A novel sialylation site onNeisseria gonorrhoeaelipooligosaccharide links heptose II lactose expression with pathogenicity

Abstract

22Sialylation of lacto-N-neotetraose (LNnT) extending from heptose I (HepI) of 23 gonococcal lipooligosaccharide (LOS) contributes to pathogenesis. Previously, 24 gonococcal LOS sialyltransterase (Lst) was shown to sialylate LOS in Triton X-100 25 extracts of strain 15253, which expresses lactose from both HepI and HepII, the minimal 26 structure required for mAb 2C7 binding. Ongoing work has shown that growth of 15253 27 in cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac)-containing media 28 enables binding to CD33/Siglec-3, a cell surface receptor that binds sialic acid, 29 suggesting that lactose termini on LOS of intact gonococci can be sialylated. Neu5Ac 30 was detected on LOSs of strains 15253 and a MS11 mutant with only lactose from HepI 31 and HepII by mass spectrometry; deleting HepII lactose rendered Neu5Ac undetectable. 32Resistance of HepII lactose Neu5Ac to desialylation by α2-3-specific neuraminidase 33 suggested an α2-6-linkage. Although not associated with increased factor H binding, 34 HepII lactose sialylation inhibited complement C3 deposition on gonococci. 15253 35 mutants that lacked Lst or HepII lactose were significantly attenuated in mice, 36 confirming the importance of HepII Neu5Ac in virulence. All 75 minimally passaged 37 clinical isolates from Nanjing, China, expressed HepII lactose, evidenced by reactivity 38 with mAb 2C7; mAb 2C7 was bactericidal against the first 62 (of 75) isolates that had 39 been collected sequentially and were sialylated before testing. mAb 2C7 effectively 40 attenuated 15253 vaginal colonization in mice. In conclusion, this novel sialylation site 41 could explain the ubiquity of gonococcal HepII lactose in vivo. Our findings reiterate the 42 candidacy of the 2C7 epitope as a vaccine antigen and mAb 2C7 as an 43 immunotherapeutic antibody. 44 79 that lactose expressed by N. gonorrhoeae LOS could also be sialylated. This study 80 describes sialylation of gonococcal LOS lactose termini and elucidates its function in 81 complement evasion and virulence. 82 83 84 5 Results 85 Neu5Ac 'caps' N. gonorrhoeae HepII lactose. LOS glycan extensions from 86 HepI and HepII for the strains used in this study are shown in Fig. 1. The ability of 87 strains 15253 and MS11 2-Hex/G+, which express lactose extending from HepI and 88 HepII, and their respective isogenic mutants, 15253/G-and MS11 2-Hex/G-, which 89 express only lactose only from HepI, to add Neu5Ac to LOS was determined by SDS-90 PAGE. MS11 4-Hex/G-(expresses LNnT LOS from HepI) was used as a positive 91 control for sialylation. An lst deletion mutant of 15253 (15253 Δlst) that lacks the ability 92to sialylate its LOS was also tested to address whether the previously described Lst 93 enzyme is also responsible for sialylation of lactose. 94 Bacteria were grown in media alone, or media supplemented with 100 µg/ml 95 CMP-Neu5Ac for maximal LOS sialylation. As shown in Fig. 2, the upper band in the '+' 96 lane of 2-Hex/G+ shows slower mobility compared to the upper band in the 97 corresponding '-' lane. Note...