Background: Gonorrhoea, caused by Neisseria gonorrhoeae, is a global health concern due to rising antimicrobial resistance and the absence of an effective vaccine. Outer membrane vesicle (OMV) vaccines from Neisseria meningitidis (MeNZB and Bexsero) confer partial cross-protection against N. gonorrhoeae, highlighting OMVs as a promising vaccine platform. While OMV protein antigens have been widely studied, the role of lipooligosaccharide (LOS), an abundant and highly variable OMV surface component, remains relatively underexplored. LOS diversity is driven by phase variation of four glycosyltransferase genes (lgtA, lgtC, lgtD and lgtG), but its variation across isolates has not been systematically characterised.
Aim/Methods: A flow cytometry-based assay was developed to profile LOS phenotypes in N. gonorrhoeae. LOS variants were identified using four probes: monoclonal antibodies 3F11, L1 and 2C7, and lectin soybean agglutinin (SBA), detecting lgtA, lgtC, lgtG and lgtD phenotypes, respectively. Twenty-four genetically diverse N. gonorrhoeaeisolates were analysed, including laboratory and clinical strains from the UK, China and Kenya. For each isolate, a ΔlgtE mutant with truncated LOS was generated as a negative control.
Results: LOS diversity was observed across the 24 N. gonorrhoeae isolates. Three dominant phenotypes were identified reflecting: (i) lgtAONlgtCOFFlgtDONlgtGOFF, (ii) lgtAONlgtCOFFlgtDONlgtGON, and (iii) lgtAONlgtCONlgtDONlgtGOFF, together accounting for 87.5% (21/24) of isolates. The lgtAON (23/24, 95.8%) and lgtDON (22/24, 91.7%) phase states were highly prevalent, while lgtC (ON: 7/24, 29.2%) and lgtG (ON: 9/24, 37.5%) showed greater variability. LOS phenotypes did not cluster with genetic lineages, consistent with phase variation. Overall, LOS diversity was primarily driven by combinatorial switching of lgtC and lgtG on a relatively conserved lgtAONlgtDON background.
Conclusions: N. gonorrhoeae LOS exhibits structural diversity, characterised by mostly conserved lgtAON and lgtDONstates, and variability in lgtC and lgtG. The widespread expression of lgtA-associated lacto-N-neotetraose has implications for vaccine targeting and design. Future work will investigate how LOS variation influences immune responses and OMV vaccine efficacy, including recognition by serum from Bexsero-immunised individuals, to inform the rational design of a N. gonorrhoeae OMV vaccine.