scholarly journals Modified defence peptides from horseshoe crab target and kill bacteria inside host cells

2021 ◽  
Author(s):  
Anna S Amiss ◽  
Jessica B von Pein ◽  
Jessica R Webb ◽  
Nicholas D Condon ◽  
Peta J Harvey ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Pathogenic Bacteria ◽  
Horseshoe Crab ◽  
Cell Penetrating Peptides ◽  
Host Cells ◽  
Therapeutic Window ◽  
Infection Model ◽  
Zinc Toxicity ◽  
Cell Penetrating ◽  
Tachyplesin I

Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two β-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogues [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell penetrating peptides are attractive alternatives to traditional small molecule antimicrobials for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.

scholarly journals The J-Domain of Heat Shock Protein 40 Can Enhance the Transduction Efficiency of Arginine-Rich Cell-Penetrating Peptides

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Tzu-Yin Lin ◽  
Yu-Hsiu Su ◽  
Kun-Hsiung Lee ◽  
Chin-Kai Chuang
Keyword(s):  
Recombinant Proteins ◽  
Mammalian Cells ◽  
Cellular Membrane ◽  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Host Cells ◽  
Soluble Form ◽  
Transduction Efficiency ◽  
E Coli ◽  
Cell Penetrating

Sense and antisense oligonucleotide pairs encoding cell-penetrating peptides PTD(Tat47–57), DPV3A, E162, pVEC, R11, and TP13 were used to construct two sets of pET22b-CPP-DsRed and pET22b-CPP-J-DsRed vectors for CPP-DsRed and CPP-J-DsRed recombinant proteins expression. PTD-DsRed, DPV3A-DsRed, PTD-J-DsRed, and DPV3A-J-DsRed recombinant proteins were expressed in a soluble form. PTD-J-DsRed and DPV3A-J-DsRed recombinant proteins were able to escape fromE. colihost cells into the culture medium. The membrane-penetrating activity of PTD-J-DsRed and DPV3A-J-DsRed recombinant proteins to mammalian cells was more effective than that of PTD-DsRed and DPV3A-DsRed. The route of the cellular membrane translocation of these recombinant proteins is suggested via macropinocytosis followed by an endosomal escape pathway.

scholarly journals Cell-penetrating peptides and antimicrobial peptides: how different are they?

2006 ◽  
Vol 399 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Sónia Troeira Henriques ◽  
Manuel Nuno Melo ◽  
Miguel A. R. B. Castanho
Keyword(s):  
Cell Membrane ◽  
Antimicrobial Peptides ◽  
Cellular Uptake ◽  
Mammalian Cells ◽  
Cell Penetrating Peptides ◽  
Host Cells ◽  
Cell Penetrating ◽  
New Perspective ◽  
Pathogenic Agents ◽  
Endocytic Pathways

Some cationic peptides, referred to as CPPs (cell-penetrating peptides), have the ability to translocate across biological membranes in a non-disruptive way and to overcome the impermeable nature of the cell membrane. They have been successfully used for drug delivery into mammalian cells; however, there is no consensus about the mechanism of cellular uptake. Both endocytic and non-endocytic pathways are supported by experimental evidence. The observation that some AMPs (antimicrobial peptides) can enter host cells without damaging their cytoplasmic membrane, as well as kill pathogenic agents, has also attracted attention. The capacity to translocate across the cell membrane has been reported for some of these AMPs. Like CPPs, AMPs are short and cationic sequences with a high affinity for membranes. Similarities between CPPs and AMPs prompted us to question if these two classes of peptides really belong to unrelated families. In this Review, a critical comparison of the mechanisms that underlie cellular uptake is undertaken. A reflection and a new perspective about CPPs and AMPs are presented.

scholarly journals Novel human-derived cell-penetrating peptides for specific subcellular delivery of therapeutic biomolecules

2005 ◽  
Vol 390 (2) ◽  
pp. 407-418 ◽  
Author(s):  
Catherine de Coupade ◽  
Antonio Fittipaldi ◽  
Vanessa Chagnas ◽  
Matthieu Michel ◽  
Sophie Carlier ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Mammalian Cells ◽  
Heparan Sulphate ◽  
Intracellular Delivery ◽  
Membrane Lipid ◽  
Cell Penetrating Peptides ◽  
Heparin Binding ◽  
Independent Manner ◽  
Cell Penetrating

