title: Modulating endotoxin activity by combinatorial bioengineering of meningococcal lipopolysaccharide.
abstract: Neisseria meningitidis contains a very potent hexa-acylated LPS that is too toxic for therapeutic applications. We used systematic molecular bioengineering of meningococcal LPS through deletion of biosynthetic enzymes in combination with induction of LPS modifying enzymes to yield a variety of novel LPS mutants with changes in both lipid A acylation and phosphorylation. Mass spectrometry was used for detailed compositional determination of the LPS molecular species, and stimulation of immune cells was done to correlate this with endotoxic activity. Removal of phosphethanolamine in lipid A by deletion of lptA slightly reduces activity of hexa-acylated LPS, but this reduction is even more evident in penta-acylated LPS. Surprisingly, expression of PagL deacylase in a penta-acylated lpxL1 mutant increased LPS activity, contradicting the general rule that tetra-acylated LPS is less active than penta-acylated LPS. Further modification included expression of lpxP, an enzyme known to add a secondary 9-hexadecenoic acid to the 2' acyl chain. The LpxP enzyme is temperature-sensitive, enabling control over the ratio of expressed modified hexa- and penta-acylated LPS by simply changing the growth temperature. These LPS derivatives display a broad range of TLR4 activity and differential cytokine induction, which can be exploited for use as vaccine adjuvant or other TLR4-based therapeutics.
authors.affiliation: Institute for Translational Vaccinology (Intravacc), Antonie van Leeuwenhoeklaan 9, 3720 AL Bilthoven, the Netherlands.|Department of Infectious Diseases and Immunology, Utrecht University, 3584 CL, Utrecht, the Netherlands.
authors.forename: Afshin
authors.initials: A
authors.lastname: Zariri
authors.affiliation: Institute for Translational Vaccinology (Intravacc), Antonie van Leeuwenhoeklaan 9, 3720 AL Bilthoven, the Netherlands.
authors.forename: Elder
authors.initials: E
authors.lastname: Pupo
authors.affiliation: Institute for Translational Vaccinology (Intravacc), Antonie van Leeuwenhoeklaan 9, 3720 AL Bilthoven, the Netherlands.
authors.forename: Elly
authors.initials: E
authors.lastname: van Riet
authors.affiliation: Department of Infectious Diseases and Immunology, Utrecht University, 3584 CL, Utrecht, the Netherlands.
authors.forename: Jos P M
authors.initials: JP
authors.lastname: van Putten
authors.affiliation: Institute for Translational Vaccinology (Intravacc), Antonie van Leeuwenhoeklaan 9, 3720 AL Bilthoven, the Netherlands.
authors.forename: Peter
authors.initials: P
authors.lastname: van der Ley
mesh_terms: D000215:Acylation; D005818:Genetic Engineering / Q000379:methods*; D008050:Lipid A / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000378:metabolism*; D015394:Molecular Structure; D009345:Neisseria meningitidis / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation
chemical_list: D008050:Lipid A; D008070:Lipopolysaccharides