Design strategies for positively charged endolysins: Insights into Artilysin development.

Endolysins are bacteriophage-encoded enzymes that can specifically degrade the peptidoglycan layer of bacterial cell wall, making them an attractive tool for the development of novel antibacterial agents. The use of genetic engineering techniques for the production and modification of endolysins offers the opportunity to customize their properties and activity against specific bacterial targets, paving the way for the development of personalized therapies for bacterial infections. Gram-negative bacteria possess an outer membrane that can hinder the action of recombinantly produced endolysins. However, certain endolysins are capable of crossing the outer membrane by virtue of segments that share properties resembling those of cationic peptides. These regions increase the affinity of the endolysin towards the bacterial surface and assist in the permeabilization of the membrane. In order to improve the bactericidal effectiveness of endolysins, approaches have been implemented to increase their net charge, including the development of Artilysins containing positively charged amino acids at one end. At present, there are no specific guidelines outlining the steps for implementing these modifications. There is an ongoing debate surrounding the optimal location of positive charge, the need for a linker region, and the specific amino acid composition of peptides for modifying endolysins. The aim of this study is to provide clarity on these topics by analyzing and comparing the most effective modifications found in previous literature.

Sequence edition of single domains modulates the final immune and antimicrobial potential of a new generation of multidomain recombinant proteins.

Combining several innate immune peptides into a single recombinant antimicrobial and immunomodulatory polypeptide has been recently demonstrated. However, the versatility of the multidomain design, the role that each domain plays and how the sequence edition of the different domains affects their final protein activity is unknown. Parental multidomain antimicrobial and immunomodulatory protein JAMF1 and several protein variants (JAMF1.2, JAMF2 and AM2) have been designed and recombinantly produced to explore how the tuning of domain sequences affects their immunomodulatory potential in epithelial cells and their antimicrobial capacity against Gram-positive and Gram-negative bacteria. The replacement of the sequence of defensin HD5 and phospholipase sPLA2 by shorter active fragments of both peptides improves the final immunomodulatory (IL-8 secretion) and antimicrobial function of the multidomain protein against antimicrobial-resistant Klebsiella pneumoniae and Enterococcus spp. Further, the presence of Jun and Fos leucine zippers in multidomain proteins is crucial in preventing toxic effects on producer cells. The generation of antimicrobial proteins based on multidomain polypeptides allows specific immunomodulatory and antimicrobial functions, which can be easily edited by modifying of each domain sequence.

Extracellular vesicles from recombinant cell factories improve the activity and efficacy of enzymes defective in lysosomal storage disorders.

In the present study the use of extracellular vesicles (EVs) as vehicles for therapeutic enzymes in lysosomal storage disorders was explored. EVs were isolated from mammalian cells overexpressing alpha-galactosidase A (GLA) or N-sulfoglucosamine sulfohydrolase (SGSH) enzymes, defective in Fabry and Sanfilippo A diseases, respectively. Direct purification of EVs from cell supernatants was found to be a simple and efficient method to obtain highly active GLA and SGSH proteins, even after EV lyophilization. Likewise, EVs carrying GLA (EV-GLA) were rapidly uptaken and reached the lysosomes in cellular models of Fabry disease, restoring lysosomal functionality much more efficiently than the recombinant enzyme in clinical use. In vivo, EVs were well tolerated and distributed among all main organs, including the brain. DiR-labelled EVs were localized in brain parenchyma 1 h after intra-arterial (internal carotid artery) or intravenous (tail vein) administrations. Moreover, a single intravenous administration of EV-GLA was able to reduce globotriaosylceramide (Gb3) substrate levels in clinically relevant tissues, such kidneys and brain. Overall, our results demonstrate that EVs from cells overexpressing lysosomal enzymes act as natural protein delivery systems, improving the activity and the efficacy of the recombinant proteins and facilitating their access to organs neglected by conventional enzyme replacement therapies.

Exploring the use of leucine zippers for the generation of a new class of inclusion bodies for pharma and biotechnological applications.

