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Antimicrobial resistance (AMR) is an escalating global health crisis, driven by the overuse and misuse of antibiotics. Multidrug-resistant Gram-negative bacteria, such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, are particularly concerning due to their high morbidity and mortality rates. In this context, endolysins, derived from bacteriophages, offer a promising alternative to traditional antibiotics. This study introduces LysJEP8, a novel endolysin derived from Escherichia phage JEP8, which exhibits remarkable antimicrobial activity against key Gram-negative members of the ESKAPE group. Comparative assessments highlight LysJEP8's superior performance in reducing bacterial survival rates compared to previously described endolysins, with the most significant impact observed against P. aeruginosa, and notable effects on A. baumannii and K. pneumoniae. The study found that LysJEP8, as predicted by in silico analysis, worked best at lower pH values but lost its effectiveness at salt concentrations close to physiological levels. Importantly, LysJEP8 exhibited remarkable efficacy in the disruption of P. aeruginosa biofilms. This research underscores the potential of LysJEP8 as a valuable candidate for the development of innovative antibacterial agents, particularly against Gram-negative pathogens, and highlights opportunities for further engineering and optimization to address AMR effectively., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Both nanostructure and multivalency enhance the biological activities of antimicrobial peptides (AMPs), whose mechanism of action is cooperative. In addition, the efficacy of a particular AMP should benefit from a steady concentration at the local place of action and, therefore, from a slow release after a dynamic repository. In the context of emerging multi-resistant bacterial infections and the urgent need for novel and effective antimicrobial drugs, we tested these concepts through the engineering of four AMPs into supramolecular complexes as pharmacological entities. For that purpose, GWH1, T22, Pt5, and PaD, produced as GFP or human nidogen-based His-tagged fusion proteins, were engineered as self-assembling oligomeric nanoparticles ranging from 10 to 70 nm and further packaged into nanoparticle-leaking submicron granules. Since these materials slowly release functional nanoparticles during their time-sustained unpacking, they are suitable for use as drug depots in vivo. In this context, a particular AMP version (GWH1-NIDO-H6) was selected for in vivo validation in a zebrafish model of a complex bacterial infection. The GWH1-NIDO-H6-secreting protein granules are protective in zebrafish against infection by the multi-resistant bacterium Stenotrophomonas maltophilia , proving the potential of innovative formulations based on nanostructured and slowly released recombinant AMPs in the fight against bacterial infections.

Background: Recombinant proteins cover a wide range of biomedical, biotechnological, and industrial needs. Although there are diverse available protocols for their purification from cell extracts or from culture media, many proteins of interest such as those containing cationic domains are difficult to purify, a fact that results in low yields of the final functional product. Unfortunately, this issue prevents the further development and industrial or clinical application of these otherwise interesting products., Results: Aiming at improving the purification of such difficult proteins, a novel procedure has been developed based on supplementing crude cell extracts with non-denaturing concentrations of the anionic detergent N-Lauroylsarcosine. The incorporation of this simple step in the downstream pipeline results in a substantial improvement of the protein capture by affinity chromatography, an increase of protein purity and an enhancement of the overall process yield, being the detergent not detectable in the final product., Conclusion: By taking this approach, which represents a smart repurposing of N-Lauroylsarcosine applied to protein downstream, the biological activity of the protein is not affected. Being technologically simple, the N-Lauroylsarcosine-assisted protein purification might represent a critical improvement in recombinant protein production with wide applicability, thus smothering the incorporation of promising proteins into the protein market., (© 2023. The Author(s).)

