Rossella Tarallo

Microbe-Host Interactions: Structure and Role of Gram-Negative Bacterial Porins

Gram negative bacteria have evolved many mechanisms of attaching to and invading host epithelial and immune cells. In particular, many outer membrane proteins (OMPs) are involved in this initial interaction between the pathogen and their host. The outer membrane (OM) of Gram-negative bacteria performs the crucial role of providing an extra layer of protection to the organism without compromising the exchange of material required for sustaining life. The OM, therefore, represents a sophisticated macromolecular assembly, whose complexity has yet to be fully elucidated. This review will summarize the structural information available for porins, a class of OMP, and highlight their role in bacterial pathogenesis and their potential as therapeutic targets. The functional role of porins in microbe-host interactions during various bacterial infections has emerged only during the last few decades, and their interaction with a variety of host tissues for adhesion to and invasion of the cell and for evasion of host-defense mechanisms have placed bacterial porins at the forefront of research in bacterial pathogenesis. This review will discuss the role that porins play in activating immunological responses, in inducing signaling pathways and their influence on antibiotic resistance mechanisms that involve modifications of the properties of the OM lipid barrier.

Shuttle‐Mediated Nanoparticle Delivery to the Blood–Brain Barrier

Many therapeutic drugs are excluded from entering the brain due to their lack of transport through the blood-brain barrier (BBB). The development of new strategies for enhancing drug delivery to the brain is of great importance in diagnostics and therapeutics of central nervous diseases. To overcome this problem, a viral fusion peptide (gH625) derived from the glycoprotein gH of Herpes simplex virus type 1 is developed, which possesses several advantages including high cell translocation potency, absence of toxicity of the peptide itself, and the feasibility as an efficient carrier for delivering therapeutics. Therefore, it is hypothesized that brain delivery of nanoparticles conjugated with gH625 should be efficiently enhanced. The surface of fluorescent aminated polystyrene nanoparticles (NPs) is functionalized with gH625 via a covalent binding procedure, and the NP uptake mechanism and permeation across in vitro BBB models are studied. At early incubation times, the uptake of NPs with gH625 by brain endothelial cells is greater than that of the NPs without the peptide, and their intracellular motion is mainly characterized by a random walk behavior. Most importantly, gH625 peptide decreases NP intracellular accumulation as large aggregates and enhances the NP BBB crossing. In summary, these results establish that surface functionalization with gH625 may change NP fate by providing a good strategy for the design of promising carriers to deliver drugs across the BBB for the treatment of brain diseases.

Clickable Functionalization of Liposomes with the gH625 Peptide from Herpes simplex Virus Type I for Intracellular Drug Delivery

Liposomes externally modified with the nineteen residues gH625 peptide, previously identified as a membrane-perturbing domain in the gH glycoprotein of Herpes simplex virus type I, have been prepared in order to improve the intracellular uptake of an encapsulated drug. An easy and versatile synthetic strategy, based on click chemistry, has been used to bind, in a controlled way, several copies of the hydrophobic gH625 peptide on the external surface of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPG)-based liposomes. Electron paramagnetic resonance studies, on liposomes derivatized with gH625 peptides, which are modified with the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) spin label in several peptide positions, confirm the positioning of the coupled peptides on the liposome external surface, whereas dynamic light scattering measurements indicate an increase of the diameter of the liposomes of approximately 30% after peptide introduction. Liposomes have been loaded with the cytotoxic drug doxorubicin and their ability to penetrate inside cells has been evaluated by confocal microscopy experiments. Results suggest that liposomes functionalized with gH625 may act as promising intracellular targeting carriers for efficient delivery of drugs, such as chemotherapeutic agents, into tumor cells.

A peptide derived from herpes simplex virus type 1 glycoprotein H: membrane translocation and applications to the delivery of quantum dots

UNLABELLED Cell membranes are impermeable to most molecules that are not actively imported by living cells, including all macromolecules and even small molecules whose physiochemical properties prevent passive membrane diffusion. However, recently, we have seen the development of increasingly sophisticated methodology for intracellular drug delivery. Cell-penetrating peptides (CPPs), short peptides believed to enter cells by penetrating cell membranes, have attracted great interest in the hope of enhancing gene therapy, vaccine development and drug delivery. Nevertheless, to achieve an efficient intracellular delivery, further strategies to bypass the endocytotic pathway must be investigated. We report on a novel peptide molecule derived from glycoprotein gH of herpes simplex type I virus that is able to traverse the membrane bilayer and to transport a cargo into the cytoplasm with novel properties in comparison with existing CPPs. We use as cargo molecule quantum dots that do not significantly traverse the membrane bilayer on their own. FROM THE CLINICAL EDITOR Cell-penetrating peptides have recently attracted great interest in optimizing gene therapy, vaccine development and drug delivery. In this study, a peptide derived from glycoprotein gH of herpes simplex I is investigated from this standpoint.

