title: Genetically manipulated phages with improved pH resistance for oral administration in veterinary medicine.
abstract: Orally administered phages to control zoonotic pathogens face important challenges, mainly related to the hostile conditions found in the gastrointestinal tract (GIT). These include temperature, salinity and primarily pH, which is exceptionally low in certain compartments. Phage survival under these conditions can be jeopardized and undermine treatment. Strategies like encapsulation have been attempted with relative success, but are typically complex and require several optimization steps. Here we report a simple and efficient alternative, consisting in the genetic engineering of phages to display lipids on their surfaces. Escherichia coli phage T7 was used as a model and the E. coli PhoE signal peptide was genetically fused to its major capsid protein (10 A), enabling phospholipid attachment to the phage capsid. The presence of phospholipids on the mutant phages was confirmed by High Performance Thin Layer Chromatography, Dynamic Light Scattering and phospholipase assays. The stability of phages was analysed in simulated GIT conditions, demonstrating improved stability of the mutant phages with survival rates 102-107pfu.mL-1higher than wild-type phages. Our work demonstrates that phage engineering can be a good strategy to improve phage tolerance to GIT conditions, having promising application for oral administration in veterinary medicine.
authors.affiliation: CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors.forename: Franklin L
authors.initials: FL
authors.lastname: Nobrega
authors.affiliation: CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors.forename: Ana Rita
authors.initials: AR
authors.lastname: Costa
authors.affiliation: CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors.forename: José F
authors.initials: JF
authors.lastname: Santos
authors.affiliation: Laboratory of Microbiology, Wageningen University and Research Centre, Stippeneng 4, 6708 WE Wageningen, The Netherlands.
authors.forename: Melvin F
authors.initials: MF
authors.lastname: Siliakus
authors.affiliation: Laboratory of Virology, Department of Plant Sciences, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Jan W M
authors.initials: JW
authors.lastname: van Lent
authors.affiliation: Laboratory of Microbiology, Wageningen University and Research Centre, Stippeneng 4, 6708 WE Wageningen, The Netherlands.
authors.forename: Servé W M
authors.initials: SW
authors.lastname: Kengen
authors.affiliation: CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Azeredo
authors.affiliation: CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors.forename: Leon D
authors.initials: LD
authors.lastname: Kluskens
mesh_terms: D000284:Administration, Oral; D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002855:Chromatography, Thin Layer; D024882:Drug Resistance, Viral; D000067493:Dynamic Light Scattering; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D041981:Gastrointestinal Tract / Q000821:virology; D005818:Genetic Engineering; D006863:Hydrogen-Ion Concentration; D046529:Microscopy, Electron, Transmission; D010743:Phospholipids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D018272:Porins / Q000737:chemistry / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D013696:Temperature; D014730:Veterinary Medicine
chemical_list: D036022:Capsid Proteins; D029968:Escherichia coli Proteins; D010743:Phospholipids; D018272:Porins; D021382:Protein Sorting Signals; C082922:PhoE protein, E coli