Short peptide sequences that are able to transport molecules across the cell membrane have been developed as tools for intracellular delivery of therapeutic molecules. This work describes a novel family of cell-penetrating peptides named Vectocell® peptides [also termed DPVs (Diatos peptide vectors)]. These peptides, originating from human heparin binding proteins and/or anti-DNA antibodies, once conjugated to a therapeutic molecule, can deliver the molecule to either the cytoplasm or the nucleus of mammalian cells. Vectocell® peptides can drive intracellular delivery of molecules of varying molecular mass, including full-length active immunoglobulins, with efficiency often greater than that of the well-characterized cell-penetrating peptide Tat. The internalization of Vectocell® peptides has been demonstrated to occur in both adherent and suspension cell lines as well as in primary cells through an energy-dependent endocytosis process, involving cell-membrane lipid rafts. This endocytosis occurs after binding of the cell-penetrating peptides to extracellular heparan sulphate proteoglycans, except for one particular peptide (DPV1047) that partially originates from an anti-DNA antibody and is internalized in a caveolar independent manner. These new therapeutic tools are currently being developed for intracellular delivery of a number of active molecules and their potentiality for in vivo transduction investigated.

scholarly journals Enhanced delivery of protein fused to cell penetrating peptides to mammalian cells

BMB Reports ◽  
2019 ◽  
Vol 52 (5) ◽  
pp. 324-329 ◽  
Author(s):  
Jung-Il Moon ◽  
Min-Joon Han ◽  
Shin-Hye Yu ◽  
Eun-Hye Lee ◽  
Sang-Mi Kim ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Penetrating Peptides ◽  
Cell Penetrating

scholarly journals Neuropilin-1 and heparan sulfate proteoglycans cooperate in cellular uptake of nanoparticles functionalized by cationic cell-penetrating peptides

2015 ◽  
Vol 1 (10) ◽  
pp. e1500821 ◽  
Author(s):  
Hong-Bo Pang ◽  
Gary B. Braun ◽  
Erkki Ruoslahti
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Vascular Permeability ◽  
Mammalian Cells ◽  
Cell Penetrating Peptides ◽  
Proteolytic Processing ◽  
Cell Surface Receptor ◽  
Neuropilin 1 ◽  
Cell Penetrating ◽  
Surface Receptor

Cell-penetrating peptides (CPPs) have been widely used to deliver nanomaterials and other types of macromolecules into mammalian cells for therapeutic and diagnostic use. Cationic CPPs that bind to heparan sulfate (HS) proteoglycans on the cell surface induce potent endocytosis; however, the role of other surface receptors in this process is unclear. We describe the convergence of an HS-dependent pathway with the C-end rule (CendR) mechanism that enables peptide ligation with neuropilin-1 (NRP1), a cell surface receptor known to be involved in angiogenesis and vascular permeability. NRP1 binds peptides carrying a positive residue at the carboxyl terminus, a feature that is compatible with cationic CPPs, either intact or after proteolytic processing. We used CPP and CendR peptides, as well as HS- and NRP1-binding motifs from semaphorins, to explore the commonalities and differences of the HS and NRP1 pathways. We show that the CendR-NRP1 interaction determines the ability of CPPs to induce vascular permeability. We also show at the ultrastructural level, using a novel cell entry synchronization method, that both the HS and NRP1 pathways can initiate a macropinocytosis-like process and visualize these CPP-cargo complexes going through various endosomal compartments. Our results provide new insights into how CPPs exploit multiple surface receptor pathways for intracellular delivery.

