Exploring the impact of the recombinant Escherichia coli strain on defensins antimicrobial activity: BL21 versus Origami strain

The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. These HDPs are classified into three categories: defensins, cathelicidins, and histatins. Traditionally, HDPs have been chemically synthesized, but this strategy often limits their application due to the high associated production costs. Alternatively, some HDPs have been recombinantly produced, but little is known about the impact of the bacterial strain in the recombinant product. This work aimed to assess the influence of the Escherichia coli strain used as cell factory to determine the activity and stability of recombinant defensins, which have 3 disulfide bonds. For that, an α-defensin [human α-defensin 5 (HD5)] and a β-defensin [bovine lingual antimicrobial peptide (LAP)] were produced in two recombinant backgrounds. The first one was an E. coli BL21 strain, which has a reducing cytoplasm, whereas the second was an E. coli Origami B, that is a strain with a more oxidizing cytoplasm. The results showed that both HD5 and LAP, fused to Green Fluorescent Protein (GFP), were successfully produced in both BL21 and Origami B strains. However, differences were observed in the HDP production yield and bactericidal activity, especially for the HD5-based protein. The HD5 protein fused to GFP was not only produced at higher yields in the E. coli BL21 strain, but it also showed a higher quality and stability than that produced in the Origami B strain. Hence, this data showed that the strain had a clear impact on both HDPs quantity and quality.info:eu-repo/semantics/publishedVersio

A Novel Generation of Tailored Antimicrobial Drugs Based on Recombinant Multidomain Proteins

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.info:eu-repo/semantics/publishedVersio

Intervals de confiança i de contrastos d'hipòtesi de dues mostres, ANOVA i el contrast de les taules de contingència

Prova d'avaluació continuada (PAC) de l'assignatura Fonaments d'estadística, dins els graus d'Administració i direcció d'empreses, Màrqueting i investigació de mercats, Turisme i Relacions laborals i ocupació.Prueba de evaluación continuada (PEC) de la asignatura Fundamentos de estadística, dentro de los grados Administración y dirección de empresas, Márketing e investigación de mercados, Turismo y Relaciones laborales y ocupación.Continuous evaluation activity (PEC) of the subject Fundamentals of Statistics, within the degrees Administration and business management, Marketing and market research, Tourism and Labour relations and employment

Intervals de confiança i de contrastos d'hipòtesi de dues mostres, ANOVA i el contrast de les taules de contingència

Prova d'avaluació continuada (PAC) de l'assignatura Fonaments d'estadística, dins els graus d'Administració i direcció d'empreses, Màrqueting i investigació de mercats, Turisme i Relacions laborals i ocupació.Prueba de evaluación continuada (PEC) de la asignatura Fundamentos de estadística, dentro de los grados Administración y dirección de empresas, Márketing e investigación de mercados, Turismo y Relaciones laborales y ocupación.Continuous evaluation activity (PEC) of the subject Fundamentals of Statistics, within the degrees Administration and business management, Marketing and market research, Tourism and Labour relations and employment

Intervals de confiança i contrastos d'hipòtesis

Prova d'avaluació continuada (PAC) de l'assignatura Fonaments d'estadística, dins els graus d'Administració i direcció d'empreses, Màrqueting i investigació de mercats, Turisme i Relacions laborals i ocupació.Prueba de evaluación continuada (PEC) de la asignatura Fundamentos de estadística, dentro de los grados Administración y dirección de empresas, Márketing e investigación de mercados, Turismo y Relaciones laborales y ocupación.Continuous evaluation activity (PEC) of the subject Fundamentals of Statistics, within the degrees Administration and business management, Marketing and market research, Tourism and Labour relations and employment

Intervals de confiança i contrastos d'hipòtesis

Prova d'avaluació continuada (PAC) de l'assignatura Fonaments d'estadística, dins els graus d'Administració i direcció d'empreses, Màrqueting i investigació de mercats, Turisme i Relacions laborals i ocupació.Prueba de evaluación continuada (PEC) de la asignatura Fundamentos de estadística, dentro de los grados Administración y dirección de empresas, Márketing e investigación de mercados, Turismo y Relaciones laborales y ocupación.Continuous evaluation activity (PEC) of the subject Fundamentals of Statistics, within the degrees Administration and business management, Marketing and market research, Tourism and Labour relations and employment

Exploring the impact of the recombinant Escherichia coli strain on defensins antimicrobial activity: BL21 versus Origami strain

The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. These HDPs are classified into three categories: defensins, cathelicidins, and histatins. Traditionally, HDPs have been chemically synthesized, but this strategy often limits their application due to the high associated production costs. Alternatively, some HDPs have been recombinantly produced, but little is known about the impact of the bacterial strain in the recombinant product. This work aimed to assess the influence of the Escherichia coli strain used as cell factory to determine the activity and stability of recombinant defensins, which have 3 disulfide bonds. For that, an α-defensin [human α-defensin 5 (HD5)] and a β-defensin [bovine lingual antimicrobial peptide (LAP)] were produced in two recombinant backgrounds. The first one was an E. coli BL21 strain, which has a reducing cytoplasm, whereas the second was an E. coli Origami B, that is a strain with a more oxidizing cytoplasm. The results showed that both HD5 and LAP, fused to Green Fluorescent Protein (GFP), were successfully produced in both BL21 and Origami B strains. However, differences were observed in the HDP production yield and bactericidal activity, especially for the HD5-based protein. The HD5 protein fused to GFP was not only produced at higher yields in the E. coli BL21 strain, but it also showed a higher quality and stability than that produced in the Origami B strain. Hence, this data showed that the strain had a clear impact on both HDPs quantity and quality.This work was funded by Ministerio de Ciencia, Innovación y Universidades Grants (PID2019-107298RB-C21) to AA and EG-F, PID2019-105622RB-I00 to IR and through the ‘‘Severo Ochoa’’ Programme for Centers of Excellence in R&D (FUNFUTURE CEX2019-000917-S and SEV-2017-0706). The authors are also grateful to Marató de TV3 foundation for the Grant 201812-30-31-32-33 to EG-F and IR, and the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). AL-C received a pre-doctoral fellowship from Generalitat de Catalunya (FI-AGAUR), EG-F a post-doctoral fellowship from INIA and JG a Ramón y Cajal fellowship from MINN (RyC-2017-22614).With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

Thermomagnetic Molecular System Based on TTF-PTM Radical: Switching the Spin and Charge Delocalization

The electron donor–acceptor dyad 1, based on a polychlorotriphenylmethyl radical subunit linked to a tetrathiafulvalene moiety, shows in DMF solution a reversible temperature-induced switching between diamagnetic dimers, observed at room temperature, and paramagnetic monomers at high temperature. Different optical and magnetic properties are observed for the two states of this supramolecular switch, related to the subtle interplay between intramolecular electron-transfer and intermolecular charge-transfer processes