Genetic and structural characterization of L11 lipooligosaccharide from Neisseria meningitidis serogroup A strains.
The lipooligosaccharide (LOS) of immunotype L11 is unique within serogroup A meningococci. In order to resolve its molecular structure, we conducted LOS genotyping by PCR analysis of genes responsible for alpha-chain sugar addition (lgtA, -B, -C, -E, -H, and -F) and inner core substituents (lgtG, lpt-3, and lpt-6). For this study, we selected seven strains belonging to subgroup III, a major clonal complex responsible for meningococcal meningitis epidemics in Africa. In addition, we sequenced the homopolymeric tract regions of three phase-variable genes (lgtA, lgtG, and lot-3) to predict gene functionality. The fine structure of the L11 LOS of each strain was determined using composition and glycosyl linkage analyses, NMR, and mass spectrometry. The masses of the dephosphorylated oligosaccharides were consistent with an oligosaccharide composed of two hexoses, one N-acetyl-hexosamine, two heptoses, and one KDO, as proposed previously. The molar composition of LOS showed two glucose residues to be present, in agreement with lgtH sequence prediction. Despite phosphoethanolaminetransferase genes lpt-3 and lpt-6 being present in all seven Neisseria meningitidis strains, phosphoethanolamine (PEtn) was found at both O-3 and O-6 of HepII among the three ST-5 strains, whereas among the four ST-7 strains, only one PEtn was found and located at O-3 of the HepII. The L11 LOS was found to be O-acetylated, as was indicated by the presence of the lot-3 gene being in-frame in all of the seven N. meningitidis strains. To our knowledge, these studies represent the first full genetic and structural characterization of the L11 LOS of N. meningitidis. These investigations also suggest the presence of further regulatory mechanisms affecting LOS structure microheterogeneity in N. meningitidis related to PEtn decoration of the inner core.
Mistretta
Noëlle
N
Sanofi Pasteur, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France.
Seguin
Delphine
D
Thiébaud
Jerôme
J
Vialle
Sandrine
S
Blanc
Frédéric
F
Brossaud
Marina
M
Talaga
Philippe
P
Norheim
Gunnstein
G
Moreau
Monique
M
Rokbi
Bachra
B
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002240:Carbohydrate Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008585:Meningitis, Meningococcal / Q000382:microbiology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009005:Monosaccharides / Q000032:analysis; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D038381:Neisseria meningitidis, Serogroup A / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D012703:Serotyping; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008070:Lipopolysaccharides; D009005:Monosaccharides; C023023:lipid-linked oligosaccharides; D016695:Glycosyltransferases; C101829:LgtC protein, Neisseria; C401409:LgtA protein, bacteria; D017351:N-Acetylglucosaminyltransferases