title: The role of oxidoreductases in determining the function of the neisserial lipid A phosphoethanolamine transferase required for resistance to polymyxin.
abstract: The decoration of the lipid A headgroups of the lipooligosaccharide (LOS) by the LOS phosphoethanolamine (PEA) transferase (LptA) in Neisseria spp. is central for resistance to polymyxin. The structure of the globular domain of LptA shows that the protein has five disulphide bonds, indicating that it is a potential substrate of the protein oxidation pathway in the bacterial periplasm. When neisserial LptA was expressed in Escherichia coli in the presence of the oxidoreductase, EcDsbA, polymyxin resistance increased 30-fold. LptA decorated one position of the E. coli lipid A headgroups with PEA. In the absence of the EcDsbA, LptA was degraded in E. coli. Neisseria spp. express three oxidoreductases, DsbA1, DsbA2 and DsbA3, each of which appear to donate disulphide bonds to different targets. Inactivation of each oxidoreductase in N. meningitidis enhanced sensitivity to polymyxin with combinatorial mutants displaying an additive increase in sensitivity to polymyxin, indicating that the oxidoreductases were required for multiple pathways leading to polymyxin resistance. Correlates were sought between polymyxin sensitivity, LptA stability or activity and the presence of each of the neisserial oxidoreductases. Only meningococcal mutants lacking DsbA3 had a measurable decrease in the amount of PEA decoration on lipid A headgroups implying that LptA stability was supported by the presence of DsbA3 but did not require DsbA1/2 even though these oxidoreductases could oxidise the protein. This is the first indication that DsbA3 acts as an oxidoreductase in vivo and that multiple oxidoreductases may be involved in oxidising the one target in N. meningitidis. In conclusion, LptA is stabilised by disulphide bonds within the protein. This effect was more pronounced when neisserial LptA was expressed in E. coli than in N. meningitidis and may reflect that other factors in the neisserial periplasm have a role in LptA stability.
authors.affiliation: School of Pathology and Laboratory Medicine, and The Marshall Center for Infectious Diseases, Research and Training, University of Western Australia, Perth, Western Australia, Australia.
authors.forename: Susannah
authors.initials: S
authors.lastname: Piek
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States of America.
authors.forename: Zhirui
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States of America.
authors.forename: Jhuma
authors.initials: J
authors.lastname: Ganguly
authors.affiliation: School of Pathology and Laboratory Medicine, and The Marshall Center for Infectious Diseases, Research and Training, University of Western Australia, Perth, Western Australia, Australia.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Lakey
authors.affiliation: School of Pathology and Laboratory Medicine, and The Marshall Center for Infectious Diseases, Research and Training, University of Western Australia, Perth, Western Australia, Australia.
authors.forename: Stephanie N
authors.initials: SN
authors.lastname: Bartley
authors.affiliation: School of Pathology and Laboratory Medicine, and The Marshall Center for Infectious Diseases, Research and Training, University of Western Australia, Perth, Western Australia, Australia.
authors.forename: Shakeel
authors.initials: S
authors.lastname: Mowlaboccus
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Perth, Western Australia, Australia.
authors.forename: Anandhi
authors.initials: A
authors.lastname: Anandan
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Perth, Western Australia, Australia.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Stubbs
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia; ARC Centre of Excellence for Coherent X-ray Science, Monash University, Melbourne, Victoria, Australia.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Scanlon
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Perth, Western Australia, Australia.
authors.forename: Alice
authors.initials: A
authors.lastname: Vrielink
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States of America.
authors.forename: Parastoo
authors.initials: P
authors.lastname: Azadi
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States of America.
authors.forename: Russell W
authors.initials: RW
authors.lastname: Carlson
authors.affiliation: School of Pathology and Laboratory Medicine, and The Marshall Center for Infectious Diseases, Research and Training, University of Western Australia, Perth, Western Australia, Australia.
authors.forename: Charlene M
authors.initials: CM
authors.lastname: Kahler
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D055162:Biocatalysis / Q000187:drug effects; D004220:Disulfides / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000187:drug effects*; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism; D004982:Ethanolaminephosphotransferase / Q000378:metabolism*; D008050:Lipid A / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D009345:Neisseria meningitidis / Q000187:drug effects / Q000201:enzymology*; D010084:Oxidation-Reduction / Q000187:drug effects; D010088:Oxidoreductases / Q000378:metabolism*; D019897:Periplasm / Q000187:drug effects / Q000378:metabolism; D011113:Polymyxins / Q000494:pharmacology*
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D008050:Lipid A; D008070:Lipopolysaccharides; D011113:Polymyxins; D010088:Oxidoreductases; D004982:Ethanolaminephosphotransferase