BACKGROUND: Inclusion bodies (IBs) are biologically active protein aggregates forming natural nanoparticles with a high stability and a slow-release behavior. Because of their nature, IBs have been explored to be used as biocatalysts, in tissue engineering, and also for human and animal therapies. To improve the production and biological efficiency of this nanomaterial, a wide range of aggregation tags have been evaluated. However, so far, the presence in the IBs of bacterial impurities such as lipids and other proteins coexisting with the recombinant product has been poorly studied. These impurities could strongly limit the potential of IB applications, being necessary to control the composition of these bacterial nanoparticles. Thus, we have explored the use of leucine zippers as alternative tags to promote not only aggregation but also the generation of a new type of IB-like protein nanoparticles with improved physicochemical properties. RESULTS: Three different protein constructs, named GFP, J-GFP-F and J/F-GFP were engineered. J-GFP-F corresponded to a GFP flanked by two leucine zippers (Jun and Fos); J/F-GFP was formed coexpressing a GFP fused to Jun leucine zipper (J-GFP) and a GFP fused to a Fos leucine zipper (F-GFP); and, finally, GFP was used as a control without any tag. All of them were expressed in Escherichia coli and formed IBs, where the aggregation tendency was especially high for J/F-GFP. Moreover, those IBs formed by J-GFP-F and J/F-GFP constructs were smaller, rougher, and more amorphous than GFP ones, increasing surface/mass ratio and, therefore, surface for protein release. Although the lipid and carbohydrate content were not reduced with the addition of leucine zippers, interesting differences were observed in the protein specific activity and conformation with the addition of Jun and Fos. Moreover, J-GFP-F and J/F-GFP nanoparticles were purer than GFP IBs in terms of protein content. CONCLUSIONS: This study proved that the use of leucine zippers strategy allows the formation of IBs with an increased aggregation ratio and protein purity, as we observed with the J/F-GFP approach, and the formation of IBs with a higher specific activity, in the case of J-GFP-F IBs. Thus, overall, the use of leucine zippers seems to be a good system for the production of IBs with more promising characteristics useful for pharma or biotech applications.

CCL21-loaded 3D hydrogels for T cell expansion and differentiation.

Recent achievements in the field of immunotherapy, such as the development of engineered T cells used in adoptive cell therapy, are introducing more efficient strategies to combat cancer. Nevertheless, there are still many limitations. For example, these T cells are challenging to manufacture, manipulate, and control. Specifically, there are limitations in producing the large amounts of therapeutic T cells needed for these therapies in a short period of time and in an economically viable manner. In this study, three-dimensional (3D) poly(ethylene) glycol (PEG) hydrogels covalently combined with low molecular weight heparin are engineered to resemble the lymph nodes, where T cells reproduce. In these hydrogels, PEG provides the needed structural and mechanical properties, whereas heparin is used as an anchor for the cytokine CCL21, which is present in the lymph nodes, and can affect cell migration and proliferation. The 3D structure of the hydrogel in combination with its loading capacity result in an increased primary human CD4+ T cell proliferation compared to the state-of-the-art expansion systems consisting of artificial antigen presenting cells. Thus, we present a new tool for adoptive cell therapy to help achieving the large numbers of cells required for therapy of selected phenotypes targeted against cancer cells, by mimicking the lymph nodes.

A new generation of recombinant polypeptides combines multiple protein domains for effective antimicrobial activity.

BACKGROUND: Although most of antimicrobial peptides (AMPs), being relatively short, are produced by chemical synthesis, several AMPs have been produced using recombinant technology. However, AMPs could be cytotoxic to the producer cell, and if small they can be easily degraded. The objective of this study was to produce a multidomain antimicrobial protein based on recombinant protein nanoclusters to increase the yield, stability and effectivity. RESULTS: A single antimicrobial polypeptide JAMF1 that combines three functional domains based on human α-defensin-5, human XII-A secreted phospholipase A2 (sPLA2), and a gelsolin-based bacterial-binding domain along with two aggregation-seeding domains based on leucine zippers was successfully produced with no toxic effects for the producer cell and mainly in a nanocluster structure. Both, the nanocluster and solubilized format of the protein showed a clear antimicrobial effect against a broad spectrum of Gram-negative and Gram-positive bacteria, including multi-resistant strains, with an optimal concentration between 1 and 10 µM. CONCLUSIONS: Our findings demonstrated that multidomain antimicrobial proteins forming nanoclusters can be efficiently produced in recombinant bacteria, being a novel and valuable strategy to create a versatile, highly stable and easily editable multidomain constructs with a broad-spectrum antimicrobial activity in both soluble and nanostructured format.