Antibiotic resistance has exponentially increased during the last years. It is necessary to develop new antimicrobial drugs to prevent and treat infectious diseases caused by multidrug- or extensively-drug resistant (MDR/XDR)-bacteria. Host Defense Peptides (HDPs) have a versatile role, acting as antimicrobial peptides and regulators of several innate immunity functions. The results shown by previous studies using synthetic HDPs are only the tip of the iceberg, since the synergistic potential of HDPs and their production as recombinant proteins are fields practically unexplored. The present study aims to move a step forward through the development of a new generation of tailored antimicrobials, using a rational design of recombinant multidomain proteins based on HDPs. This strategy is based on a two-phase process, starting with the construction of the first generation molecules using single HDPs and further selecting those HDPs with higher bactericidal efficiencies to be combined in the second generation of broad-spectrum antimicrobials. As a proof of concept, we have designed three new antimicrobials, named D5L37βD3, D5L37D5L37 and D5LAL37βD3. After an in-depth exploration, we found D5L37D5L37 to be the most promising one, since it was equally effective against four relevant pathogens in healthcare-associated infections, such as methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus , methicillin-resistant Staphylococcus epidermidis (MRSE) and MDR Pseudomonas aeruginosa , being MRSA, MRSE and P. aeruginosa MDR strains. The low MIC values and versatile activity against planktonic and biofilm forms reinforce the use of this platform to isolate and produce unlimited HDP combinations as new antimicrobial drugs by effective means.

Bacterial inclusion bodies (IBs) are discrete macromolecular complexes that appear in recombinant prokaryotic cells under stress conditions. These structures are often discarded for biotechnological uses given the difficulty in recovering proteins of interest from them in a soluble form. However, recent approaches have revealed the potential of these protein clusters as biomaterials to promote cell growth and as protein depots for the release of recombinant proteins for biotechnological and biomedical applications. Although these kinds of natural supramolecular complexes have attracted great interest, no comprehensive study of their toxicity in cell cultures has been carried out. In this study, caco-2 cells were exposed to natural IBs, soluble protein-only nanoparticles (NPs), and non-assembled versions of the same protein for comparative purposes. Cytotoxicity, oxidative stress, and genotoxicity were analyzed for all these protein formats. Natural IBs and soluble protein formats demonstrated their safety in eukaryotic cells. No cytotoxicity, genotoxicity, or oxidative stress was detected in caco-2 cells exposed to the protein samples in any of the experimental conditions evaluated, which covered protein concentrations used in previous biological activity assays. These conditions evaluated the activity of protein samples obtained from three prokaryotic hosts [ Escherichia coli and the endotoxin-free expression systems Lactococcus lactis and ClearColi® BL21 (DE3)]. Our results demonstrate that natural IBs and soluble protein nanoparticles are non-toxic materials for eukaryotic cells and that this may represent an interesting alternative to the classical unassembled format of recombinant proteins for certain applications in biotechnology and biomedicine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Barguilla, Unzueta, Carratalá, Cano-Garrido, Villaverde, Hernández and Ferrer-Miralles.)

The coordination between histidine-rich peptides and divalent cations supports the formation of nano- and micro-scale protein biomaterials, including toxic and non-toxic functional amyloids, which can be adapted as drug delivery systems. Among them, inclusion bodies (IBs) formed in recombinant bacteria have shown promise as protein depots for time-sustained protein release. We have demonstrated here that the hexahistidine (H6) tag, fused to recombinant proteins, impacts both on the formation of bacterial IBs and on the conformation of the IB-forming protein, which shows a higher content of cross-beta intermolecular interactions in H6-tagged versions. Additionally, the addition of EDTA during the spontaneous disintegration of isolated IBs largely affects the protein leakage rate, again protein release being stimulated in His-tagged materials. This event depends on the number of His residues but irrespective of the location of the tag in the protein, as it occurs in either C-tagged or N-tagged proteins. The architectonic role of H6 in the formation of bacterial IBs, probably through coordination with divalent cations, offers an easy approach to manipulate protein leakage and to tailor the applicability of this material as a secretory amyloidal depot in different biomedical interfaces. In addition, the findings also offer a model to finely investigate, in a simple set-up, the mechanics of protein release from functional secretory amyloids.

