Characterization of MspA, an Immunogenic Autotransporter Protein That Mediates Adhesion to Epithelial and Endothelial Cells in Neisseria men
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感染与免疫杂志 2006年第5期
Molecular Biology and Immunology Group, Institute of Infections, Immunity and Inflammation, School of Molecular Medical Sciences, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom
ABSTRACT
A novel putative autotransporter protein (NMB1998) was identified in the available genomic sequence of meningococcal strain MC58 (ET-5; ST-32). The mspA gene is absent from the genomic sequences of meningococcal strain Z2491 (ET-IV; ST-4) and the gonococcal strain FA1090. An orthologue is present in the meningococcal strain FAM18 (ET-37; ST-11), but the sequence contains a premature stop codon, suggesting that the protein may not be expressed in this strain. MspA is predicted to be a 157-kDa protein with low cysteine content, and it exhibits 36 and 33% identity to the meningococcal autotransporter proteins immunoglobulin A1 (IgA1) protease and App, respectively. Search of the Pfam database predicts the presence of IgA1 protease and autotransporter -barrel domains. MspA was cloned, and a recombinant protein of the expected size was expressed and after being affinity purified was used to raise rabbit polyclonal monospecific antiserum. Immunoblot studies showed that ca. 125- and 95-kDa fragments of MspA are secreted in meningococcal strain MC58, which are absent from the isogenic mutant. Secretion of MspA was shown to be modified in an AspA isogenic mutant. A strain survey showed that MspA is expressed by all ST-32 and ST-41/44 (lineage 3) strains, but none of the ST-8 (A4) strains examined. Sera from patients convalescing from meningococcal disease were shown to contain MspA-specific antibodies. In bactericidal assays, anti-MspA serum was shown to kill the homologous strain (MC58) and another ST-32 strain. Escherichia coli-expressing recombinant MspA was shown to adhere to both human bronchial epithelial cells and brain microvascular endothelial cells.
INTRODUCTION
Neisseria meningitidis is found in the human nasopharynx in about 10% of the population and is normally a harmless commensal. Colonization of the nasopharynx is a complex and incompletely understood process, which involves long-range attachment to host epithelial cells via type IV pili, remodeling of the meningococcal outer membrane, and interactions between several bacterial adhesins, including Opa and OpcA, and various host cell molecules. Occasionally, in susceptible individuals, the bacterium gains access to the bloodstream and may cause septicemia, meningitis, or localized infection at other sites. The majority of meningococcal infections are caused by five serogroups: A, B, C, Y, and W135. The serogroup C polysaccharide-protein conjugate vaccine has virtually eliminated childhood disease due to this serogroup in the United Kingdom (24); a quadrivalent conjugate vaccine against serogroup A, C, Y, and W135 strains (MCV-4; Menactra) has recently been licensed in the United States. In contrast, the capsular polysaccharide of the serogroup B meningococcus, which is now the most common cause of meningococcal disease in the United Kingdom and many other developed countries, is poorly immunogenic in humans (19) and consequently is unlikely to provide the basis for a protective vaccine.
As an alternative to the serogroup B polysaccharide capsule, outer membrane proteins (OMPs) have been widely investigated for their vaccine potential, either as undefined complex mixtures, as in outer membrane vesicle (OMV) preparations, or as highly characterized individual proteins (19, 27, 41). Clinical trials using OMV-based vaccines, which generate bactericidal responses predominantly against the hypervariable subtyping antigen PorA, have failed to demonstrate sufficient efficacy (particularly in children of <4 years of age, the age group at highest risk of serogroup B disease) to recommend their widespread use. OMV-based vaccines may, however, have a role in curtailing long-term outbreaks caused by a single serosubtype of serogroup B meningococci, as in New Zealand (19). A hexavalent recombinant PorA vaccine was developed to provide protection against the six commonest PorA types, but in a phase II clinical trial the bactericidal response elicited was either moderate or low for four of the six PorA types (9). Furthermore, a hexavalent PorA vaccine would, at best, provide protection against less than half of the endemic serogroup B strains in some countries (34).
Other OMPs which are under investigation as possible vaccine antigens include the transferrin-binding proteins A and B (40) and neisserial surface protein A (NspA) (17, 22). The latter protein is characterized by high sequence conservation and low abundance in the outer membrane. Although NspA elicits a bactericidal response that confers passive protection in animal models of serogroup B bacteremia, the protein does not appear to be accessible on the surface of some encapsulated meningococcal strains (25).
More recently, the availability of the meningococcal genome sequences has led to the discovery of a number of previously unrecognized OMP antigens, which are promising vaccine candidates. These include GNA33, a mimetic of a surface-exposed loop of PorA (18); two lipoproteins, GNA2132 (38) and 1870 (23, 39); and the autotransporter proteins NadA (4, 6) and NhhA (29). We and others have previously identified and characterized three additional meningococcal autotransporter proteins which are possible vaccine candidates, namely AutA (2), App (1, 31, 36), and autotransported serine protease A (AspA; also designated NalP) (35, 37). From a functional perspective, App has recently been shown to mediate adhesion to epithelial cells and may be important in nasopharyngeal colonization (31). In this report, we describe the molecular, immunological, and functional characterization of another meningococcal autotransporter protein, which shares homology to immunoglobulin A1 (IgA1) protease and App, here designated MspA (for meningococcal serine protease A). We show that MspA is expressed and secreted by ST-32 and ST-41/44 (lineage 3) strains and is an immunogenic protein with adhesive properties.
MATERIALS AND METHODS
Sequence analysis. The predicted amino acid sequence of MspA (NMB1998) was used to perform searches of nonredundant protein databases (using the BLAST program available at http://www.ncbi.nlm.nih.gov/BLAST/), the genome sequence of Neisseria gonorrhoeae strain FA1090 (University of Oklahoma; http://www.genome.ou.edu/gono/html), and the genome sequence of Neisseria lactamica (http://www.sanger.ac.uk/projects/N_lactamica/). The DNAMAN programs (Lynnon Biosoft) were used to analyze putative peptides and their encoding DNA sequences. The promoter prediction by neural network program (http://www.fruitfly.org/seq_tools/promoter.html) and GeneMark (http://opal.biology.gatech.edu/GeneMark/) were used to examine the region upstream of the putative mspA gene for a promoter sequence and ribosome-binding site. The SignalP (http://www.cbs.dtu.dk/services//SignalP/) program was used to predict the presence of a signal sequence, and the Prosite program (http://www.expasy.ch/prosite/) was used to search for conserved amino acid motifs.
Bacterial strains, growth conditions, and media. Escherichia coli strains JM109 (Promega), XL10GOLD (Stratagene), TOP10F', and BL21(DE3)pLysS (Invitrogen) were grown at 37°C in Luria-Bertani (LB) broth or on LB agar supplemented, where appropriate, with ampicillin (50 μg ml–1), chloramphenicol (34 μg ml–1), or kanamycin (25 μg ml–1). N. meningitidis isolates were grown at 37°C on chocolate agar or on Mueller-Hinton agar supplemented with Vitox at the concentration suggested by the manufacturer (Oxoid) in an atmosphere of 5% CO2 or in Mueller-Hinton broth supplemented with Vitox). Where appropriate, streptomycin (50 to 100 μg ml–1) and spectinomycin (50 to 100 μg ml–1) were added to the Mueller-Hinton agar supplemented with Vitox or Mueller-Hinton broth supplemented with Vitox for selection of meningococcal mutants. All meningococcal strains were clinical isolates belonging to different serogroups and types and included representative isolates of recognized hypervirulent lineages. These were fully characterized by multilocus enzyme sequence typing (21).