Recombinant Protein-Based Nanoparticles: Elucidating their Inflammatory Effects In Vivo and their Potential as a New Therapeutic Format.

Bacterial inclusion bodies (IBs) are protein-based nanoparticles of a few hundred nanometers formed during recombinant protein production processes in different bacterial hosts. IBs contain active protein in a mechanically stable nanostructured format that has been broadly characterized, showing promising potential in different fields such as tissue engineering, protein replacement therapies, cancer, and biotechnology. For immunomodulatory purposes, however, the interference of the format immunogenic properties-intrinsic to IBs-with the specific effects of the therapeutic protein is still an uncovered gap. For that, active and inactive forms of the catalytic domain of a matrix metalloproteinase-9 (MMP-9 and mutMMP-9, respectively) have been produced as IBs and compared with the soluble form for dermal inflammatory effects in mmp9 knock-out mice. After protein injections in air-pouches in the mouse model, MMP-9 IBs induce local neutrophil recruitment and increase pro-inflammatory chemokine levels, lasting for at least two days, whereas the effects triggered by the soluble MMP-9 format fade out after 3 h. Interestingly, the IB intrinsic effects (mutMMP-9 IBs) do not last more than 24 h. Therefore, it may be concluded that IBs could be used for the delivery of therapeutic proteins, such as immunomodulating proteins while preserving their stability in the specific tissue and without triggering important unspecific inflammatory responses due to the protein format.

Stable anchoring of bacteria-based protein nanoparticles for surface enhanced cell guidance.

In tissue engineering, biological, physical, and chemical inputs have to be combined to properly mimic cellular environments and successfully build artificial tissues which can be designed to fulfill different biomedical needs such as the shortage of organ donors or the development of in vitro disease models for drug testing. Inclusion body-like protein nanoparticles (pNPs) can simultaneously provide such physical and biochemical stimuli to cells when attached to surfaces. However, this attachment has only been made by physisorption. To provide a stable anchoring, a covalent binding of lactic acid bacteria (LAB) produced pNPs, which lack the innate pyrogenic impurities of Gram-negative bacteria like Escherichia coli, is presented. The reported micropatterns feature a robust nanoscale topography with an unprecedented mechanical stability. In addition, they are denser and more capable of influencing cell morphology and orientation. The increased stability and the absence of pyrogenic impurities represent a step forward towards the translation of this material to a clinical setting.

Aggregation-prone peptides modulate activity of bovine interferon gamma released from naturally occurring protein nanoparticles.

Efficient protocols for the production of recombinant proteins are indispensable for the development of the biopharmaceutical sector. Accumulation of recombinant proteins in naturally-occurring protein aggregates is detrimental to biopharmaceutical development. In recent years, the view of protein aggregates has changed with the recognition that they are a valuable source of functional recombinant proteins. In this study, bovine interferon-gamma (rBoIFN-γ) was engineered to enhance the formation of protein aggregates, also known as protein nanoparticles (NPs), by the addition of aggregation-prone peptides (APPs) in the generally recognized as safe (GRAS) bacterial Lactococcus lactis expression system. The L6K2, HALRU and CYOB peptides were selected to assess their intrinsic aggregation capability to nucleate protein aggregation. These APPs enhanced the tendency of the resulting protein to aggregate at the expense of total protein yield. However, fine physico-chemical characterization of the resulting intracellular protein NPs, the protein released from them and the protein purified from the soluble cell fraction indicated that the compactability of protein conformations was directly related to the biological activity of variants of IFN-γ, used here as a model protein with therapeutic potential. APPs enhanced the aggregation tendency of fused rBoIFN-γ while increasing compactability of protein species. Biological activity of rBoIFN-γ was favored in more compacted conformations. Naturally-occurring protein aggregates can be produced in GRAS microorganisms as protein depots of releasable active protein. The addition of APPs to enhance the aggregation tendency has a positive impact in overall compactability and functionality of resulting protein conformers.

