title: Differential expression and transcriptional analysis of the alpha-2,3-sialyltransferase gene in pathogenic Neisseria spp.
abstract: Alpha-2,3-sialyltransferase (Lst) is expressed on the outer membrane of Neisseria gonorrhoeae and Neisseria meningitidis and sialylates surface lipooligosaccharide (LOS), facilitating resistance to complement-mediated killing. The enzyme is constitutively expressed from a single gene (lst) and does not undergo antigenic or phase variation. We observed that Triton X-100 extracts of N. gonorrhoeae strain F62 contain about fivefold more sialyltransferase (Stase) activity than extracts of N. meningitidis strain MC58 [symbol: see text]3 a serogroup B acapsulate mutant. We confirmed and expanded upon this observation by showing that extracts of 16 random N. gonorrhoeae isolates contain various amounts of Stase activity, but, on average, 2.2-fold-more Stase activity than extracts of 16 N. meningitidis clinical isolates, representing several serogroups and nongroupable strains. Northern and real-time reverse transcription-PCR analysis of lst transcript levels in N. gonorrhoeae and N. meningitidis revealed that N. gonorrhoeae strains express more lst transcript than N. meningitidis strains. Although transcript levels correlate with average Stase activity observed in the two species, there was not a direct correlation between lst transcript levels and Stase activity among individual isolates of each species. Comparison of lst upstream (5'lst) regions of N. gonorrhoeae and N. meningitidis revealed striking sequence differences characteristic of the two pathogens. N. gonorrhoeae 5'lst regions possess 30-bp and 13-bp elements present as single elements or as tandem repeats that exist only as single elements in the 5'lst regions of N. meningitidis isolates. In addition, the 5'lst regions of N. meningitidis strains have 105-bp transposon-like Correia elements which are absent in N. gonorrhoeae. Chromosomal N. gonorrhoeae 5'lst::lacZ translational fusions expressed 4.75 +/- 0.09-fold (n = 4) higher beta-galactosidase (beta-gal) activity than N. meningitidis 5'lst::lacZ fusions in a host-independent manner, indicating differential expression is governed at least in part by sequence variations in the 5'lst regions. Reporter fusion assays and promoter-mapping analysis revealed that N. gonorrhoeae and N. meningitidis use different promoters with different strengths to transcribe lst. In N. gonorrhoeae, a strong sigma 70 promoter 80 bp upstream of the translational start site is used to transcribe lst, whereas this promoter is inactive in N. meningitidis. In N. meningitidis, a weak sigma 70 promoter at the 3' terminus of a 105-bp Correia repeat-enclosed element 99 bp upstream of the translational start site is used to transcribe lst. We conclude that differential Stase expression between N. gonorrhoeae and N. meningitidis is due at least in part to differential lst gene transcription.
authors.affiliation: Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA.
authors.forename: Mathanraj
authors.initials: M
authors.lastname: Packiam
authors.forename: Dawn M
authors.initials: DM
authors.lastname: Shell
authors.forename: Shi V
authors.initials: SV
authors.lastname: Liu
authors.forename: Yao-Bin
authors.initials: YB
authors.lastname: Liu
authors.forename: David J
authors.initials: DJ
authors.lastname: McGee
authors.forename: Ranjana
authors.initials: R
authors.lastname: Srivastava
authors.forename: Samar
authors.initials: S
authors.lastname: Seal
authors.forename: Richard F
authors.initials: RF
authors.lastname: Rest
mesh_terms: D001483:Base Sequence; D015152:Blotting, Northern; D008969:Molecular Sequence Data; D009344:Neisseria gonorrhoeae / Q000201:enzymology* / Q000235:genetics; D009345:Neisseria meningitidis / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D012799:Sialyltransferases / Q000235:genetics*; D014158:Transcription, Genetic; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
chemical_list: D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase