924ca461f52a72ce42a9d4d263072c4c pharmaceutics_a2020v12n12p1217.pdf f941e7d0fe4af0b2d33b66576ffa8fcbca613185 pharmaceutics_a2020v12n12p1217.pdf bdd8ff4881d4e2c4c38757be45d870b5348d5b6abb88b74621338cca8709437a pharmaceutics_a2020v12n12p1217.pdf Title: In Vivo Bactericidal Efficacy of GWH1 Antimicrobial Peptide Displayed on Protein Nanoparticles, a Potential Alternative to Antibiotics Subject: 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- 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- 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. Keywords: mouse mastitis model; antimicrobial peptide; protein nanoparticle; inclusion body; recombinant protein; Escherichia coli; Staphylococcus aureus; therapeutic protein Author: Jose V. Carratalá, Eric Brouillette, Naroa Serna, Alejandro Sánchez-Chardi, Julieta M. Sánchez, Antonio Villaverde, Anna Arís, Elena Garcia-Fruitós, Neus Ferrer-Miralles and François Malouin Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Fri Dec 18 10:17:26 2020 CET ModDate: Fri Dec 18 10:30:04 2020 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3816929 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- USXBDB+Pxsy Type 1 Builtin yes yes no 10 0 ARGBYC+VnURWPalladioL Type 1 Custom yes yes no 14 0 VDKNAR+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 XDRYRW+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 RCFYRP+Rpxb Type 1 Builtin yes yes no 28 0 TYGGCI+URWPalladioL-Roma Type 1 Custom yes yes no 32 0 HAKARX+Rpxr Type 1 Builtin yes yes no 36 0 QNWFPZ+EURM10 Type 1 Builtin yes 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Purification Destination: subsection.2.2 Item: Title: Nanoparticle Size Characterization Destination: subsection.2.3 Item: Title: Bactericidal Activity Assay Destination: subsection.2.4 Item: Title: Disassembling of Soluble Nanoparticles Destination: subsection.2.5 Item: Title: Protein Solubilization from IBs Destination: subsection.2.6 Item: Title: Determination of Murine IFN- Biological Activity Destination: subsection.2.7 Item: Title: Mouse Model of Infectious Mastitis Destination: subsection.2.8 Item: Title: Statistical Analysis Destination: subsection.2.9 Item: Title: Results Destination: section.3 Children: Item: Title: Characterization of Self-Assembling Nanoparticles Destination: subsection.3.1 Item: Title: In Vitro Evaluation of Antibacterial Activity Destination: subsection.3.2 Item: Title: Importance of Nanoparticle Format on Antibacterial Activity Destination: subsection.3.3 Item: Title: Bioactivity Evaluation of IFN Based Proteins Destination: subsection.3.4 Item: Title: Growth Kinetics of E. coli in the Mastitis Mice Model Destination: subsection.3.5 Item: Title: Characterization of IBs Destination: subsection.3.6 Item: Title: Therapeutic Effect of PNPs and IBs in the Mouse Mastitis Model Destination: subsection.3.7 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: In Vivo Bactericidal Efficacy of GWH1 Antimicrobial Peptide Displayed on Protein Nanoparticles, a Potential Alternative to Antibiotics Author: Jose V. Carratalá, Eric Brouillette, Naroa Serna, Alejandro Sánchez-Chardi, Julieta M. Sánchez, Antonio Villaverde, Anna Arís, Elena Garcia-Fruitós, Neus Ferrer-Miralles and François Malouin Subject: 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- 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- 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. 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