Stereochemistry as a determining factor for the effect of a cell-penetrating peptide on cellular viability and epithelial integrity

2018 ◽  
Vol 475 (10) ◽  
pp. 1773-1788 ◽  
Author(s):  
Ditlev Birch ◽  
Malene V. Christensen ◽  
Dan Staerk ◽  
Henrik Franzyk ◽  
Hanne Mørck Nielsen
Keyword(s):  
Amino Acids ◽  
Cell Viability ◽  
Mammalian Cells ◽  
Membrane Integrity ◽  
Cell Penetrating Peptides ◽  
Proliferating Cells ◽  
Epithelial Integrity ◽  
Cell Penetrating ◽  
Culture Models ◽  
Well Differentiated

Cell-penetrating peptides (CPPs) comprise efficient peptide-based delivery vectors. Owing to the inherent poor enzymatic stability of peptides, CPPs displaying partial or full replacement of l-amino acids with the corresponding d-amino acids might possess advantages as delivery vectors. Thus, the present study aims to elucidate the membrane- and metabolism-associated effects of l-Penetratin (l-PEN) and its corresponding all-d analog (d-PEN). These effects were investigated when exerted on hepatocellular (HepG2) or intestinal (Caco-2 and IEC-6) cell culture models. The head-to-head comparison of these enantiomeric CPPs included evaluation of their effects on cell viability and morphology, epithelial membrane integrity, and cellular ultrastructure. In all investigated cell models, a rapid decrease in cell viability, pronounced membrane perturbation and an altered ultrastructure were detected upon exposure to d-PEN. At equimolar concentrations, these observations were less pronounced or even absent for cells exposed to l-PEN. Both CPPs remained stable for at least 2 h during exposure to proliferating cells (cultured for 24 h), although d-PEN exhibited a longer half-life when compared with that of l-PEN when exposed to well-differentiated cell monolayers (cultured for 18–20 days). Thus, the stereochemistry of the CPP penetratin significantly influences its effects on cell viability and epithelial integrity when profiled against a panel of mammalian cells.

High-throughput assays of phagocytosis, phagosome maturation, and bacterial invasion

2007 ◽  
Vol 292 (2) ◽  
pp. C945-C952 ◽  
Author(s):  
Benjamin E. Steinberg ◽  
Cameron C. Scott ◽  
Sergio Grinstein
Keyword(s):  
High Throughput ◽  
Mammalian Cells ◽  
Pathogenic Bacteria ◽  
Host Cells ◽  
Bacterial Invasion ◽  
Small Interference Rna ◽  
Phagosomal Maturation ◽  
Large Populations ◽  
Intracellular Proliferation ◽  
Efficient Screening

Ingestion of foreign particles by macrophages and neutrophils and the fate of the vacuole that contains the ingested material are generally monitored by optical microscopy. Invasion of host cells by pathogenic bacteria and their intracellular proliferation are similarly studied by microscopy or by plating assays. These labor-intensive and time-consuming methods limit the number of assays that can be performed. The effort required to test multiple reagents or conditions can be prohibitive. We describe high-throughput assays of phagocytosis and of phagosomal maturation. An automated fluorescence microscope-based platform and associated analysis software were used to study Fcγ receptor-mediated phagocytosis of IgG-opsonized particles by cultured murine macrophages. Phagosomal acidification was measured as an index of maturation. The same platform was similarly used to implement high-throughput assays of invasion of mammalian cells by pathogenic bacteria. The invasion of HeLa cells by Salmonella and the subsequent intracellular proliferation of the bacteria were measured rapidly and reliably in large populations of cells. These high-throughput methods are ideally suited for the efficient screening of chemical libraries to select potential drugs and of small interference RNA libraries to identify essential molecules involved in critical steps of the immune response.

scholarly journals Transportan Peptide Stimulates the Nanomaterial Internalization into Mammalian Cells in the Bystander Manner through Macropinocytosis

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 552
Author(s):  
Yue-Xuan Li ◽  
Yushuang Wei ◽  
Rui Zhong ◽  
Ling Li ◽  
Hong-Bo Pang
Keyword(s):  
Mammalian Cells ◽  
Ex Vivo ◽  
Cell Penetrating Peptides ◽  
Cell Entry ◽  
Physical Interaction ◽  
Chemical Modifications ◽  
Cell Penetrating ◽  
Common Strategy ◽  
Entry Mechanism