Patient sera. Serum samples were obtained from patients who were convalescing from invasive meningococcal infection for use in immunoblots as previously described (35). In this study, sera from six convalescent-phase patients (infected with serogroup B strains) were used. Sera were used at a dilution of 1:50.
PCR amplification, cloning, and expression of MspA. Genomic DNA was isolated from meningococcal strain MC58 using the method described by Chen and Kuo (5). Plasmid DNA was isolated using the Qiaprep spin miniprep kit (QIAGEN). The mspA gene was amplified by PCR from the first ATG initiation codon using primers MspAFor (5'-CGCGGATCCATGCGCTTCACACACACCAC-3') and MspARev (5'-CGCGTCGACTTACCAGTTGTAGCCTATTTTGATTCC-3'), as shown in Fig. 1. Taq polymerase used in PCR was purchased from Boehringer Mannheim.
Plasmid pTOPOMspA was constructed by ligating gel-purified, undigested mspA PCR product to pCRT7/NT-TOPO (Invitrogen) according to the manufacturer's instructions. pTOPOMspA was used to transform E. coli strain TOP10F' by heat shock, and plasmid preparations from the resultant transformants were analyzed by restriction enzyme digestion to ascertain the orientation of mspA within the constructs. A construct in which mspA was appropriately orientated for translation was chosen and used to transform E. coli strain BL21(DE3)pLysS by heat shock according to the manufacturer's instructions. Plasmid pQEMspA was constructed by ligation of the BamHI/SalI-digested mspA DNA fragment from pTOPOMspA to the expression vector pQE30 (QIAGEN) cut with the same enzymes. The resulting plasmid was transformed into E. coli JM109 (Promega), according to the manufacturer's recommendations.
Construction of the MspA mutant. A 2,034-bp region of DNA, encompassing ca. 1 kb of upstream DNA and the first ca. 1 kb of mspA, was amplified by PCR from meningococcal strain MC58 using the primers MspAfl-1 (5'-GCGTCGACCCTTCCGAACCC-3') and MspAfl-2 (5'-GCCGGCATCTTCAGACGGCATG-3'), the latter containing a complete neisserial uptake sequence (Fig. 1). This DNA fragment was cloned into pCRT7/NT-TOPO (Invitrogen) according to the manufacturer's instructions. The resulting plasmid (pTOPOMspAfl) was used as a template for inverse PCR using primers MspAup (5'-CGCGGTACCGGCACATTATCTCTTTTAGTTGC-3') and MspAdo (5'-CGGGGTACCGGACGAATGAGTAGGTAACAG-3'), and the resulting product was digested overnight at 25°C with KpnI before being self ligated. The resulting plasmid (pTOPOMspAfl) was digested with KpnI and ligated to the 2-kb element of plasmid pHP45 (30), excised with the same enzyme. The resulting plasmid (pTOPOMspAfl) was used to mutate N. meningitidis strain MC58 by natural transformation and allelic exchange as previously described (1, 35). The mutation was confirmed by PCR.
Purification of the histidine-tagged recombinant MspA. E. coli strain JM109 containing pQEMspA was grown overnight in 10 ml of LB medium containing ampicillin. One milliliter of the culture was added to 50 ml of fresh LB broth and allowed to grow to an optical density at 600 nm of 0.5 to 0.6 before the addition of isopropyl--D-thiogalactopyranoside (IPTG) to 1 mM. After 3 h, cells were harvested and the recombinant protein was purified under denaturing conditions as previously described (2). Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, as previously described (20).
Rabbit antiserum to purified MspA. Rabbit monospecific polyclonal antibodies to MspA (RMspA) were raised against the denatured purified protein in a New Zealand White female rabbit as previously described (35). Preimmune serum was obtained before immunization. The serum was stored at –20°C. RMspA was used in immunoblots at a dilution of 1:500.
Preparation of meningococcal supernatant proteins. N. meningitidis cells were grown overnight in Dulbecco's modified Eagle's Medium (DMEM; Gibco) at 37°C with shaking. The cells were harvested by centrifugation (11,000 x g for 10 min), and the supernatant was filtered using a 0.2-μm Minisart filter. Supernatant proteins were concentrated by ultrafiltration using a Vivaspin-2 protein concentrator (30,000 molecular weight cutoff; Vivascience) according to the manufacturer's instructions.
Serum bactericidal assays. The bactericidal assay was performed as previously described (1, 3). Briefly, 20 μl of RMspA serum (preadsorbed with MC58MspA and decomplemented by heating to 56°C for 30 min), 10 μl of a bacterial suspension (grown to exponential phase and diluted to ca. 800 CFU per well), and 10 μl of sterile baby rabbit serum (Pel-Freeze) were added sequentially to a sterile 96-well microtiter plate. Plates were covered and incubated on a shaker (150 rpm) at 37°C for 1 h. Ten-microliter aliquots were plated on blood agar plates at time zero and after 1 h of incubation. Serial dilutions were performed to determine the number of CFU in each microtiter well, and the serum bactericidal titers were reported as the reciprocal of the serum dilution yielding 50% killing. An antiserum to meningococcal whole-cell proteins and the preimmune serum corresponding to RMspA were used as positive and negative controls, respectively. Additional control wells included a complement control (to determine that organisms were viable in complement in the absence of antibody), an inactive complement control (complement heated to 56°C for 30 min), and an antibody control (to determine that organisms were viable in antibody in the absence of complement).
Adhesion assays. Human bronchial epithelial cells (BEAS-2B; ATCC CRL-9609) and human brain microvascular endothelial cells (HBME; a gift from S. Murphy, University of Nottingham, United Kingdom) were grown in DMEM supplemented with 10% fetal calf serum (FCS; Gibco) and penicillin-streptomycin (Sigma) in 5% CO2 at 37°C. The cells were seeded onto glass coverslips in 24-well plates, grown to full confluence, and washed in DMEM supplemented with 2% FCS. E. coli strains expressing either App or MspA or control strains were grown in LB broth as previously described. Five hundred microliters of each of the overnight cultures was added to 10 ml fresh LB broth. After 2 h incubation at 37°C, expression of the recombinant proteins was induced by the addition of IPTG for 30 min. The optical density at 600 nm was determined, and ca. 107 CFU was added to each cell monolayer in 500 μl DMEM supplemented with 2% FCS, followed by incubation for 4 h in 5% CO2 at 37°C. The cell monolayers were then washed three times in phosphate-buffered saline (PBS), fixed with 500 μl of 2% paraformaldehyde for 20 min, and washed again. Monolayers were stored overnight in PBS at 4°C. For immunostaining, the monolayers were incubated in 0.3% bovine serum albumin (BSA; Sigma) in PBS for 1 h, followed by washing as before. One milliliter of 0.1% saponin in 0.3% BSA-PBS was added to the wells for 10 min, and the wells were again washed. The monolayers were then incubated with 1 ml of a 1:500 dilution of polyclonal rabbit IgG anti-E. coli (DakoCytomation) for 1 h. The monolayer was washed four times in PBS before the addition of anti-rabbit IgG conjugated to fluorescein isothiocyanate (Sigma) and 1% Evans blue (DiaMedix) in 0.3% BSA-PBS for 1 h in the dark. The monolayers were finally washed in PBS, and the glass coverslips were mounted on glass slides. Adherent bacteria were counted with a fluorescent microscope (Axiovert S100; Zeiss Axiocam), and the data were analyzed by the Mann-Whitney U test. Experiments were repeated three times.