Bacterial Inclusion Bodies: Discovering Their Better Half.

Bacterial inclusion bodies (IBs) are functional, non-toxic amyloids occurring in recombinant bacteria showing analogies with secretory granules of the mammalian endocrine system. The scientific interest in these mesoscale protein aggregates has been historically masked by their status as a hurdle in recombinant protein production. However, progressive understanding of how the cell handles the quality of recombinant polypeptides and the main features of their intriguing molecular organization has stimulated the interest in inclusion bodies and spurred their use in diverse technological fields. The engineering and tailoring of IBs as functional protein particles for materials science and biomedicine is a good example of how formerly undesired bacterial byproducts can be rediscovered as promising functional materials for a broad spectrum of applications.

Engineering tumor cell targeting in nanoscale amyloidal materials.

Bacterial inclusion bodies are non-toxic, mechanically stable and functional protein amyloids within the nanoscale size range that are able to naturally penetrate into mammalian cells, where they deliver the embedded protein in a functional form. The potential use of inclusion bodies in protein delivery or protein replacement therapies is strongly impaired by the absence of specificity in cell binding and penetration, thus preventing targeting. To address this issue, we have here explored whether the genetic fusion of two tumor-homing peptides, the CXCR4 ligands R9 and T22, to an inclusion body-forming green fluorescent protein (GFP), would keep the interaction potential and the functionality of the fused peptides and then confer CXCR4 specificity in cell binding and further uptake of the materials. The fusion proteins have been well produced in Escherichia coli in their full-length form, keeping the potential for fluorescence emission of the partner GFP. By using specific inhibitors of CXCR4 binding, we have demonstrated that the engineered protein particles are able to penetrate CXCR4+ cells, in a receptor-mediated way, without toxicity or visible cytopathic effects, proving the availability of the peptide ligands on the surface of inclusion bodies. Since no further modification is required upon their purification, the biological production of genetically targeted inclusion bodies opens a plethora of cost-effective possibilities in the tissue-specific intracellular transfer of functional proteins through the use of structurally and functionally tailored soft materials.

Nanostructured recombinant cytokines: A highly stable alternative to short-lived prophylactics.

Cytokines have been widely used as adjuvants and therapeutic agents in treatments of human diseases. Despite their recognized potential as drugs, the medical use of cytokines has considerable drawbacks, mainly related to their low stability and short half-life. Such intrinsic limitations imply the administration of high doses, often prompting toxicity, undesirable side effects and greater production costs. Here, we describe a new category of mechanically stable nanostructured cytokines (TNFα and CCL4/MIP-1ß) that resist harsh physicochemical conditions in vitro (pH and temperature), while maintaining functionality. These bio-functional materials are produced in recombinant cell factories through cost-effective and fully scalable processes. Notably, we demonstrate their prophylactic potential in vivo showing they protect zebrafish from a lethal infection by Pseudomonas aeruginosa.

CXCR4(+)-targeted protein nanoparticles produced in the food-grade bacterium Lactococcus lactis.

AIM: Lactococcus lactis is a Gram-positive (endotoxin-free) food-grade bacteria exploited as alternative to Escherichia coli for recombinant protein production. We have explored here for the first time the ability of this platform as producer of complex, self-assembling protein materials. MATERIALS & METHODS: Biophysical properties, cell penetrability and in vivo biodistribution upon systemic administration of tumor-targeted protein nanoparticles produced in L. lactis have been compared with the equivalent material produced in E. coli. RESULTS: Protein nanoparticles have been efficiently produced in L. lactis, showing the desired size, internalization properties and biodistribution. CONCLUSION: In vitro and in vivo data confirm the potential and robustness of the production platform, pointing out L. lactis as a fascinating cell factory for the biofabrication of protein materials intended for therapeutic applications.

