Neisseria gonorrhoeae is an obligate human pathogen of global public health concern. Periplasmic disulfide bond (Dsb) oxidoreductases are facilitators of virulence in gram-negative bacteria and are considered excellent targets for drug development. Neisserial oxidoreductases DsbA1 and DsbA3 obtain reducing power from the inner membrane protein DsbB. This enables the oxidoreductase to donate disulfide bonds to substrates in the periplasm. Disulfide bonds are important for stabilising proteins to protect them from degradation by periplasmic proteases and therefore play important roles during responses to periplasmic stress.
Previous work has shown that DsbA1 and DsbA3 have differing specificities, with DsbA1 being involved in pilin biogenesis while DsbA3 is needed for folding the periplasmic lipid A phosphoethanolamine transferase, EptA. Based upon an in silico analysis of the proteomes of three pathogenic Neisseria closed genomes, a further 140 periplasmic proteins with predicted disulfide bonds have been identified, including the enrichment of a network required for oxidative stress repair in the periplasm. To test this association, dsbA1 and dsbA3 singleton mutants were shown to be more sensitive to oxidative stress than wild type (over 20-fold and 5-fold, respectively). Any mutation combination lacking DsbA1 (dsbA1A3, dsbBA1, and dsbBA1A3) were more sensitive to oxidative stress (over 100-fold reduction) and had significantly reduced intracellular survival in macrophages (<10% survival). DsbA3 did not have a significant role in oxidative stress or survival in macrophages. However, DsbA3 but not DsbA1 was essential for signalling via the two-component system MisRS, suggesting a significant role in periplasmic protein homeostasis.
Together, this work emphasises that the two gonococcal oxidoreductases, DsbA1 and DsbA3, have evolved distinct specificities and are involved in different pathways in the periplasm. DsbA1 is a lipid-linked inner membrane protein involved in pilin biogenesis, resistance to oxidative stress and is necessary for gonococcal survival in macrophages. DsbA3 has a role in general protein homeostasis as it is free in the periplasm. The structures of DsbA1 and DsbA3 have been solved and can be used in further studies examining the potency of DsbA inhibitors in models of gonococcal infection.