RESULTS
Sequence analysis of the mspA gene and flanking DNA. In N. meningitidis strain MC58, the mspA gene is 4,293 bp long and has a G+C content of 56.6%. A predicted ribosomal-binding site (nucleotides –14 to –9) and promoter sequence (nucleotides –89 to –40) were identified upstream of the putative initiation codon (Fig. 1). The predicted transcriptional start site was at nucleotide –49. A 9-bp polycytidine tract was present in mspA (nucleotides 2690 to 2698), raising the possibility that expression of MspA was phase variable. The mspA gene is downstream of a gene (gloB; NMB1997) encoding a putative glyoxalase II enzyme (hydroxyacylglutathione hydrolase), which catalyzes the second of two sequential reactions in the conversion of methylglyoxal to D-lactate (33), and upstream of mgtE (NMB1999) encoding a putative magnesium transporter (26). The mspA gene is absent from the genomic sequence of the serogroup A meningococcal strain Z2491, the gonococcal strain FA1090, and the sequenced N. lactamica strain (ST-640). In Z2491, the 5' end of the gloB gene was highly conserved with respect to its orthologue in strain MC58, but there was marked variation at the 3' end immediately following an uptake sequence, suggesting a possible site of recombination. The gloB/mgtE intergenic region in strain Z2491 contained two complete copies of the uptake sequence separated by nine nucleotides, forming an inverted repeat. In strain MC58, the mspA gene was located 16 nucleotides upstream of the first copy of the uptake sequence, which was disrupted by a single nucleotide mutation (A to G) at position nine. The remainder of the downstream intergenic region and the mgtE gene were highly conserved in the two strains. In the sequenced serogroup C meningococcal strain FAM18 (ET-37), the mspA orthologue contained a 180-bp insertion (nucleotides 1900 to 2079) and a single nucleotide mutation (C to T) at nucleotide 3217, which places a premature TAG stop codon in frame.
The mspA gene encodes a putative polypeptide of 1,431 amino acids, with a calculated molecular weight of 157 kDa. A 26-amino-acid signal peptide was predicted, which contains a single cysteine residue (10C). An additional two pairs of cysteine residues were present in the predicted passenger domain; the first pair was separated by ten residues (768C to 779C) and the second pair was separated by nine residues (1056C to 1066C). The predicted passenger domain contained a putative serine protease (chymotrypsin) catalytic triad (100H-135D-241S), an RGD motif (residues 799 to 801), and a glutamine-rich region (residues 989 to 1041). A predicted C-terminal 263-amino-acid -barrel domain, with an extreme C-terminal motif characteristic of an autotransporter protein, was also identified. A search of the Pfam database predicted the presence of IgA1 protease (residues 18 to 842) and an autotransporter (residues 1179 to 1426) domain. The autotransporter intramolecular chaperone domain (PD002475), which is required for folding of the passenger domain, was also predicted (residues 834 to 964) (28). The terminal three amino acids of MspA (YNW) are similar to those of App and Hap (YRW), and in the latter they have been shown to be essential for outer membrane localization and protein stability (15). Overall, MspA was 36 and 33% identical to IgA1 protease and App, respectively. Furthermore, MspA and App possessed an identical putative autocleavage site (870FNTL873 in MspA) (31).
Cloning of the mspA gene and expression of MspA. The mspA gene was initially cloned into the expression vector pCRT7/NT-TOPO, but we were unable to detect expression of a recombinant protein after induction of cells containing this clone with IPTG (not shown). The mspA gene was subsequently subcloned to the expression vector pQE30. After induction with IPTG, cells containing this new plasmid construct expressed a ca. 160-kDa protein, which included the six-histidine tag encoded by the vector (Fig. 2).
Mutagenesis of mspA. We amplified a region of DNA containing ca. 1 kb of DNA both upstream and downstream of the start codon of mspA (Fig. 1). This fragment was cloned into pCRT7/NT-TOPO to yield plasmid pTOPOMspAfl. This construct was used as a substrate for inverse PCR, in which a region of DNA from nucleotides –9 to + 63 with respect to the start codon was deleted and a new unique KpnI site was introduced to generate plasmid pTOPOMspAfl. A cassette encoding resistance to spectinomycin and streptomycin was ligated into the KpnI site, and the resulting construct (pTOPOMspAfl) was used to mutate meningococcal strain MC58 by natural transformation and allelic exchange to yield MC58MspA.
Secretion of MspA in meningococci. To determine whether MspA, analogous to App, is cleaved and secreted, meningococci were grown overnight in DMEM, and the spent culture medium was concentrated by ultrafiltration after removal of cells by centrifugation and filtration. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with RMspA. Strongly immunoreactive ca. 125- and 95-kDa bands were detected in the supernatant from strain MC58 (ET-5; ST-32) but not the isogenic mutant (Fig. 3). These bands were absent from Z2491 (ET-IV; ST-4), which lacks the mspA gene, and Z4181 (ET-37; ST-11) (data not shown). In the AspA/NalP isogenic mutant, an additional ca. 45-kDa immunoreactive protein was detected, which was absent from the MC58 parent strain (Fig. 3). No immunoreactive proteins were detected using the preimmune serum corresponding to RMspA. To control for outer membrane contamination of the supernatant protein preparations, we probed the fractions in immunoblots with a monoclonal antibody to the outer membrane protein PorA. PorA was undetectable in the supernatant protein fractions but was readily detected in outer membrane protein preparations (not shown).
Strain survey for MspA expression. To determine whether MspA is expressed in a range of clinical isolates of meningococci, we used RMspA to probe concentrated culture supernatants from strains from various hypervirulent lineages (Table 1). Overall, MspA was expressed in 18 of 30 meningococcal strains examined. MspA was expressed by all strains belonging to clonal groups ET-5 (ST-32) and lineage 3 (ST-41/44) but was not expressed by any of the strains examined belonging to the A4 clonal group (ST-8). In addition, MspA was expressed by the single isolates of serogroup W135, 29E, and Z examined but not by the serogroup X or Y isolates. MspA was not expressed by the single isolates of N. lactamica and Neisseria polysacchareae that were examined.
MspA is expressed in vivo and is antigenic. To assess whether MspA is expressed in vivo during meningococcal disease and is immunogenic, purified rMspA was probed by immunoblotting with sera from convalescent-phase patients. Convalescent-phase sera from six patients infected with serogroup B strains recognized purified recombinant MspA (rMspA) (typical examples are shown in Fig. 4). Conversely, convalescent-phase serum from a patient infected with a serogroup C strain (not expressing MspA) was unreactive (Fig. 4). Acute-phase sera were unreactive (data not shown).
Antibodies to MspA are bactericidal. The antiserum raised against MspA was examined for its ability to kill meningococci in the presence of complement. The RMspA antiserum killed 50% of the homologous strain (MC58) at a concentration of 1:32 and strain Z4664 (ET-5; ST-32) at a concentration of 1:8. In contrast, the corresponding preimmune serum failed to produce detectable bactericidal activity at a concentration of 1:4. The antiserum also failed to kill the MspA mutant, confirming that the observed bactericidal activity was dependent on the presence of MspA. However, the RMspA antiserum failed to kill strains Z6421 (lineage 3; ST-41/44) and Z4242 (ET-37; ST-11), despite immunoblot confirmation that these strains expressed MspA.
MspA has adhesive properties. Given the homology of MspA to App, which has been demonstrated to mediate adhesion to epithelial cells, we wished to determine whether MspA also has adhesive properties. E. coli strains expressing either MspA or App were shown to adhere to BEAS-2B cells significantly more (P < 0.0001) than control strains (JM109 and XL10GOLD, respectively), which contained the plasmid vector without an insert (Table 2 and Fig. 5). Although App was previously shown not to mediate adhesion to human umbilical vein endothelial cells (HUVECs), we decided to assess adhesion to human brain microvascular endothelial cells. Interestingly, E. coli expressing either App or MspA adhered significantly more (P < 0.0001) than their respective negative control strains (Table 2 and Fig. 5). Of note, E. coli cells expressing MspA did not show an autoagglutination phenotype (not shown).
