Neisseria gonorrhoeae (Ngo) is the causative agent of the sexually transmitted infection gonorrhoea and is a major global public health concern. There is an urgent need to focus on the development of novel therapeutics to treat increasing drug resistant Ngo. The disulfide bond (Dsb) formation oxidoreductase enzymes involved in the periplasmic protein folding pathways are considered excellent targets for the development of anti-virulence therapeutics. The periplasmic oxidation pathway consists of the disulfide oxidase DsbA and the disulfide reductase DsbB. The activity of DsbA has been shown to be necessary for pathogenesis phenotypes dependent on the folding of extracytoplasmic proteins such as Type IV pilin. To understand the entire Dsb-dependent protein network, bioinformatics was used to create a protein interaction dataset which was mined for new virulence-associated phenotypes.
Methods: DeepTMHMM v1.0.24 and SignalP v6.0 were used to predict membrane topology and subcellular localisation of the proteome of Ngo strains FA1090 and FA19. Proteins with ≤1 cysteine residues were excluded and AlphaFold v2.3.2 was then used to predict disulfide connectivity. Strain FA190 and FA19 were chosen to examine the roles of the oxidoreductases as they differ in the presence of the gonococcal genetic island which encodes a Type IV secretion system. Mutants dsbA1, dsbA2, dsbA1/A3, dsbB, and dsbB/A1/A3 were constructed.
Results: Strain FA1090 and FA19 contained 113 extracytoplasmic proteins with predicted disulfide bonds. Multiple components of the Type IV pili machinery were predicted to be involved, including PilE, PilC1, PilI, PilJ, PilX, PilW, and PilQ. Additional functional categories included cell wall biogenesis, energy production and conversion, and inorganic ion transport and metabolism. Multiple elements of the pathway to repair oxidised cysteine and methionine were identified as a potential novel catalytic pathway requiring disulfide bond exchange. To test this hypothesis, we found that dsbA1, dsbA3, and dsbB mutants had significantly reduced survival to hydrogen peroxide compared to wild type.
Further work will aim to investigate the possible folding partners impaired in a dsbA mutant by tagging and tracking the folding of proteins that came up in our analysis and are known to be involved in resistance to oxidative stress.