Covalent coupling with cell-penetrating peptides (CPPs) has been a common strategy to facilitate the cell entry of nanomaterial and other macromolecules. Though efficient, this strategy requires chemical modifications on nanomaterials, which is not always desired for their applications. Recent studies on a few cationic CPPs have revealed that they can stimulate the cellular uptake of nanoparticles (NPs) simply via co-administration (bystander manner), which bypasses the requirement of chemical modification. In this study, we investigated the other classes of CPPs and discovered that transportan (TP) peptide, an amphiphilic CPP, also exhibited such bystander activities. When simply co-administered, TP peptide enabled the cells to engulf a variety of NPs, as well as common solute tracers, while these payloads had little or no ability to enter the cells by themselves. This result was validated in vitro and ex vivo, and TP peptide showed no physical interaction with co-administered NPs (bystander cargo). We further explored the cell entry mechanism for TP peptide and its bystander cargo, and showed that it was mediated by a receptor-dependent macropinocytosis process. Together, our findings improve the understanding of TP-assisted cell entry, and open up a new avenue to apply this peptide for nanomaterial delivery.

scholarly journals Engineering Cell-Permeable Proteins through Insertion of Cell-Penetrating Motifs into Surface Loops

2020 ◽  
Author(s):  
Kuangyu Chen ◽  
Dehua Pei
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Tyrosine Phosphatase ◽  
Structural Features ◽  
Cell Penetrating Peptides ◽  
Biologically Active ◽  
Enzyme Replacement ◽  
Loop Region ◽  
Wide Range ◽  
Cell Penetrating

ABSTRACTEffective delivery of proteins into the cytosol and nucleus of mammalian cells would open the door to a wide range of applications including treatment of many currently intractable diseases. However, despite great efforts from numerous investigators and the development of a variety of innovative methods, effective protein delivery in a clinical setting is yet to be accomplished. Herein we report a potentially general approach to engineering cell-permeable proteins by genetically grafting a short cell-penetrating peptide to an exposed loop region of a protein of interest. The grafted peptide is conformationally constrained by the protein structure, sharing the structural features of cyclic cell-penetrating peptides and exhibiting enhanced proteolytic stability and cellular entry efficiency. Insertion of an amphipathic motif, Arg-Arg-Arg-Arg-Trp-Trp-Trp, into the loop regions of enhanced green fluorescent protein (EGFP), protein-tyrosine phosphatase 1B (PTP1B), and purine nucleoside phosphorylase (PNP) rendered all three proteins cell-permeable and biologically active in cellular assays. When added into growth medium, the engineered PTP1B dose-dependently reduced the phosphotyrosine levels of intracellular proteins, while the modified PNP protected PNP-deficient mouse T lymphocytes (NSU-1) against toxic levels of deoxyguanosine, providing a potential enzyme replacement therapy for a rare genetic disease.

scholarly journals Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Marta Gomarasca ◽  
Thaynan F. C. Martins ◽  
Lilo Greune ◽  
Philip R. Hardwidge ◽  
M. Alexander Schmidt ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Shigella Flexneri ◽  
Cell Penetrating Peptides ◽  
Biologically Active ◽  
Intracellular Pathogens ◽  
Content Type ◽  
Cell Penetrating ◽  
Serovar Typhimurium ◽  
Therapeutic Tools

ABSTRACT Commonly used antimicrobials show poor cellular uptake and often have limited access to intracellular targets, resulting in low antimicrobial activity against intracellular pathogens. An efficient delivery system to transport these drugs to the intracellular site of action is needed. Cell-penetrating peptides (CPPs) mediate the internalization of biologically active molecules into the cytoplasm. Here, we characterized two CPPs, α1H and α2H, derived from the Yersinia enterocolitica YopM effector protein. These CPPs, as well as Tat (trans-activator of transcription) from HIV-1, were used to deliver the antibiotic gentamicin to target intracellular bacteria. The YopM-derived CPPs penetrated different endothelial and epithelial cells to the same extent as Tat. CPPs were covalently conjugated to gentamicin, and CPP-gentamicin conjugates were used to target infected cells to kill multiple intracellular Gram-negative pathogenic bacteria, including Escherichia coli K1, Salmonella enterica serovar Typhimurium, and Shigella flexneri. Taken together, CPPs show great potential as delivery vehicles for antimicrobial agents and may contribute to the generation of new therapeutic tools to treat infectious diseases caused by intracellular pathogens.

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