the hydrophilic region of the membrane and the density of the (2)H nuclei in the layer. DEER was used to probe oligomerization through the dipolar interaction between two spin−labels on different peptides. We observed that the center of WALP23 does not coincide with the bilayer midplane and its N−terminus is more buried than the C−terminus. In addition, the ESEEM data fitting yielded a (2)H layer density that was much lower than expected. The DEER experiments revealed the presence of oligomers, the presence of which was attributable to the negative mismatch and the electrostatic dipole of the peptide. A discussion of a possible arrangement of the individual helices in the oligomers that is consistent with the ESEEM and DEER data is presented

Multidrug-resistant bacteria are a growing problem worldwide. One extensively studied resistance mechanism is biofilm colonization-microbial colonies formed by many Gram-positive and Gram-negative bacteria species. Cationic antimicrobial peptides (AMPs) are innate immune system molecules serving as a first line of defense in fighting invading pathogens. The AMPs' underlying mechanism and biophysical properties required for anti-biofilm activity are not fully known. Here we present protocols for investigating AMPs' biological activity against major stages of biofilm life cycle, namely, planktonic stage (MIC assay), initial adhesion to surfaces (bacterial attachment assay), and formation or degradation of sessile microcolonies (biofilm formation and degradation assays). Furthermore, we demonstrate experiments that allow determination and comparison between peptide biophysical properties (secondary structure, hydrophobicity, and oligomerization) and how they affect their mechanism (peptide-binding assays) of anti-biofilm activity.

Multidrug-resistant bacteria are a growing problem worldwide. One extensively studied resistance mechanism is biofilm colonization-microbial colonies formed by many Gram-positive and Gram-negative bacteria species. Cationic antimicrobial peptides (AMPs) are innate immune system molecules serving as a first line of defense in fighting invading pathogens. The AMPs' underlying mechanism and biophysical properties required for anti-biofilm activity are not fully known. Here we present protocols for investigating AMPs' biological activity against major stages of biofilm life cycle, namely, planktonic stage (MIC assay), initial adhesion to surfaces (bacterial attachment assay), and formation or degradation of sessile microcolonies (biofilm formation and degradation assays). Furthermore, we demonstrate experiments that allow determination and comparison between peptide biophysical properties (secondary structure, hydrophobicity, and oligomerization) and how they affect their mechanism (peptide-binding assays) of anti-biofilm activity.

The ErbB family of tyrosine kinase receptors is a key element in preserving cell growth homeostasis. This family is comprised of four single-transmembrane domain proteins designated ErbB-1-4. Ligand binding initiates dimerization followed by tyrosine phosphorylation and signaling, which when uncontrolled lead to cancer. Accordingly, extensive research has been devoted to finding ErbB-intercepting agents, directed against ErbB-1 and ErbB-2, but so far, no inhibitor has targeted the transmembrane domain (TMD), which is instrumental for receptor dimerization and activation. Moreover, no antitumor agents targeted ErbB-3, which although it cannot generate signals in isolation, its heterodimerization with ErbB-2 leads to the most powerful and oncogenic signaling unit in the ErbB family. Here, to further elucidate the role of the interactions between the TMDs of the ErbB family in cancer, we investigated peptides derived from the TMDs of ErbB-1 and ErbB-2. We then focused on the C-terminal domains (B2C) and their analogue, named B2C-D, that contains both d- and l-amino acids. Both peptides incorporated the distal GXXXG dimerization motif to target the TMD of ErbB-3. Our results revealed that B2C-D is highly active both in vitro and in vivo. It significantly inhibits neuregulin- and EGF-induced ErbB activation, impedes the proliferation of a battery of human cancer cell lines, and retards tumor growth in vivo. Notably, combining low concentrations of B2C-D and gemcitabine chemotherapy completely arrested proliferation of pancreatic cancer cells. Biochemical and in vitro interaction studies suggest direct interference with the assembly of the wild-type ErbB-2-ErbB-3 heterodimer as the underlying mode of action. To the best of our knowledge, this is the first agent to target the TMDs of ErbB to delay tumor growth and signaling.

The persistence of HIV in resting memory CD4+ T cells at a latent state is considered as the major barrier on the path to achieve a cure for HIV. Proteasome inhibitors (PIs) were previously reported as latency reversing agents (LRAs) but the mechanism underlying this function is yet unclear. Here we demonstrate that PIs reactivate latent HIV ex vivo without global T cell activation, and may facilitate host innate immune responses. Mechanistically, latent HIV reactivation induced by PIs is mediated by heat shock factor 1 (HSF1) via the recruitment of the heat shock protein (HSP) 90-positive transcriptional elongation factor b (p-TEFb) complex. Specifically, HSP90 downstream HSF1 gives positive feedback to the reactivation process through binding to cyclin-dependent kinase 9 (CDK9) and preventing it from undergoing degradation by the proteasome. Overall, these findings suggest proteasome inhibitors as potential latency reversing agents. In addition, HSF1/HSP90 involved in HIV transcription elongation, may serve as therapeutic targets in HIV eradication.