DISCUSSION
NMB1998 is a novel autotransporter protein detected in silico in the genome of the serogroup B meningococcal strain MC58, due to its homology to App, IgA1 protease, and other known autotransporter proteins (1, 36). Interestingly, the gene encoding NMB1998 is absent from the genome sequences of meningococcal strain Z2491 and the gonococcal strain FA1090, suggesting that it may only be present in a subset of meningococcal clonal lineages. Given the homology of NMB1998 to IgA1 protease and App, which are both chymotrypsin serine proteases, we have designated the protein MspA, for meningococcal serine protease A. Curiously, MspA is located in the serogroup B (MC58) meningococcal genome just four open reading frames downstream of another autotransporter protein, NadA (NMB1994), which is also absent from the serogroup A (Z2491) genome (6). This may be a mere coincidence, however, since the gene encoding NadA contains a low G+C content with respect to the average for the genome, suggesting that the gene was acquired by horizontal transfer from another organism, which does not appear to be the case for MspA. Furthermore, the 5.6-kb region that separates nadA and mspA contains three conserved genes, which are present in both Z2491 and MC58.
We have demonstrated that MspA, which possesses the characteristic three-domain structure of an autotransporter protein, is naturally expressed in N. meningitidis and is cleaved and secreted into the external milieu. The autotransporter protein family is large and diverse and has been the subject of several reviews (10, 12-14). Proteins belonging to this family represent one of the most important groups of secreted proteins in gram-negative bacteria and mediate a variety of interactions between gram-negative bacteria and their environmental niches. Indeed, in most gram-negative pathogens studied so far, autotransporter proteins make a significant contribution to pathogenesis and are responsible for characteristics such as adhesion, toxigenicity, serum resistance, intracellular movement, and proteolysis (12). Interestingly, N. meningitidis, Haemophilus influenzae, and Bordetella pertussis, which all colonize the upper respiratory tract in humans, possess multiple autotransporter proteins, some of which share considerable homology. For example, H. influenzae contains IgA1 protease, Hap, and Hia/Hsf, which are 54, 56, and 53/57% identical to their respective homologues, IgA1 protease, App, and NhhA in N. meningitidis (1, 29). Both Hap and App have been shown to have a role in adhesion of their respective organisms to host cells, and Hap is important in the invasion of host cells (11, 16, 31, 32). In addition, we previously reported the identification and characterization of AspA, an outer membrane protein, and secreted subtilase in N. meningitidis, which shares homology with SphB1 in B. pertussis (7). SphB1 is a surface-anchored maturation protease, which following autocleavage specifically catalyzes the conversion of the filamentous hemagglutinin precursor molecule to mature, secreted filamentous hemagglutinin (7, 8). Recently, AspA was demonstrated to alter the secretion profile of App and, in some strains, IgA1 protease (37). In the presence of AspA/NalP, two forms of App (ca. 100 and 140 kDa, respectively) are secreted, while in the aspA/nalP mutant the lower-molecular-weight form predominates (37; our unpublished data). Consistent with these observations, we have also detected an anti-App-reactive ca. 40-kDa band in concentrated supernatant proteins prepared from the AspA/NalP mutant, which is absent from the parent MC58 strain (our unpublished data). The homology of MspA with App and IgA1 protease suggested that MspA might represent an additional substrate for AspA/NalP. To investigate this possibility, secreted proteins prepared from the AspA/NalP mutant were probed with the MspA-specific antiserum. Analogous to App, a smaller (ca. 45-kDa) anti-MspA-reactive protein was detected, which was absent from the MC58 parent strain. This suggests that MspA is an additional substrate for AspA/NalP.
We have previously shown that App is constitutively expressed by meningococcal strains representing the clonal groups that are responsible for the majority of meningococcal infections worldwide (1). Furthermore, App was shown to be immunogenic and expressed in vivo during natural infection (1). Having shown that MspA is secreted in meningococcal strain MC58, we wished to determine whether MspA is similarly expressed in diverse meningococcal lineages. A strain survey demonstrated that MspA is expressed by ST-32 and ST-41/44, which contain the majority of hypervirulent serogroup B strains, but not by ST-8. Interestingly, isolates from ST-11 gave discrepant results, with some isolates expressing MspA and some not. Of note, in the sequenced ST-11 strain (FAM18), MspA contains a premature stop codon at the 3' end of the gene, which presumably results in the gene not being expressed. Despite the proximity of the encoding genes in the MC58 genome sequence, the strain distribution of MspA is clearly distinct from that of NadA; the latter was expressed by ST-8 but not ST-41/44 strains.
In vivo expression and B-cell immunogenicity of MspA were confirmed by the detection of specific anti-MspA antibodies in sera from patients convalescing from serogroup B invasive meningococcal disease. In view of the immunogenicity of MspA and its homology to App, which generates a bactericidal response, we investigated the bactericidal activity of anti-MspA antibodies. Anti-MspA antibodies were shown to kill the homologous strain MC58 and another meningococcal strain belonging to the same sequence type (ST-5), but strains Z6421 and Z4242 (representing ST-41/44 and ST-11, respectively) were insensitive to killing. The bactericidal response elicited by anti-MspA antibodies suggests that, analogous to App, MspA is also accessible to antibodies on the bacterial surface. The insensitivity of strains belonging to heterogeneous lineages to complement-mediated killing was also noted in respect to anti-App antibodies, and, as suggested for App (1), may result from differences in the processing and surface exposure of MspA. It is possible that antibodies raised against the native form of the protein may result in the binding of surface-exposed conformational epitopes on a wider range of strains.
App was recently shown to mediate in vitro adherence to Chang (human conjunctiva) epithelial cells when expressed on the surface of E. coli, although adhesion to HUVECs could not be demonstrated (31). In addition, purified App was shown to bind to Chang and HepG2 (human liver carcinoma) cells at a high level but not to other epithelial cell lines including HEC-1-B (human endometrium), Hep-2 (human laryngeal epidermoid carcinoma), or 16HBE14o (human bronchial epithelial) cells. In contrast, we found that E. coli expressing either App or MspA adhered to BEAS-2B and HBME cells, suggesting that MspA may also play a role in adhesion to host cells. The adhesion to HBME cells but not HUVECs may be explained by differential expression of eukaryotic surface ligands, as has been suggested for the differential binding of App to epithelial cell lines (31). Work is currently in progress to identify the host ligand(s) for both MspA and App. Once the ligand(s) has been identified, the hypothesis of differential cellular expression can be tested.
In conclusion, we have characterized a novel meningococcal autotransporter protein, which is expressed by the majority of serogroup B strains in vitro, is immunogenic in humans following invasive infection, and is capable of generating a bactericidal response, albeit in a limited range of strains. MspA, like App, may also possess a role in colonization and provide a selective advantage to strains that express both proteins. The proposed role in colonization, however, does not exclude an additional function for the secreted fragment of the protein, which may cleave a host protein, analogous to IgA1 protease. We propose that an effective antibody response to MspA, in addition to App, may interfere with the normal interaction of this protein with host molecules and contribute to the prevention of meningococcal disease.
ACKNOWLEDGMENTS
We thank Dominique Caugant for kindly providing the N. meningitidis isolates representing different hypervirulent lineages, Ray Borrow for providing the baby rabbit complement, and Sean Murphy for providing the HBME cells.
