Almughrbi, Ali M A and UNSPECIFIED
(2025)
Investigation of NGO1152 and NGO0206 as potential vaccine antigens against Neisseria gonorrhoeae.
PhD thesis, University of Nottingham.
Abstract
Gonorrhoea, caused by Neisseria gonorrhoeae, is the second most common bacterial STI worldwide, with the WHO estimating ~82 million new cases in 2020 among individuals aged 15–49 years. Cases in England continue to rise, and surveillance in 2025 reported a sharp increase in ceftriaxone-resistant strains. Growing resistance and the absence of a vaccine complicate disease control. Vaccine development is limited by the scarcity of conserved antigens, lack of human correlates of protection, antigenic variability, and inadequate animal models. Moreover, differences between in vivo and in vitro gene expression complicate antigen evaluation.
ATP-binding cassette (ABC) transporters are a highly conserved protein family in prokaryotes and eukaryotes. Using ATP hydrolysis, they transport substrates such as metal ions and proteins across membranes, acting as toxin exporters or nutrient importers. Their conservation, essential roles, and surface accessibility make them strong vaccine candidates. NGO1152 and NGO0206 are predicted SBPs of distinct ABC transporters in N. gonorrhoeae FA1090. NGO1152 (~30 kDa) is a putative histidine-binding protein with a meningococcal orthologue (NMB1612/NMC1533), while NGO0206 (~41 kDa) is a putative polyamine-binding SBP homologous to NMB0623/NMC0567. Both genes were previously deleted from FA1090, and recombinant His-tagged proteins were expressed, purified, and used to generate rabbit polyclonal antibodies. Although SBPs are typically periplasmic, evidence from N. meningitidis MetQ indicates some may be surface exposed. This research evaluated the vaccine potential of NGO1152 and NGO0206.
Hence, Δngo1152 or Δngo0206 mutant strains were complemented by reintroducing ngo1152 or ngo0206 at the iga-trpB intergenic region under either a constitutive PopaB (pMR32) or an IPTG-inducible promoter (pMR33). PCR confirmed genotypes. Phenotypic analysis by immunoblotting with anti-NGO1152 and anti-NGO0206 antibodies revealed reactive bands at 30 kDa and 46 kDa, respectively, consistent with NGO1152 and NGO0206. IPTG induction restored protein expression in MR331152 and MR330206 to WT-FA1090 levels within 2 h. Unexpectedly, PopaB-driven strains failed to express protein due to upstream transcription defects. In vitro growth analyses showed no significant differences among the WT-FA1090, mutant, and complemented strains, irrespective of IPTG induction.
To evaluate conservation and prevalence, immunoblotting confirmed NGO1152 and NGO0206 expression across 28 clinical isolates. Complementary bioinformatic analysis of 7,327 clinical-isolate genomes from PubMLST found ngo1152 in 100% of isolates (99.97% predicted to encode a functional protein), whereas ngo0206 was present in 87.9% (87.8% predicted functional); despite its lower prevalence, ngo0206 exhibited limited sequence variability. Pairwise alignment showed limited overall similarity between NGO1152 and NGO0206 (246 residues overlap; 24% identity, 36.6% similarity, 32.9% gaps), but conservation of the N-terminal signal peptide, lipobox and short motifs (e.g. YAVPYF/FSDPYF, GFDVDL/GKSGYD), which likely support folding, epitope presentation and reciprocal cross-reactivity of anti-NGO1152 and anti-NGO0206 antibodies. NGO1152 is nearly identical to its N. meningitidis orthologue NMB1612 (98.1% identity), confirming its conserved role as an ABC transporter SBP and highlighting its potential as a cross-species vaccine target. By contrast, NGO0206 is more divergent, retaining N-terminal conservation and suggesting functional specialisation relative to NGO1152 and NMB1612.
Surface exposure of NGO1152 and NGO0206 was confirmed by immuno-dot blot analysis, which showed antibody binding to intact WT-FA1090 and IPTG-induced complemented strains, but not to the corresponding mutants. Subcellular fractionation coupled with immunoblotting detected NGO1152 and NGO0206 in outer membrane (OM), cytoplasmic (C), and cytoplasmic membrane (CM) fractions, but not in the periplasmic (PP) fraction. Both proteins were expressed in IPTG-induced complemented strains at levels comparable to WT-FA109 and were absent in mutant and uninduced strains. Whole-cell ELISA (WC-ELISA) showed strong antibody binding to WT-FA1090 and complemented strains, with significantly reduced binding in mutant and uninduced strains and similar patterns were observed across 7 additional clinical isolates. Finally, serum bactericidal activity (SBA) revealed that antisera against NGO1152 and NGO0206 mediated human complement-dependent killing of WT-FA1090. Antisera against NGO0206 exhibited titres of 1:1024–1:2048, whereas antisera against NGO1152 induced even higher titres, up to 1:8192. In both cases, bactericidal activity was markedly reduced against the corresponding mutant strains. Collectively, these findings demonstrate that NGO1152 and NGO0206 are surface-accessible, conserved, and capable of eliciting human complement-mediated killing of N. gonorrhoeae, strongly supporting their potential as vaccine candidates.
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