Structural and functional features of self-assembling protein nanoparticles produced in endotoxin-free Escherichia coli.

BACKGROUND: Production of recombinant drugs in process-friendly endotoxin-free bacterial factories targets to a lessened complexity of the purification process combined with minimized biological hazards during product application. The development of nanostructured recombinant materials in innovative nanomedical activities expands such a need beyond plain functional polypeptides to complex protein assemblies. While Escherichia coli has been recently modified for the production of endotoxin-free proteins, no data has been so far recorded regarding how the system performs in the fabrication of smart nanostructured materials. RESULTS: We have here explored the nanoarchitecture and in vitro and in vivo functionalities of CXCR4-targeted, self-assembling protein nanoparticles intended for intracellular delivery of drugs and imaging agents in colorectal cancer. Interestingly, endotoxin-free materials exhibit a distinguishable architecture and altered size and target cell penetrability than counterparts produced in conventional E. coli strains. These variant nanoparticles show an eventual proper biodistribution and highly specific and exclusive accumulation in tumor upon administration in colorectal cancer mice models, indicating a convenient display and function of the tumor homing peptides and high particle stability under physiological conditions. DISCUSSION: The observations made here support the emerging endotoxin-free E. coli system as a robust protein material producer but are also indicative of a particular conformational status and organization of either building blocks or oligomers. This appears to be promoted by multifactorial stress-inducing conditions upon engineering of the E. coli cell envelope, which impacts on the protein quality control of the cell factory.

Cellular uptake and intracellular fate of protein releasing bacterial amyloids in mammalian cells.

Bacterial Inclusion Bodies (IBs) are amyloidal protein deposits that functionally mimic secretory granules from the endocrine system. When formed by therapeutically relevant proteins, they complement missing intracellular activities in jeopardized cell cultures, offering an intriguing platform for protein drug delivery in substitutive therapies. Despite the therapeutic potential of IBs, their capability to interact with eukaryotic cells, cross the cell membrane and release their functional building blocks into the cytosolic space remains essentially unexplored. We have systematically dissected the process by which bacterial amyloids interact with mammalian cells. An early and tight cell membrane anchorage of IBs is followed by cellular uptake of single or grouped IBs of variable sizes by macropinocytosis. Although an important fraction of the penetrating particles is led to lysosomal degradation, biologically significant amounts of protein are released into the cytosol. In addition, our data suggest the involvement of the bacterial cell folding modulator DnaK in the release of functional proteins from these amyloidal reservoirs. The mechanisms supporting the internalization of disintegrable protein nanoparticles revealed here offer clues to implement novel approaches for protein drug delivery based on controlled protein packaging as bacterial IBs.

Bottom-Up Instructive Quality Control in the Biofabrication of Smart Protein Materials.

The impact of cell factory quality control on material properties is a neglected but critical issue in the fabrication of protein biomaterials, which are unique in merging structure and function. The molecular chaperoning of protein conformational status is revealed here as a potent molecular instructor of the macroscopic properties of self-assembling, cell-targeted protein nanoparticles, including biodistribution upon in vivo administration.

Lactic acid bacteria: reviewing the potential of a promising delivery live vector for biomedical purposes.

Lactic acid bacteria (LAB) have a long history of safe exploitation by humans, being used for centuries in food production and preservation and as probiotic agents to promote human health. Interestingly, some species of these Gram-positive bacteria, which are generally recognized as safe organisms by the US Food and Drug Administration (FDA), are able to survive through the gastrointestinal tract (GIT), being capable to reach and colonize the intestine, where they play an important role. Besides, during the last decades, an important effort has been done for the development of tools to use LAB as microbial cell factories for the production of proteins of interest. Given the need to develop effective strategies for the delivery of prophylactic and therapeutic molecules, LAB have appeared as an appealing option for the oral, intranasal and vaginal delivery of such molecules. So far, these genetically modified organisms have been successfully used as vehicles for delivering functional proteins to mucosal tissues in the treatment of many different pathologies including GIT related pathologies, diabetes, cancer and viral infections, among others. Interestingly, the administration of such microorganisms would suppose a significant decrease in the production cost of the treatments agents since being live organisms, such vectors would be able to autonomously amplify and produce and deliver the protein of interest. In this context, this review aims to provide an overview of the use of LAB engineered as a promising alternative as well as a safety delivery platform of recombinant proteins for the treatment of a wide range of diseases.