Despite substantial development of production and purification protocols for heterologous recombinant proteins, some proteins are difficult to produce or, when produced, are accumulated in inclusion bodies (IBs). Nondenaturing protocols can be used to recover the entrapped protein from these protein aggregates. In this chapter, we provide a detailed procedure to analyze the physicochemical properties of one of those proteins produced in prokaryotic expression systems. Serum amyloid A3 (SAA3) was recovered from inclusion bodies (IBs) and its secondary structure associated to thermal stability and size was determined by circular dichroism (CD) and dynamic light scattering (DLS), respectively. These techniques were also applied to evaluate the SAA3 interaction with model membranes. These results show the importance of the structural analysis of proteins released from inclusion bodies under nondenaturing procedures, although similar approaches can be extended to any type of recombinant protein preparation., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

CXCR4 is a cytokine receptor used by HIV during cell attachment and infection. Overexpressed in the cancer stem cells of more than 20 human neoplasias, CXCR4 is a convenient antitumoral drug target. T22 is a polyphemusin-derived peptide and an effective CXCR4 ligand. Its highly selective CXCR4 binding can be exploited as an agent for the cell-targeted delivery and internalization of associated antitumor drugs. Sharing chemical and structural traits with antimicrobial peptides (AMPs), the capability of T22 as an antibacterial agent remains unexplored. Here, we have detected T22-associated antimicrobial activity and biofilm formation inhibition over Escherichia coli , Staphylococcus aureus and Pseudomonas aeruginosa , in a spectrum broader than the reference AMP GWH1. In contrast to GWH1, T22 shows neither cytotoxicity over mammalian cells nor hemolytic activity and is active when displayed on protein-only nanoparticles through genetic fusion. Under the pushing need for novel antimicrobial agents, the discovery of T22 as an AMP is particularly appealing, not only as its mere addition to the expanding catalogue of antibacterial drugs. The recognized clinical uses of T22 might allow its combined and multivalent application in complex clinical conditions, such as colorectal cancer, that might benefit from the synchronous destruction of cancer stem cells and local bacterial biofilms.

We have developed a simple, robust, and fully transversal approach for the a-la-carte fabrication of functional multimeric nanoparticles with potential biomedical applications, validated here by a set of diverse and unrelated polypeptides. The proposed concept is based on the controlled coordination between Zn 2+ ions and His residues in His-tagged proteins. This approach results in a spontaneous and reproducible protein assembly as nanoscale oligomers that keep the original functionalities of the protein building blocks. The assembly of these materials is not linked to particular polypeptide features, and it is based on an environmentally friendly and sustainable approach. The resulting nanoparticles, with dimensions ranging between 10 and 15 nm, are regular in size, are architecturally stable, are fully functional, and serve as intermediates in a more complex assembly process, resulting in the formation of microscale protein materials. Since most of the recombinant proteins produced by biochemical and biotechnological industries and intended for biomedical research are His-tagged, the green biofabrication procedure proposed here can be straightforwardly applied to a huge spectrum of protein species for their conversion into their respective nanostructured formats., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

A detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies-IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli ) and Lactococcus lactis, mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations.

Background: Protein aggregation is a biological event observed in expression systems in which the recombinant protein is produced under stressful conditions surpassing the homeostasis of the protein quality control system. In addition, protein aggregation is also related to conformational diseases in animals as transmissible prion diseases or non-transmissible neurodegenerative diseases including Alzheimer, Parkinson's disease, amyloidosis and multiple system atrophy among others. At the molecular level, the presence of aggregation-prone domains in protein molecules act as seeding igniters to induce the accumulation of protein molecules in protease-resistant clusters by intermolecular interactions., Results: In this work we have studied the aggregating-prone performance of a small peptide (L6K2) with additional antimicrobial activity and we have elucidated the relevance of the accompanying scaffold protein to enhance the aggregating profile of the fusion protein. Furthermore, we demonstrated that the fusion of L6K2 to highly soluble recombinant proteins directs the protein to inclusion bodies (IBs) in E. coli through stereospecific interactions in the presence of an insoluble protein displaying the same aggregating-prone peptide (APP)., Conclusions: These data suggest that the molecular bases of protein aggregation are related to the net balance of protein aggregation potential and not only to the presence of APPs. This is then presented as a generic platform to generate hybrid protein aggregates in microbial cell factories for biopharmaceutical and biotechnological applications.

