Bacteriophages inject DNA into giant unilamellar vesicles mimicking Gram-negative lipopolysaccharide outer membranes

ABSTRACT Giant unilamellar vesicles (GUVs) are a versatile platform to study cell membrane functions. We have constructed phospholipid GUVs presenting lipopolysaccharide on their external surface (LPS-GUVs) to mimic the outer membrane (OM) of Gram-negative bacteria. GUVs allow for adjusting a defined OM composition, unlike the dynamic changes of LPS structures typically observed in vivo. The OM is a major control point for the viral genome transfer from bacteriophages into bacterial hosts. We found that siphovirus 9NA specifically binds to the surface of GUVs when presenting its Salmonella Typhimurium LPS phage receptor. Using LPS-GUVs filled with DNA-sensitive dyes we show that after surface fixation, the bacteriophage particle opens and injects its DNA into the GUV lumen. No OM proteins were included in the LPS-GUV membrane, emphasizing that the presence of the LPS membrane glycolipid assembly alone is sufficient to trigger the start of bacteriophage genome transfer. LPS-GUVs thus open a sustainable route to systematic studies of viral infection mechanisms at the host envelope and provide a cell-free platform to study surfaces of pathogenic bacteria. This is an important prerequisite for developing effective antimicrobial therapies based on bacteriophages that target Gram-negative pathogens. Competing Interest Statement The authors have declared no competing interest. ABBREVIATIONS - GUV - giant unilamellar vesicles - LPS - lipopolysaccharide - OM - outer membrane - PL - phospholipid.