Autotransported Serine Protease A of Neisseria meningitidis : an Immunogenic, Surface-Exposed Outer Membrane, and Secreted Protein

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Abstract
Several autotransporter proteins have previously been identified in Neisseria meningitidis. Using molecular features common to most members of the autotransporter family of proteins, we have identified an additional novel ca. 112-kDa autotransporter protein in the meningococcal genomic sequence data. This protein, designated autotransported serine protease A (AspA), has significant N-terminal homology to the secreted serine proteases (subtilases) from several organisms and contains a serine protease catalytic triad. The amino acid sequence of AspA is well-conserved in serogroup A, B, and C meningococci. In Neisseria gonorrhoeae, the AspA homologue appears to be a pseudogene. The gene encoding AspA was cloned and expressed from meningococcal strain MC58 (B15:P1.16b). Anti-AspA antibodies were detected in patients' convalescent-phase sera, suggesting that AspA is expressed in vivo during infection and is immunogenic and cross-reactive. Rabbit polyclonal monospecific anti-AspA serum was used to probe whole-cell proteins from a panel of wild-type meningococcal strains and two AspA mutant strains. Expression of the ca. 112-kDa precursor polypeptide was detected in 12 of 20 wild-type meningococcal strains examined, suggesting that AspA expression is phase variable. Immunogold electron microscopy and cellular fractionation studies showed that the AspA precursor is transported to the outer membrane and remains surface exposed. Western blot experiments confirmed that smaller, ca. 68- or 70-kDa components of AspA (AspA68 and AspA70, respectively) are then secreted into the meningococcal culture supernatant. Site-directed mutagenesis of S426 abolished secretion of both rAspA68 and rAspA70 in Escherichia coli, confirming that AspA is an autocleaved autotransporter protein. In conclusion, we characterized a novel, surface-exposed and secreted, immunogenic, meningococcal autotransporter protein.