Three homologues, including two membrane-bound proteins, of the disulfide oxidoreductase DsbA in Neisseria meningitidis -: Effects on bacterial growth and biogenesis of functional type IV pili

Many proteins, especially membrane and exported proteins, are stabilized by intramolecular disulfide bridges between cysteine residues without which they fail to attain their native functional conformation. The formation of these bonds is catalyzed in Gram-negative bacteria by enzymes of the Dsb system. Thus, the activity of DsbA has been shown to be necessary for many phenotypes dependent on exported proteins, including adhesion, invasion, and intracellular survival of various pathogens. The Dsb system in Neisseria meningitidis, the causative agent of cerebrospinal meningitis, has not, however, been studied. In a previous work where genes specific to N. meningitidis and not present in the other pathogenic Neisseria were isolated, a meningococcus-specific dsbA gene was brought to light (Tinsley, C. R., and Nassif, X. ( 1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11109 - 11114). Inactivation of this gene, however, did not result in deficits in the phenotypes commonly associated with DsbA. A search of available genome data revealed that the meningococcus contains three dsbA genes encoding proteins with different predicted subcellular locations, i.e. a soluble periplasmic enzyme and two membrane-bound lipoproteins. Cell fractionation experiments confirmed the localization in the inner membrane of the latter two, which include the previously identified meningococcus-specific enzyme. Mutational analysis demonstrated that the deletion of any single enzyme was compensated by the action of the remaining two on bacterial growth, whereas the triple mutant was unable to grow at 37 C. Remarkably, however, the combined absence of the two membrane-bound enzymes led to a phenotype of sensitivity to reducing agents and loss of functionality of the pili. Although in many species a single periplasmic DsbA is sufficient for the correct folding of various proteins, in the meningococcus a membrane-associated DsbA is required for a wild type DsbA+ phenotype even in the presence of a functional periplasmic DsbA.