Part of this work was performed during the tenure of a Medical Council Research Fellowship awarded to D.P.J.T. (grant G84/5370).
These authors contributed equally to this work.
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ABSTRACT
A novel putative autotransporter protein (NMB1998) was identified in the available genomic sequence of meningococcal strain MC58 (ET-5; ST-32). The mspA gene is absent from the genomic sequences of meningococcal strain Z2491 (ET-IV; ST-4) and the gonococcal strain FA1090. An orthologue is present in the meningococcal strain FAM18 (ET-37; ST-11), but the sequence contains a premature stop codon, suggesting that the protein may not be expressed in this strain. MspA is predicted to be a 157-kDa protein with low cysteine content, and it exhibits 36 and 33% identity to the meningococcal autotransporter proteins immunoglobulin A1 (IgA1) protease and App, respectively. Search of the Pfam database predicts the presence of IgA1 protease and autotransporter -barrel domains. MspA was cloned, and a recombinant protein of the expected size was expressed and after being affinity purified was used to raise rabbit polyclonal monospecific antiserum. Immunoblot studies showed that ca. 125- and 95-kDa fragments of MspA are secreted in meningococcal strain MC58, which are absent from the isogenic mutant. Secretion of MspA was shown to be modified in an AspA isogenic mutant. A strain survey showed that MspA is expressed by all ST-32 and ST-41/44 (lineage 3) strains, but none of the ST-8 (A4) strains examined. Sera from patients convalescing from meningococcal disease were shown to contain MspA-specific antibodies. In bactericidal assays, anti-MspA serum was shown to kill the homologous strain (MC58) and another ST-32 strain. Escherichia coli-expressing recombinant MspA was shown to adhere to both human bronchial epithelial cells and brain microvascular endothelial cells.
INTRODUCTION
Neisseria meningitidis is found in the human nasopharynx in about 10% of the population and is normally a harmless commensal. Colonization of the nasopharynx is a complex and incompletely understood process, which involves long-range attachment to host epithelial cells via type IV pili, remodeling of the meningococcal outer membrane, and interactions between several bacterial adhesins, including Opa and OpcA, and various host cell molecules. Occasionally, in susceptible individuals, the bacterium gains access to the bloodstream and may cause septicemia, meningitis, or localized infection at other sites. The majority of meningococcal infections are caused by five serogroups: A, B, C, Y, and W135. The serogroup C polysaccharide-protein conjugate vaccine has virtually eliminated childhood disease due to this serogroup in the United Kingdom (24); a quadrivalent conjugate vaccine against serogroup A, C, Y, and W135 strains (MCV-4; Menactra) has recently been licensed in the United States. In contrast, the capsular polysaccharide of the serogroup B meningococcus, which is now the most common cause of meningococcal disease in the United Kingdom and many other developed countries, is poorly immunogenic in humans (19) and consequently is unlikely to provide the basis for a protective vaccine.
As an alternative to the serogroup B polysaccharide capsule, outer membrane proteins (OMPs) have been widely investigated for their vaccine potential, either as undefined complex mixtures, as in outer membrane vesicle (OMV) preparations, or as highly characterized individual proteins (19, 27, 41). Clinical trials using OMV-based vaccines, which generate bactericidal responses predominantly against the hypervariable subtyping antigen PorA, have failed to demonstrate sufficient efficacy (particularly in children of <4 years of age, the age group at highest risk of serogroup B disease) to recommend their widespread use. OMV-based vaccines may, however, have a role in curtailing long-term outbreaks caused by a single serosubtype of serogroup B meningococci, as in New Zealand (19). A hexavalent recombinant PorA vaccine was developed to provide protection against the six commonest PorA types, but in a phase II clinical trial the bactericidal response elicited was either moderate or low for four of the six PorA types (9). Furthermore, a hexavalent PorA vaccine would, at best, provide protection against less than half of the endemic serogroup B strains in some countries (34).
Other OMPs which are under investigation as possible vaccine antigens include the transferrin-binding proteins A and B (40) and neisserial surface protein A (NspA) (17, 22). The latter protein is characterized by high sequence conservation and low abundance in the outer membrane. Although NspA elicits a bactericidal response that confers passive protection in animal models of serogroup B bacteremia, the protein does not appear to be accessible on the surface of some encapsulated meningococcal strains (25).
More recently, the availability of the meningococcal genome sequences has led to the discovery of a number of previously unrecognized OMP antigens, which are promising vaccine candidates. These include GNA33, a mimetic of a surface-exposed loop of PorA (18); two lipoproteins, GNA2132 (38) and 1870 (23, 39); and the autotransporter proteins NadA (4, 6) and NhhA (29). We and others have previously identified and characterized three additional meningococcal autotransporter proteins which are possible vaccine candidates, namely AutA (2), App (1, 31, 36), and autotransported serine protease A (AspA; also designated NalP) (35, 37). From a functional perspective, App has recently been shown to mediate adhesion to epithelial cells and may be important in nasopharyngeal colonization (31). In this report, we describe the molecular, immunological, and functional characterization of another meningococcal autotransporter protein, which shares homology to immunoglobulin A1 (IgA1) protease and App, here designated MspA (for meningococcal serine protease A). We show that MspA is expressed and secreted by ST-32 and ST-41/44 (lineage 3) strains and is an immunogenic protein with adhesive properties.
MATERIALS AND METHODS
Sequence analysis. The predicted amino acid sequence of MspA (NMB1998) was used to perform searches of nonredundant protein databases (using the BLAST program available at http://www.ncbi.nlm.nih.gov/BLAST/), the genome sequence of Neisseria gonorrhoeae strain FA1090 (University of Oklahoma; http://www.genome.ou.edu/gono/html), and the genome sequence of Neisseria lactamica (http://www.sanger.ac.uk/projects/N_lactamica/). The DNAMAN programs (Lynnon Biosoft) were used to analyze putative peptides and their encoding DNA sequences. The promoter prediction by neural network program (http://www.fruitfly.org/seq_tools/promoter.html) and GeneMark (http://opal.biology.gatech.edu/GeneMark/) were used to examine the region upstream of the putative mspA gene for a promoter sequence and ribosome-binding site. The SignalP (http://www.cbs.dtu.dk/services//SignalP/) program was used to predict the presence of a signal sequence, and the Prosite program (http://www.expasy.ch/prosite/) was used to search for conserved amino acid motifs.
Bacterial strains, growth conditions, and media. Escherichia coli strains JM109 (Promega), XL10GOLD (Stratagene), TOP10F', and BL21(DE3)pLysS (Invitrogen) were grown at 37°C in Luria-Bertani (LB) broth or on LB agar supplemented, where appropriate, with ampicillin (50 μg ml–1), chloramphenicol (34 μg ml–1), or kanamycin (25 μg ml–1). N. meningitidis isolates were grown at 37°C on chocolate agar or on Mueller-Hinton agar supplemented with Vitox at the concentration suggested by the manufacturer (Oxoid) in an atmosphere of 5% CO2 or in Mueller-Hinton broth supplemented with Vitox). Where appropriate, streptomycin (50 to 100 μg ml–1) and spectinomycin (50 to 100 μg ml–1) were added to the Mueller-Hinton agar supplemented with Vitox or Mueller-Hinton broth supplemented with Vitox for selection of meningococcal mutants. All meningococcal strains were clinical isolates belonging to different serogroups and types and included representative isolates of recognized hypervirulent lineages. These were fully characterized by multilocus enzyme sequence typing (21).
Patient sera. Serum samples were obtained from patients who were convalescing from invasive meningococcal infection for use in immunoblots as previously described (35). In this study, sera from six convalescent-phase patients (infected with serogroup B strains) were used. Sera were used at a dilution of 1:50.