A novel concept about bifunctional antimicrobial drugs, based on self-assembling protein nanoparticles, has been evaluated here over two biofilm-forming pathogens, namely Pseudomonas aeruginosa and Staphylococcus aureus . Two structurally different antimicrobial peptides (GWH1 and PaDBS1R1) were engineered to form regular nanoparticles of around 35 nm, to which the small molecular weight drug Floxuridine was covalently conjugated. Both the assembled peptides and the chemical, a conventional cytotoxic drug used in oncotherapy, showed potent antimicrobial activities that were enhanced by the combination of both molecules in single pharmacological entities. Therefore, the resulting prototypes show promises as innovative nanomedicines, being potential alternatives to conventional antibiotics. The biological performance and easy fabrication of these materials fully support the design of protein-based hybrid constructs for combined molecular therapies, expected to have broad applicability beyond antimicrobial medicines. In addition, the approach taken here validates the functional exploration and repurposing of antitumoral drugs, which at low concentrations perform well as unexpected biofilm-inhibiting agents.

Oligomerization of antimicrobial peptides into nanosized supramolecular complexes produced in biological systems (inclusion bodies and self-assembling nanoparticles) seems an appealing alternative to conventional antibiotics. In this work, the antimicrobial peptide, GWH1, was N-terminally fused to two different scaffold proteins, namely, GFP and IFN-γ for its bacterial production in the form of such recombinant protein complexes. Protein self-assembling as regular soluble protein nanoparticles was achieved in the case of GWH1-GFP, while oligomerization into bacterial inclusion bodies was reached in both constructions. Among all these types of therapeutic proteins, protein nanoparticles of GWH1-GFP showed the highest bactericidal effect in an in vitro assay against Escherichia coli , whereas non-oligomerized GWH1-GFP and GWH1-IFN-γ only displayed a moderate bactericidal activity. These results indicate that the biological activity of GWH1 is specifically enhanced in the form of regular multi-display configurations. Those in vitro observations were fully validated against a bacterial infection using a mouse mastitis model, in which the GWH1-GFP soluble nanoparticles were able to effectively reduce bacterial loads.

Peptide drugs hold great potential for the treatment of infectious diseases due to their unconventional mechanisms of action, biocompatibility, biodegradability and ease of synthesis and modification. The increasing rising of bacterial strains resistant to classical antibiotics have pushed the development of new peptide-based antimicrobial therapies. In this context, over the past few years, different approaches have reached a clinical approval. Furthermore, the application of nanotechnological principles to the design of antimicrobial peptide-based composites increases even more the already known benefits of antimicrobial peptides as competent protein drugs. Then, we provide here an overview of the current strategies for antimicrobial peptide discovery and modification and the status of such peptides already under clinical development. In addition, we summarize the innovative formulation strategies for their application, focusing on the controlled self-assembly for the fabrication of antimicrobial nanostructures without the assistance of external nanocarriers, and with emphasis on bioengineering, design of ultra-short peptides and rising insights in bacterial selectivity., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The cow dry period is a non-milking interval where the mammary gland involutes and regenerates to guarantee an optimal milk production in the subsequent lactation. Important bottlenecks such as the high risk of intramammary infections complicate the process. Antibiotics have been routinely used as a preventive treatment but the concerns about potential antibiotic resistance open a new scenario in which alternative strategies have to be developed. Matrix metalloproteinase-9 (MMP-9) is an enzyme able to degrade the extracellular matrix, triggering the involution and immune function of cow mammary gland. We have studied the infusion into the mammary gland of MMP-9 inclusion bodies as protein-based nanoparticles, demonstrating that 1.2 mg of MMP-9 enhanced the involution and immune function of the cow mammary gland. However, the comparison of the effects triggered by the administration of an active and an inactive form of MMP-9 led to conclude that the response observed in the bovine mammary gland was mainly due to the protein format but not to the biological activity of the MMP-9 embedded in the inclusion body. This study provides relevant information on the future use of protein inclusion bodies in cow mammary gland and the role of MMP-9 at dry-off.