Topographically targeted osteogenesis of mesenchymal stem cells stimulated by inclusion bodies attached to polycaprolactone surfaces.

AIM: Bacterial inclusion bodies (IBs) are nanostructured (submicron), pseudospherical proteinaceous particles produced in recombinant bacteria resulting from ordered protein aggregation. Being mechanically stable, several physicochemical and biological properties of IBs can be tuned by appropriate selection of the producer strain and of culture conditions. It has been previously shown that IBs favor cell adhesion and surface colonization by mammalian cell lines upon decoration on materials surfaces, but how these biomaterials could influence the behavior of mesenchymal stem cells remains to be explored. MATERIALS & METHODS: Here, the authors vary topography, stiffness and wettability using the IBs to decorate polycaprolactone surfaces on which mesenchymal stem cells are cultured. RESULTS: The authors show that these topographies can be used to specifically target osteogenesis from mesenchymal stem cells, and through metabolomics, they show that the cells have increased energy demand during this bone-related differentiation. CONCLUSION: IBs as topographies can be used not only to direct cell proliferation but also to target differentiation of mesenchymal stem cells.

Sheltering DNA in self-organizing, protein-only nano-shells as artificial viruses for gene delivery.

By recruiting functional domains supporting DNA condensation, cell binding, internalization, endosomal escape and nuclear transport, modular single-chain polypeptides can be tailored to associate with cargo DNA for cell-targeted gene therapy. Recently, an emerging architectonic principle at the nanoscale has permitted tagging protein monomers for self-organization as protein-only nanoparticles. We have studied here the accommodation of plasmid DNA into protein nanoparticles assembled with the synergistic assistance of end terminal poly-arginines (R9) and poly-histidines (H6). Data indicate a virus-like organization of the complexes, in which a DNA core is surrounded by a solvent-exposed protein layer. This finding validates end-terminal cationic peptides as pleiotropic tags in protein building blocks for the mimicry of viral architecture in artificial viruses, representing a promising alternative to the conventional use of viruses and virus-like particles for nanomedicine and gene therapy. FROM THE CLINICAL EDITOR: Finding efficient gene delivery methods still represents a challenge and is one of the bottlenecks to the more widespread application of gene therapy. The findings presented in this paper validate the application of end-terminal cationic peptides as pleiotropic tags in protein building blocks for "viral architecture mimicking" in artificial viruses, representing a promising alternative to the use of viruses and virus-like particles for gene delivery.

Improving protein delivery of fibroblast growth factor-2 from bacterial inclusion bodies used as cell culture substrates.

Bacterial inclusion bodies (IBs) have recently been used to generate biocompatible cell culture interfaces, with diverse effects on cultured cells such as cell adhesion enhancement, stimulation of cell growth or induction of mesenchymal stem cell differentiation. Additionally, novel applications of IBs as sustained protein delivery systems with potential applications in regenerative medicine have been successfully explored. In this scenario, with IBs gaining significance in the biomedical field, the fine tuning of this functional biomaterial is crucial. In this work, the effect of temperature on fibroblast growth factor-2 (FGF-2) IB production and performance has been evaluated. FGF-2 was overexpressed in Escherichia coli at 25 and 37 °C, producing IBs with differences in size, particle structure and biological activity. Cell culture topographies made with FGF-2 IBs biofabricated at 25 °C showed higher levels of biological activity as well as a looser supramolecular structure, enabling a higher protein release from the particles. In addition, the controlled use of FGF-2 protein particles enabled the generation of functional topographies with multiple biological activities being effective on diverse cell types.