PCR amplification, cloning, and expression of MspA. Genomic DNA was isolated from meningococcal strain MC58 using the method described by Chen and Kuo (5). Plasmid DNA was isolated using the Qiaprep spin miniprep kit (QIAGEN). The mspA gene was amplified by PCR from the first ATG initiation codon using primers MspAFor (5'-CGCGGATCCATGCGCTTCACACACACCAC-3') and MspARev (5'-CGCGTCGACTTACCAGTTGTAGCCTATTTTGATTCC-3'), as shown in Fig. 1. Taq polymerase used in PCR was purchased from Boehringer Mannheim.
Plasmid pTOPOMspA was constructed by ligating gel-purified, undigested mspA PCR product to pCRT7/NT-TOPO (Invitrogen) according to the manufacturer's instructions. pTOPOMspA was used to transform E. coli strain TOP10F' by heat shock, and plasmid preparations from the resultant transformants were analyzed by restriction enzyme digestion to ascertain the orientation of mspA within the constructs. A construct in which mspA was appropriately orientated for translation was chosen and used to transform E. coli strain BL21(DE3)pLysS by heat shock according to the manufacturer's instructions. Plasmid pQEMspA was constructed by ligation of the BamHI/SalI-digested mspA DNA fragment from pTOPOMspA to the expression vector pQE30 (QIAGEN) cut with the same enzymes. The resulting plasmid was transformed into E. coli JM109 (Promega), according to the manufacturer's recommendations.
Construction of the MspA mutant. A 2,034-bp region of DNA, encompassing ca. 1 kb of upstream DNA and the first ca. 1 kb of mspA, was amplified by PCR from meningococcal strain MC58 using the primers MspAfl-1 (5'-GCGTCGACCCTTCCGAACCC-3') and MspAfl-2 (5'-GCCGGCATCTTCAGACGGCATG-3'), the latter containing a complete neisserial uptake sequence (Fig. 1). This DNA fragment was cloned into pCRT7/NT-TOPO (Invitrogen) according to the manufacturer's instructions. The resulting plasmid (pTOPOMspAfl) was used as a template for inverse PCR using primers MspAup (5'-CGCGGTACCGGCACATTATCTCTTTTAGTTGC-3') and MspAdo (5'-CGGGGTACCGGACGAATGAGTAGGTAACAG-3'), and the resulting product was digested overnight at 25°C with KpnI before being self ligated. The resulting plasmid (pTOPOMspAfl) was digested with KpnI and ligated to the 2-kb element of plasmid pHP45 (30), excised with the same enzyme. The resulting plasmid (pTOPOMspAfl) was used to mutate N. meningitidis strain MC58 by natural transformation and allelic exchange as previously described (1, 35). The mutation was confirmed by PCR.
Purification of the histidine-tagged recombinant MspA. E. coli strain JM109 containing pQEMspA was grown overnight in 10 ml of LB medium containing ampicillin. One milliliter of the culture was added to 50 ml of fresh LB broth and allowed to grow to an optical density at 600 nm of 0.5 to 0.6 before the addition of isopropyl--D-thiogalactopyranoside (IPTG) to 1 mM. After 3 h, cells were harvested and the recombinant protein was purified under denaturing conditions as previously described (2). Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, as previously described (20).
Rabbit antiserum to purified MspA. Rabbit monospecific polyclonal antibodies to MspA (RMspA) were raised against the denatured purified protein in a New Zealand White female rabbit as previously described (35). Preimmune serum was obtained before immunization. The serum was stored at –20°C. RMspA was used in immunoblots at a dilution of 1:500.
Preparation of meningococcal supernatant proteins. N. meningitidis cells were grown overnight in Dulbecco's modified Eagle's Medium (DMEM; Gibco) at 37°C with shaking. The cells were harvested by centrifugation (11,000 x g for 10 min), and the supernatant was filtered using a 0.2-μm Minisart filter. Supernatant proteins were concentrated by ultrafiltration using a Vivaspin-2 protein concentrator (30,000 molecular weight cutoff; Vivascience) according to the manufacturer's instructions.
Serum bactericidal assays. The bactericidal assay was performed as previously described (1, 3). Briefly, 20 μl of RMspA serum (preadsorbed with MC58MspA and decomplemented by heating to 56°C for 30 min), 10 μl of a bacterial suspension (grown to exponential phase and diluted to ca. 800 CFU per well), and 10 μl of sterile baby rabbit serum (Pel-Freeze) were added sequentially to a sterile 96-well microtiter plate. Plates were covered and incubated on a shaker (150 rpm) at 37°C for 1 h. Ten-microliter aliquots were plated on blood agar plates at time zero and after 1 h of incubation. Serial dilutions were performed to determine the number of CFU in each microtiter well, and the serum bactericidal titers were reported as the reciprocal of the serum dilution yielding 50% killing. An antiserum to meningococcal whole-cell proteins and the preimmune serum corresponding to RMspA were used as positive and negative controls, respectively. Additional control wells included a complement control (to determine that organisms were viable in complement in the absence of antibody), an inactive complement control (complement heated to 56°C for 30 min), and an antibody control (to determine that organisms were viable in antibody in the absence of complement).
Adhesion assays. Human bronchial epithelial cells (BEAS-2B; ATCC CRL-9609) and human brain microvascular endothelial cells (HBME; a gift from S. Murphy, University of Nottingham, United Kingdom) were grown in DMEM supplemented with 10% fetal calf serum (FCS; Gibco) and penicillin-streptomycin (Sigma) in 5% CO2 at 37°C. The cells were seeded onto glass coverslips in 24-well plates, grown to full confluence, and washed in DMEM supplemented with 2% FCS. E. coli strains expressing either App or MspA or control strains were grown in LB broth as previously described. Five hundred microliters of each of the overnight cultures was added to 10 ml fresh LB broth. After 2 h incubation at 37°C, expression of the recombinant proteins was induced by the addition of IPTG for 30 min. The optical density at 600 nm was determined, and ca. 107 CFU was added to each cell monolayer in 500 μl DMEM supplemented with 2% FCS, followed by incubation for 4 h in 5% CO2 at 37°C. The cell monolayers were then washed three times in phosphate-buffered saline (PBS), fixed with 500 μl of 2% paraformaldehyde for 20 min, and washed again. Monolayers were stored overnight in PBS at 4°C. For immunostaining, the monolayers were incubated in 0.3% bovine serum albumin (BSA; Sigma) in PBS for 1 h, followed by washing as before. One milliliter of 0.1% saponin in 0.3% BSA-PBS was added to the wells for 10 min, and the wells were again washed. The monolayers were then incubated with 1 ml of a 1:500 dilution of polyclonal rabbit IgG anti-E. coli (DakoCytomation) for 1 h. The monolayer was washed four times in PBS before the addition of anti-rabbit IgG conjugated to fluorescein isothiocyanate (Sigma) and 1% Evans blue (DiaMedix) in 0.3% BSA-PBS for 1 h in the dark. The monolayers were finally washed in PBS, and the glass coverslips were mounted on glass slides. Adherent bacteria were counted with a fluorescent microscope (Axiovert S100; Zeiss Axiocam), and the data were analyzed by the Mann-Whitney U test. Experiments were repeated three times.
