971f74f2733801d708e9aa28ccc5f437 pmc_10146347.pdf 1beba27a73a3a1bdaeb3918f84e5b522057c4e79 pmc_10146347.pdf 8df223c41621961efd687805303622b5b3be1bca61f32fd05fcbb793a9129b65 pmc_10146347.pdf Title: A Novel Generation of Tailored Antimicrobial Drugs Based on Recombinant Multidomain Proteins Subject: 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 D5L37D3, D5L37D5L37 and D5LAL37D3. 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. Keywords: host defense peptides; antimicrobial; recombinant; nosocomial infections Author: Adrià López-Cano, Neus Ferrer-Miralles, Julieta Sánchez, Jose Vicente Carratalá, Xavier Rodriguez Rodriguez, Imma Ratera, Judith Guasch, Oscar Q. Pich, Paula Bierge, Cristina Garcia-de-la-Maria, Jose M. Miro, Elena Garcia-Fruitós and Anna Arís Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sun Mar 26 10:26:05 2023 CEST ModDate: Wed Mar 29 07:30:30 2023 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 8993683 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- MWHIBA+VnURWPalladioL Type 1 Custom yes yes no 10 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 15 0 JXMSIK+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 GOHGAM+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 NLCYVB+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 HSNKGA+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 IAFAOG+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 51 0 HIFMEI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 89 0 HIFMEK+PalatinoLinotype TrueType WinAnsi 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HIFMEI+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 425 0 HIFMEJ+PalatinoLinotype CID TrueType Identity-H yes yes yes 428 0 HIFMEK+PalatinoLinotype TrueType WinAnsi yes yes no 434 0 HIFMFM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 437 0 HIFNEH+PalatinoLinotype TrueType MacRoman yes yes no 440 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-02-02 02:35:24 CET RepresentationInformation: pmc_10146347.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-09-05 02:22:09 CEST Size: 8993683 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 644 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Methods Destination: section.2 Children: Item: Title: Bacterial Strains Destination: subsection.2.1 Item: Title: Genetic Construct Design Destination: subsection.2.2 Item: Title: Protein Production and Purification Destination: subsection.2.3 Item: Title: Evaluation of Antimicrobial Activity Destination: subsection.2.4 Item: Title: Protein Cytotoxicity Assay Destination: subsection.2.5 Item: Title: SEM Imaging of Antimicrobial Effects Destination: subsection.2.6 Item: Title: Dynamic Light Scattering Measurements Destination: subsection.2.7 Item: Title: Statistical Analysis Destination: subsection.2.8 Item: Title: Results Destination: section.3 Children: Item: Title: First Generation of HDP-Based Antimicrobial Proteins Destination: subsection.3.1 Item: Title: Second Generation of HDP-Based Antimicrobial Proteins Destination: subsection.3.2 Item: Title: Physicochemical Characterization of First and Second Generation of Antimicrobials Destination: subsection.3.3 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.A. Info: Title: A Novel Generation of Tailored Antimicrobial Drugs Based on Recombinant Multidomain Proteins Author: Adrià López-Cano, Neus Ferrer-Miralles, Julieta Sánchez, Jose Vicente Carratalá, Xavier Rodriguez Rodriguez, Imma Ratera, Judith Guasch, Oscar Q. Pich, Paula Bierge, Cristina Garcia-de-la-Maria, Jose M. Miro, Elena Garcia-Fruitós and Anna Arís Subject: 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 D5L37D3, D5L37D5L37 and D5LAL37D3. 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. 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