Autotransported serine protease A of Neisseria meningitidis: an immunogenic, surface-exposed outer membrane, and secreted protein

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.

FIG. 1.

aspA locus and mutagenesis of aspA. The aspA gene is flanked by two open reading frames in the same orientation as aspA. Correia elements (CE) are present between these open reading frames and the aspA gene. Two uptake sequences (USs), a putative promoter sequence (P) and a ribosome-binding site (R) are indicated upstream of aspA. A putative transcription termination signal (TS) is indicated downstream of aspA. The predicted signal sequence is shaded black. A 10-bp polycytidine tract (C) is present in aspA. The primers used in this study are indicated. AspAFor and AspARev were used to amplify aspA from meningococcal strain SD and MC58 for cloning and sequencing. tr-AspAFor and AspARev were used to amplify a 5′-truncated aspA from meningococcal strain MC58 for cloning into the expression vector pCRT7/NT-TOPO. The primers AspAfl-1 and AspAfl-2 were used to amplify the region indicated (containing a single, naturally occurring KpnI restriction site at position 137 in the aspA gene), which was cloned in pGEM-T Easy. The Ω cassette, amplified by using the primers indicated and cloned in pGEM-T Easy, was inserted into the KpnI restriction site. The complementary primers, AspAS426A-1.2 and AspAS426A-2.0, were used for the site-directed mutagenesis of S426.

FIG. 2.

Alignment of the deduced amino acid sequences of AspA from meningococcal strains Z2491 (Sanger Centre), MC58 (TIGR), and Z4181 (this study). CDS and accession numbers are as follows: Z2491, NMA0478; MC58, NMB1969; and Z4181, AJ311654.

FIG. 3.

Schematic diagram of the AspA protein showing the putative domains and conserved motifs (see the text), based on homology to PrtS from S. marcescens (accession number P09489). SP, signal peptide; JR, junctional region.

FIG. 4.

Coomassie brilliant blue-stained SDS-polyacrylamide gel (left) and Western blot probed with RαAspA (right). Lanes 1 and 3, molecular mass markers; lanes 2 and 4, affinity-purified tr-AspA (∼108 kDa) and the copurifying proteins of ca. 95 and 75 kDa, respectively (the ∼75 kDa is not visible on the SDS gel); lane 5, elute from the host E. coli strain not expressing tr-AspA. The numbers indicate the size in kilodaltons.

FIG. 5.

Western blot of RαAspA probed against whole-cell proteins from E. coli BL21(DE3)pLysS containing pCRT7/NT-TOPO (with no insert; lanes 2 and 3), pTOPOAspA (lanes 4 and 5), and pTOPOAspAS426A (lanes 6 and 7). Lanes 2, 4, and 6 without IPTG and lanes 3, 5, and 7 with IPTG induction. Lane 1, molecular mass markers. (The numbers indicate the size in kilodaltons.) Arrows to the right indicate the ∼112-kDa rAspA precursor protein (together with a ∼75-kDa breakdown product).

FIG. 6.

Western blot of RαAspA probed against concentrated supernatant (secreted) proteins from E. coli BL21(DE3)pLysS containing pCRT7/NT-TOPO (no insert; lanes 1 and 2), pTOPOAspA (lanes 3 and 4), and pTOPOAspAS426A (lanes 5 and 6). Lanes 1, 3, and 5 without IPTG and lanes 2, 4 and 6 with IPTG induction. Lane 7, molecular weight markers. The numbers indicate the size in kilodaltons. Arrows to the right indicate the secreted forms of rAspA (68 and 70 kDa) and the 112-kDa precursor protein.

FIG. 7.

Western blots of RαAspA probed against whole-cell proteins from meningococcal strains Z1035 (a), Z1503 (b), Z5035 (c), Z4696 (d), Z6424 (e), 6414 (f), Z4181 (g), and Z4181AspA (h). The AspA precursor protein (∼112 kDa) is present in the wild-type stains but absent from Z4181AspA.

FIG. 8.

Western blot of RαAspA probed against outer-membrane-enriched (OM), cytoplasmic-membrane-enriched (CM), cytosolic-periplasmic (C/P), and concentrated supernatant (SN) fractions from meningococcal strains MC58 (w) and MC58AspA (m). The AspA precursor protein (∼112 kDa) is only present in the outer-membrane-enriched fraction of meningococcal strain MC58. The secreted form of AspA (∼68 kDa) is present in the concentrated supernatant fraction from MC58 but not MC58AspA.

FIG. 9.

Electron micrographs of meningococcal cells (A, strain MC58; B, strain MC58AspA) after incubation in RαAspA and labeling with anti-rabbit immunoglobulin G conjugated to 5-nm gold particles (magnification, ×80,000).

FIG. 10.

Western blots of denatured, affinity-purified tr-AspA probed with convalescent-phase serum from patients 2, 3, 5, and 6 (see Table 3). Arrows to the right indicate the position of tr-AspA (or major breakdown products). Sera from patients 1 and 3 (not shown) and patient 5 were unreactive. None of the sera reacted to the control, which consisted of an elute from the host E. coli strain not expressing tr-AspA (not shown). Numbers refer to sizes in kilodaltons.

FIG. 11.

Dot immunoblots of tr-AspA purified under nondenaturing conditions (A) and elutes from the host E. coli strain not expressing tr-AspA (B), probed with convalescent-phase sera from patients 1 to 6 (Table 3).