title: Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics.
abstract: Neisseria gonorrhoeae deploys a novel immune evasion strategy wherein the lacto-N-neotetraose (LNnT) structure of lipooligosaccharide (LOS) is capped by the bacterial sialyltransferase, using host cytidine-5'-monophosphate (CMP)-activated forms of the nine-carbon nonulosonate (NulO) sugar N-acetyl-neuraminic acid (Neu5Ac), a sialic acid (Sia) abundant in humans. This allows evasion of complement-mediated killing by recruiting factor H (FH), an inhibitor of the alternative complement pathway, and by limiting classical pathway activation ("serum-resistance"). We utilized CMP salts of six additional natural or synthetic NulOs, Neu5Gc, Neu5Gc8Me, Neu5Ac9Ac, Neu5Ac9Az, legionaminic acid (Leg5Ac7Ac) and pseudaminic acid (Pse5Ac7Ac), to define structural requirements of Sia-mediated serum-resistance. While all NulOs except Pse5Ac7Ac were incorporated into the LNnT-LOS, only Neu5Gc incorporation yielded high-level serum-resistance and FH binding that was comparable to Neu5Ac, whereas Neu5Ac9Az and Leg5Ac7Ac incorporation left bacteria fully serum-sensitive and did not enhance FH binding. Neu5Ac9Ac and Neu5Gc8Me rendered bacteria resistant only to low serum concentrations. While serum-resistance mediated by Neu5Ac was associated with classical pathway inhibition (decreased IgG binding and C4 deposition), Leg5Ac7Ac and Neu5Ac9Az incorporation did not inhibit the classical pathway. Remarkably, CMP-Neu5Ac9Az and CMP-Leg5Ac7Ac each prevented serum-resistance despite a 100-fold molar excess of CMP-Neu5Ac in growth media. The concomitant presence of Leg5Ac7Ac and Neu5Ac on LOS resulted in uninhibited classical pathway activation. Surprisingly, despite near-maximal FH binding in this instance, the alternative pathway was not regulated and factor Bb remained associated with bacteria. Intravaginal administration of CMP-Leg5Ac7Ac to BALB/c mice infected with gonorrhea (including a multidrug-resistant isolate) reduced clearance times and infection burden. Bacteria recovered from CMP-Leg5Ac7Ac-treated mice were sensitive to human complement ex vivo, simulating in vitro findings. These data reveal critical roles for the Sia exocyclic side-chain in gonococcal serum-resistance. Such CMP-NulO analogs may provide a novel therapeutic strategy against the global threat of multidrug-resistant gonorrhea.
authors.affiliation: Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
authors.forename: Sunita
authors.initials: S
authors.lastname: Gulati
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council of Canada, Ottawa, Ontario, Canada.
authors.forename: Ian C
authors.initials: IC
authors.lastname: Schoenhofen
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council of Canada, Ottawa, Ontario, Canada.
authors.forename: Dennis M
authors.initials: DM
authors.lastname: Whitfield
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council of Canada, Ottawa, Ontario, Canada.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Cox
authors.affiliation: Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Li
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council of Canada, Ottawa, Ontario, Canada.
authors.forename: Frank
authors.initials: F
authors.lastname: St Michael
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council of Canada, Ottawa, Ontario, Canada.
authors.forename: Evgeny V
authors.initials: EV
authors.lastname: Vinogradov
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council of Canada, Ottawa, Ontario, Canada.
authors.forename: Jacek
authors.initials: J
authors.lastname: Stupak
authors.affiliation: Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
authors.forename: Bo
authors.initials: B
authors.lastname: Zheng
authors.affiliation: National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ohnishi
authors.affiliation: WHO Collaborating Centre for Gonorrhoea and Other STIs, Department of Laboratory Medicine, Microbiology, Örebro University Hospital, Örebro, Sweden.
authors.forename: Magnus
authors.initials: M
authors.lastname: Unemo
authors.affiliation: Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Lewis
authors.affiliation: Biomedical Sciences Graduate Program, Departments of Medicine and Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California, United States of America.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Taylor
authors.affiliation: Biomedical Sciences Graduate Program, Departments of Medicine and Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California, United States of America.
authors.forename: Corinna S
authors.initials: CS
authors.lastname: Landig
authors.affiliation: Biomedical Sciences Graduate Program, Departments of Medicine and Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California, United States of America.
authors.forename: Sandra
authors.initials: S
authors.lastname: Diaz
authors.affiliation: Preventive and Behavioral Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
authors.forename: George W
authors.initials: GW
authors.lastname: Reed
authors.affiliation: Biomedical Sciences Graduate Program, Departments of Medicine and Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California, United States of America.
authors.forename: Ajit
authors.initials: A
authors.lastname: Varki
authors.affiliation: Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Rice
authors.affiliation: Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Ram
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003165:Complement System Proteins / Q000276:immunology; D003568:Cytidine Monophosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000031:analogs & derivatives; D004352:Drug Resistance, Microbial / Q000276:immunology*; D018432:Drug Resistance, Multiple / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D006069:Gonorrhea / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000276:immunology / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D009344:Neisseria gonorrhoeae / Q000276:immunology / Q000378:metabolism; D012794:Sialic Acids / Q000494:pharmacology*
chemical_list: D008070:Lipopolysaccharides; D012794:Sialic Acids; C561601:cytidine-5'-monophosphosialic acid; C023023:lipid-linked oligosaccharides; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D003165:Complement System Proteins; D003568:Cytidine Monophosphate