RESULTS
Sequence analysis of the mspA gene and flanking DNA. In N. meningitidis strain MC58, the mspA gene is 4,293 bp long and has a G+C content of 56.6%. A predicted ribosomal-binding site (nucleotides –14 to –9) and promoter sequence (nucleotides –89 to –40) were identified upstream of the putative initiation codon (Fig. 1). The predicted transcriptional start site was at nucleotide –49. A 9-bp polycytidine tract was present in mspA (nucleotides 2690 to 2698), raising the possibility that expression of MspA was phase variable. The mspA gene is downstream of a gene (gloB; NMB1997) encoding a putative glyoxalase II enzyme (hydroxyacylglutathione hydrolase), which catalyzes the second of two sequential reactions in the conversion of methylglyoxal to D-lactate (33), and upstream of mgtE (NMB1999) encoding a putative magnesium transporter (26). The mspA gene is absent from the genomic sequence of the serogroup A meningococcal strain Z2491, the gonococcal strain FA1090, and the sequenced N. lactamica strain (ST-640). In Z2491, the 5' end of the gloB gene was highly conserved with respect to its orthologue in strain MC58, but there was marked variation at the 3' end immediately following an uptake sequence, suggesting a possible site of recombination. The gloB/mgtE intergenic region in strain Z2491 contained two complete copies of the uptake sequence separated by nine nucleotides, forming an inverted repeat. In strain MC58, the mspA gene was located 16 nucleotides upstream of the first copy of the uptake sequence, which was disrupted by a single nucleotide mutation (A to G) at position nine. The remainder of the downstream intergenic region and the mgtE gene were highly conserved in the two strains. In the sequenced serogroup C meningococcal strain FAM18 (ET-37), the mspA orthologue contained a 180-bp insertion (nucleotides 1900 to 2079) and a single nucleotide mutation (C to T) at nucleotide 3217, which places a premature TAG stop codon in frame.
The mspA gene encodes a putative polypeptide of 1,431 amino acids, with a calculated molecular weight of 157 kDa. A 26-amino-acid signal peptide was predicted, which contains a single cysteine residue (10C). An additional two pairs of cysteine residues were present in the predicted passenger domain; the first pair was separated by ten residues (768C to 779C) and the second pair was separated by nine residues (1056C to 1066C). The predicted passenger domain contained a putative serine protease (chymotrypsin) catalytic triad (100H-135D-241S), an RGD motif (residues 799 to 801), and a glutamine-rich region (residues 989 to 1041). A predicted C-terminal 263-amino-acid -barrel domain, with an extreme C-terminal motif characteristic of an autotransporter protein, was also identified. A search of the Pfam database predicted the presence of IgA1 protease (residues 18 to 842) and an autotransporter (residues 1179 to 1426) domain. The autotransporter intramolecular chaperone domain (PD002475), which is required for folding of the passenger domain, was also predicted (residues 834 to 964) (28). The terminal three amino acids of MspA (YNW) are similar to those of App and Hap (YRW), and in the latter they have been shown to be essential for outer membrane localization and protein stability (15). Overall, MspA was 36 and 33% identical to IgA1 protease and App, respectively. Furthermore, MspA and App possessed an identical putative autocleavage site (870FNTL873 in MspA) (31).
Cloning of the mspA gene and expression of MspA. The mspA gene was initially cloned into the expression vector pCRT7/NT-TOPO, but we were unable to detect expression of a recombinant protein after induction of cells containing this clone with IPTG (not shown). The mspA gene was subsequently subcloned to the expression vector pQE30. After induction with IPTG, cells containing this new plasmid construct expressed a ca. 160-kDa protein, which included the six-histidine tag encoded by the vector (Fig. 2).
Mutagenesis of mspA. We amplified a region of DNA containing ca. 1 kb of DNA both upstream and downstream of the start codon of mspA (Fig. 1). This fragment was cloned into pCRT7/NT-TOPO to yield plasmid pTOPOMspAfl. This construct was used as a substrate for inverse PCR, in which a region of DNA from nucleotides –9 to + 63 with respect to the start codon was deleted and a new unique KpnI site was introduced to generate plasmid pTOPOMspAfl. A cassette encoding resistance to spectinomycin and streptomycin was ligated into the KpnI site, and the resulting construct (pTOPOMspAfl) was used to mutate meningococcal strain MC58 by natural transformation and allelic exchange to yield MC58MspA.
Secretion of MspA in meningococci. To determine whether MspA, analogous to App, is cleaved and secreted, meningococci were grown overnight in DMEM, and the spent culture medium was concentrated by ultrafiltration after removal of cells by centrifugation and filtration. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with RMspA. Strongly immunoreactive ca. 125- and 95-kDa bands were detected in the supernatant from strain MC58 (ET-5; ST-32) but not the isogenic mutant (Fig. 3). These bands were absent from Z2491 (ET-IV; ST-4), which lacks the mspA gene, and Z4181 (ET-37; ST-11) (data not shown). In the AspA/NalP isogenic mutant, an additional ca. 45-kDa immunoreactive protein was detected, which was absent from the MC58 parent strain (Fig. 3). No immunoreactive proteins were detected using the preimmune serum corresponding to RMspA. To control for outer membrane contamination of the supernatant protein preparations, we probed the fractions in immunoblots with a monoclonal antibody to the outer membrane protein PorA. PorA was undetectable in the supernatant protein fractions but was readily detected in outer membrane protein preparations (not shown).
Strain survey for MspA expression. To determine whether MspA is expressed in a range of clinical isolates of meningococci, we used RMspA to probe concentrated culture supernatants from strains from various hypervirulent lineages (Table 1). Overall, MspA was expressed in 18 of 30 meningococcal strains examined. MspA was expressed by all strains belonging to clonal groups ET-5 (ST-32) and lineage 3 (ST-41/44) but was not expressed by any of the strains examined belonging to the A4 clonal group (ST-8). In addition, MspA was expressed by the single isolates of serogroup W135, 29E, and Z examined but not by the serogroup X or Y isolates. MspA was not expressed by the single isolates of N. lactamica and Neisseria polysacchareae that were examined.
MspA is expressed in vivo and is antigenic. To assess whether MspA is expressed in vivo during meningococcal disease and is immunogenic, purified rMspA was probed by immunoblotting with sera from convalescent-phase patients. Convalescent-phase sera from six patients infected with serogroup B strains recognized purified recombinant MspA (rMspA) (typical examples are shown in Fig. 4). Conversely, convalescent-phase serum from a patient infected with a serogroup C strain (not expressing MspA) was unreactive (Fig. 4). Acute-phase sera were unreactive (data not shown).
Antibodies to MspA are bactericidal. The antiserum raised against MspA was examined for its ability to kill meningococci in the presence of complement. The RMspA antiserum killed 50% of the homologous strain (MC58) at a concentration of 1:32 and strain Z4664 (ET-5; ST-32) at a concentration of 1:8. In contrast, the corresponding preimmune serum failed to produce detectable bactericidal activity at a concentration of 1:4. The antiserum also failed to kill the MspA mutant, confirming that the observed bactericidal activity was dependent on the presence of MspA. However, the RMspA antiserum failed to kill strains Z6421 (lineage 3; ST-41/44) and Z4242 (ET-37; ST-11), despite immunoblot confirmation that these strains expressed MspA.
MspA has adhesive properties. Given the homology of MspA to App, which has been demonstrated to mediate adhesion to epithelial cells, we wished to determine whether MspA also has adhesive properties. E. coli strains expressing either MspA or App were shown to adhere to BEAS-2B cells significantly more (P < 0.0001) than control strains (JM109 and XL10GOLD, respectively), which contained the plasmid vector without an insert (Table 2 and Fig. 5). Although App was previously shown not to mediate adhesion to human umbilical vein endothelial cells (HUVECs), we decided to assess adhesion to human brain microvascular endothelial cells. Interestingly, E. coli expressing either App or MspA adhered significantly more (P < 0.0001) than their respective negative control strains (Table 2 and Fig. 5). Of note, E. coli cells expressing MspA did not show an autoagglutination phenotype (not shown).