Dendrimer Functionalization with a Membrane-Interacting Domain of Herpes Simplex Virus Type 1: Towards Intracellular Delivery

A poly(amide)-based dendrimer was synthesized and functionalized with the membrane-interacting peptide gH(625-644) (gH625) derived from the herpes simplex virus type 1 (HSV-1) envelope glycoprotein H, which has previously been shown to assist in delivering large cargoes across the cellular membrane. We demonstrate that the attachment of the gH625 peptide sequence to the termini of a dendrimer allows the conjugate to penetrate into the cellular matrix, whereas the unfunctionalized dendrimer is excluded from translocation. The peptide-functionalized dendrimer is rapidly taken into the cells mainly through a non-active translocation mechanism. Our results suggest that the presented peptidodendrimeric scaffold may be a promising material for efficient drug delivery.

Peptide inhibitors against herpes simplex virus infections

Herpes simplex virus (HSV) is a significant human pathogen causing mucocutaneous lesions primarily in the oral or genital mucosa. Although acyclovir (ACV) and related nucleoside analogs provide successful treatment, HSV remains highly prevalent worldwide and is a major cofactor for the spread of human immunodeficiency virus. Encephalitis, meningitis, and blinding keratitis are among the most severe diseases caused by HSV. ACV resistance poses an important problem for immunocompromised patients and highlights the need for new safe and effective agents; therefore, the development of novel strategies to eradicate HSV is a global public health priority. Despite the continued global epidemic of HSV and extensive research, there have been few major breakthroughs in the treatment or prevention of the virus since the introduction of ACV in the 1980s. A therapeutic strategy at the moment not fully addressed is the use of small peptide molecules. These can be either modeled on viral proteins or derived from antimicrobial peptides. Any peptide that interrupts protein–protein or viral protein–host cell membrane interactions is potentially a novel antiviral drug and may be a useful tool for elucidating the mechanisms of viral entry. This review summarizes current knowledge and strategies in the development of synthetic and natural peptides to inhibit HSV infectivity. Copyright

Dendrimers functionalized with membrane-interacting peptides for viral inhibition

This contribution reports the synthesis of a poly(amide)-based dendrimer functionalized at the termini with a membrane-interacting peptide derived from the herpes simplex virus (HSV) type 1 glycoprotein H, namely gH625–644. This peptide has been shown to interact with model membranes and to inhibit viral infectivity. The peptidodendrimer inhibits both HSV-1 and HSV-2 at a very early stage of the entry process, most likely through an interaction with the viral envelope glycoproteins; thus, preventing the virus from coming into close contact with cellular membranes, a prerequisite of viral internalization. The 50% inhibitory concentration was 100 and 300 nM against HSV-1 and HSV-2 respectively, with no evidence of cell toxicity at these concentrations. These results show that the functionalization of a dendrimer with the peptide sequence derived from an HSV glycoprotein shows promising inhibitory activity towards viruses of the Herpesviridae family.

Elucidation of the Interaction Mechanism with Liposomes of gH625-Peptide Functionalized Dendrimers

We have demonstrated that amide-based dendrimers functionalized with the membrane-interacting peptide gH625 derived from the herpes simplex virus type 1 (HSV-1) envelope glycoprotein H enter cells mainly through a non-active translocation mechanism. Herein, we investigate the interaction between the peptide-functionalized dendrimer and liposomes composed of PC/Chol using fluorescence spectroscopy, isothermal titration calorimetry, and surface plasmon resonance to get insights into the mechanism of internalization. The affinity for the membrane bilayer is very high and the interaction between the peptide-dendrimer and liposomes took place without evidence of pore formation. These results suggest that the presented peptidodendrimeric scaffold may be a promising material for efficient drug delivery.

Review of a viral peptide nanosystem for intracellular delivery

Abstract. The internalization of bioactive molecules is one of the most critical problems to overcome in theranostics. In order to improve pharmacokinetic and pharmacodynamic properties, synthetic transporters are widely investigated. A new nanotechnological transporter, gH625, is based on a viral peptide sequence derived from the herpes simplex virus type 1 glycoprotein H (gH) that has proved to be a useful delivery vehicle, due to its intrinsic properties of inducing membrane perturbation. The peptide functionalization with several kinds of nanoparticles like quantum dots, dendrimers, and liposomes could be of particular interest in biomedical applications to improve drug release within cells, to increase site-specific action, and eventually to reduce related cytotoxicity.

The intriguing journey of gH625-dendrimers

The knowledge of the mechanism used by vectors to gain access to cell interiors is key to the development of effective drug delivery tools for different pathologies. The role of the initial interaction with the membrane bilayer is widely recognized, although not fully understood. We use neutron reflectivity experiments and internalization studies with cells to reveal the extent of interaction of dendrimers functionalized with the peptide gH625 with biomimetic membranes. We further investigate the internalization by use of Caco-2 cells for assessing the membrane permeability properties of the peptide–dendrimer construct. Neutron reflectivity allowed for the hypothesis that the peptide–dendrimer is able to pass across the bilayer which was confirmed via permeability studies. We find that gH625-dendrimers interact more strongly with cholesterol containing membranes. The advances in our understanding of the mechanism of drug uptake are extremely useful to push further the design of new drug delivery systems.