DISCUSSION
NMB1998 is a novel autotransporter protein detected in silico in the genome of the serogroup B meningococcal strain MC58, due to its homology to App, IgA1 protease, and other known autotransporter proteins (1, 36). Interestingly, the gene encoding NMB1998 is absent from the genome sequences of meningococcal strain Z2491 and the gonococcal strain FA1090, suggesting that it may only be present in a subset of meningococcal clonal lineages. Given the homology of NMB1998 to IgA1 protease and App, which are both chymotrypsin serine proteases, we have designated the protein MspA, for meningococcal serine protease A. Curiously, MspA is located in the serogroup B (MC58) meningococcal genome just four open reading frames downstream of another autotransporter protein, NadA (NMB1994), which is also absent from the serogroup A (Z2491) genome (6). This may be a mere coincidence, however, since the gene encoding NadA contains a low G+C content with respect to the average for the genome, suggesting that the gene was acquired by horizontal transfer from another organism, which does not appear to be the case for MspA. Furthermore, the 5.6-kb region that separates nadA and mspA contains three conserved genes, which are present in both Z2491 and MC58.
We have demonstrated that MspA, which possesses the characteristic three-domain structure of an autotransporter protein, is naturally expressed in N. meningitidis and is cleaved and secreted into the external milieu. The autotransporter protein family is large and diverse and has been the subject of several reviews (10, 12-14). Proteins belonging to this family represent one of the most important groups of secreted proteins in gram-negative bacteria and mediate a variety of interactions between gram-negative bacteria and their environmental niches. Indeed, in most gram-negative pathogens studied so far, autotransporter proteins make a significant contribution to pathogenesis and are responsible for characteristics such as adhesion, toxigenicity, serum resistance, intracellular movement, and proteolysis (12). Interestingly, N. meningitidis, Haemophilus influenzae, and Bordetella pertussis, which all colonize the upper respiratory tract in humans, possess multiple autotransporter proteins, some of which share considerable homology. For example, H. influenzae contains IgA1 protease, Hap, and Hia/Hsf, which are 54, 56, and 53/57% identical to their respective homologues, IgA1 protease, App, and NhhA in N. meningitidis (1, 29). Both Hap and App have been shown to have a role in adhesion of their respective organisms to host cells, and Hap is important in the invasion of host cells (11, 16, 31, 32). In addition, we previously reported the identification and characterization of AspA, an outer membrane protein, and secreted subtilase in N. meningitidis, which shares homology with SphB1 in B. pertussis (7). SphB1 is a surface-anchored maturation protease, which following autocleavage specifically catalyzes the conversion of the filamentous hemagglutinin precursor molecule to mature, secreted filamentous hemagglutinin (7, 8). Recently, AspA was demonstrated to alter the secretion profile of App and, in some strains, IgA1 protease (37). In the presence of AspA/NalP, two forms of App (ca. 100 and 140 kDa, respectively) are secreted, while in the aspA/nalP mutant the lower-molecular-weight form predominates (37; our unpublished data). Consistent with these observations, we have also detected an anti-App-reactive ca. 40-kDa band in concentrated supernatant proteins prepared from the AspA/NalP mutant, which is absent from the parent MC58 strain (our unpublished data). The homology of MspA with App and IgA1 protease suggested that MspA might represent an additional substrate for AspA/NalP. To investigate this possibility, secreted proteins prepared from the AspA/NalP mutant were probed with the MspA-specific antiserum. Analogous to App, a smaller (ca. 45-kDa) anti-MspA-reactive protein was detected, which was absent from the MC58 parent strain. This suggests that MspA is an additional substrate for AspA/NalP.
We have previously shown that App is constitutively expressed by meningococcal strains representing the clonal groups that are responsible for the majority of meningococcal infections worldwide (1). Furthermore, App was shown to be immunogenic and expressed in vivo during natural infection (1). Having shown that MspA is secreted in meningococcal strain MC58, we wished to determine whether MspA is similarly expressed in diverse meningococcal lineages. A strain survey demonstrated that MspA is expressed by ST-32 and ST-41/44, which contain the majority of hypervirulent serogroup B strains, but not by ST-8. Interestingly, isolates from ST-11 gave discrepant results, with some isolates expressing MspA and some not. Of note, in the sequenced ST-11 strain (FAM18), MspA contains a premature stop codon at the 3' end of the gene, which presumably results in the gene not being expressed. Despite the proximity of the encoding genes in the MC58 genome sequence, the strain distribution of MspA is clearly distinct from that of NadA; the latter was expressed by ST-8 but not ST-41/44 strains.
In vivo expression and B-cell immunogenicity of MspA were confirmed by the detection of specific anti-MspA antibodies in sera from patients convalescing from serogroup B invasive meningococcal disease. In view of the immunogenicity of MspA and its homology to App, which generates a bactericidal response, we investigated the bactericidal activity of anti-MspA antibodies. Anti-MspA antibodies were shown to kill the homologous strain MC58 and another meningococcal strain belonging to the same sequence type (ST-5), but strains Z6421 and Z4242 (representing ST-41/44 and ST-11, respectively) were insensitive to killing. The bactericidal response elicited by anti-MspA antibodies suggests that, analogous to App, MspA is also accessible to antibodies on the bacterial surface. The insensitivity of strains belonging to heterogeneous lineages to complement-mediated killing was also noted in respect to anti-App antibodies, and, as suggested for App (1), may result from differences in the processing and surface exposure of MspA. It is possible that antibodies raised against the native form of the protein may result in the binding of surface-exposed conformational epitopes on a wider range of strains.
App was recently shown to mediate in vitro adherence to Chang (human conjunctiva) epithelial cells when expressed on the surface of E. coli, although adhesion to HUVECs could not be demonstrated (31). In addition, purified App was shown to bind to Chang and HepG2 (human liver carcinoma) cells at a high level but not to other epithelial cell lines including HEC-1-B (human endometrium), Hep-2 (human laryngeal epidermoid carcinoma), or 16HBE14o (human bronchial epithelial) cells. In contrast, we found that E. coli expressing either App or MspA adhered to BEAS-2B and HBME cells, suggesting that MspA may also play a role in adhesion to host cells. The adhesion to HBME cells but not HUVECs may be explained by differential expression of eukaryotic surface ligands, as has been suggested for the differential binding of App to epithelial cell lines (31). Work is currently in progress to identify the host ligand(s) for both MspA and App. Once the ligand(s) has been identified, the hypothesis of differential cellular expression can be tested.
In conclusion, we have characterized a novel meningococcal autotransporter protein, which is expressed by the majority of serogroup B strains in vitro, is immunogenic in humans following invasive infection, and is capable of generating a bactericidal response, albeit in a limited range of strains. MspA, like App, may also possess a role in colonization and provide a selective advantage to strains that express both proteins. The proposed role in colonization, however, does not exclude an additional function for the secreted fragment of the protein, which may cleave a host protein, analogous to IgA1 protease. We propose that an effective antibody response to MspA, in addition to App, may interfere with the normal interaction of this protein with host molecules and contribute to the prevention of meningococcal disease.
ACKNOWLEDGMENTS
We thank Dominique Caugant for kindly providing the N. meningitidis isolates representing different hypervirulent lineages, Ray Borrow for providing the baby rabbit complement, and Sean Murphy for providing the HBME cells.
Part of this work was performed during the tenure of a Medical Council Research Fellowship awarded to D.P.J.T. (grant G84/5370).
These authors contributed equally to this work.
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