Identification and characterization of App: an ... - Wiley Online Library

Molecular Microbiology (2001) 41(3), 611–623

Identification and characterization of App: an immunogenic autotransporter protein of Neisseria meningitidis Hoda Abdel Hadi, Karl G. Wooldridge, Karen Robinson and Dlawer A. A. Ala’Aldeen* Molecular Bacteriology and Immunology Group, Division of Microbiology, School of Clinical Laboratory Sciences, University of Nottingham, Nottingham NG7 2UH, UK. Summary In a search for immunogenic virulence factors in Neisseria meningitidis, we have identified a gene encoding a predicted 160 kDa protein with homology to the autotransporter family of proteins. Members of this family are secreted or surface exposed and are often associated with virulence in Gram-negative bacterial pathogens. We named the gene adhesion and penetration protein (app ), because of its extensive homology to the hap gene of Haemophilus influenzae. We reconstructed the gene with reference to genomic sequence data and cloned and expressed the protein in Escherichia coli. Rabbit antiserum raised against recombinant App reacted with proteins in all meningococcal isolates examined, which represented clonal groups responsible for the majority of meningococcal invasive disease. Antibodies to the protein were detected in the sera of patients convalescing from meningococcal infection. Purified App had strong stimulating activity for T cells isolated from a number of healthy donors and from one convalescent patient. We confirmed that App is surface localized, cleaved and secreted by N. meningitidis. Importantly, the rabbit anti-App serum killed the organism in the presence of complement. Thus, App is conserved among meningococci, immunogenic in humans and potentially involved in virulence. It therefore merits further investigation as a component of a future multivalent vaccine.

Introduction Neisseria meningitidis, normally residing as a harmless commensal in the human nasopharynx, is the cause of life-threatening disease. Thirteen serogroups of the

Q 2001 Blackwell Science Ltd

bacterium are recognized, which are determined by the chemical composition of the polysaccharide capsule. Serogroup A is responsible for epidemic meningitis in sub-Saharan Africa, whereas serogroups B and C are responsible for the majority of meningococcal disease in industrialized countries (Achtman, 1995). The outcome of meningococcal infection ranges from asymptomatic carriage to meningitis and fulminant meningococcaemia (Ala’Aldeen, 1997). The onset of meningococcal infection can be extremely rapid, leading to high morbidity and mortality despite antimicrobial chemotherapy and modern intensive care. In addition, both meningitis and meningococcaemia can result in permanent debilitating sequelae. Therefore, vaccination provides the best strategy for combating meningococcal disease. Vaccines based on meningococcal capsular polysaccharide (group A, C, Y and W135) have been used widely for a number of years (Ala’Aldeen and Griffiths, 1995). A new generation of vaccines based on capsular polysaccharide conjugated to protein carriers is currently being introduced and is showing promising early results (Richmond et al., 1999; 2000; Campagne et al., 2000). However, the capsular polysaccharide of serogroup B meningococci is very poorly immunogenic in humans. This is thought to be because of the chemical similarity between this antigen and the polysialic acid moieties of developmentally regulated glycoproteins in neural and other tissues (Finne et al., 1987). Serogroup B meningococci are responsible for the majority of meningococcal disease in industrialized countries (Achtman, 1995). Furthermore, it has been suggested that the incidence of serogroup B infection may increase in the wake of a reduction in group C carriage, which may result from the introduction of the new generation of polysaccharide conjugate vaccines (Maiden and Spratt, 1999). Patients who recover from group B meningococcal infection have serum bactericidal antibodies to noncapsular surface antigens (Frasch and Chapman, 1973; Jones and Eldridge, 1979). A number of groups have investigated non-capsular vaccine candidates including outer membrane proteins (OMPs), either in crude vesicle preparations or as purified recombinant proteins. Clinical trials with Cuban and Norwegian vaccines based on crude vesicle preparations have yielded mixed results and demonstrated relatively poor protection of infants below

612 H. Abdel Hadi, K. G. Wooldridge, K. Robinson and D. A. A. Ala’Aldeen 2 years of age (Ala’Aldeen, 1997). Serum bactericidal responses elicited by the Norwegian vaccine were directed largely against the hypervariable subtyping antigen PorA. A recombinant hexavalent vaccine containing the six most common isotypes of PorA has been developed, but serum bactericidal antibodies elicited were rather modest, and there appeared to be antigenic interference between some of the antigens (Rumke et al., 1998). Other OMPs that have been investigated include the transferrin-binding proteins, the class 5 proteins (Opc and Opa) and a Neisseria-specific surface protein, NspA. In addition, we have recently described TspA and AutA, two highly conserved Neisseria-specific proteins that are potent T-cell-stimulating antigens (Kizil et al., 1999; Ait-Tahar et al., 2000). It is likely that a successful vaccine will need to contain multiple, but well-characterized antigens. Here, we describe the identification and initial immunological characterization of another conserved, secreted autotransporter protein of N. meningitidis that is antigenic to human B cells and T cells.

Results Identification, cloning and expression of the app gene of N. meningitidis By screening a previously described meningococcal genomic expression library (in lzapII), using a murine polyclonal serum raised against an OMP-enriched fraction of N. meningitidis (strain SD; B:15:P1.16, Table 1), we detected a number of immunoreactive clones. One such clone was sequenced and found to contain an open reading frame (ORF) with homology to the cvaB gene of a pathogenic Escherichia coli isolate (Otto et al., 1998). This

Table 1. Neisseria meningitidis strains used in this study. Strain

Serogroup

Clonal subgroup

Origin

MC58 Z2491 SD UN4210 UN3501 UN4512 UN2015 Z1035 Z5010 Z3515 Z1269 Z1001 Z5035 Z6414 Z4181 Z4701 Z4671 Z6426

B A B Y W135 C 29E A A A A A A C C B B B

ET-5 IV-1 ET-5 NDa ND ND ND I II III IV-1 IV-2 V A4 ET-37 ET-37 A4 Lineage 3

UK Gambia UK UK UK UK UK Pakistan Djibouti Saudi Arabia Burkina Faso USA China New Zealand Mali Norway Holland New Zealand

a . ND, not determined.

gene was located on a large ColV plasmid close to the hbp gene, which encodes a large protein belonging to the autotransporter family. The hbp gene sequence was used to search the N. meningitidis (strain Z2491) genome database (which was partially completed at the time). We found two large ORFs with a high degree of homology with the hbp gene. One of these was the previously described iga gene, which encodes the meningococcal IgA protease (Lomholt et al., 1995). The second ORF, which displayed extensive homology with the iga gene, had not been described previously. This second gene has extensive homology with a number of proteins belonging to the autotransporter family of proteins (Fig. 1). In particular, the gene was highly homologous to the recently described hap (haemophilus adhesion and penetration) gene of Haemophilus influenzae (St Geme et al., 1994). To correspond with the name of hap, we called the meningococcal homologue app (adhesion and penetration protein). The entire ORF of the app gene was amplified from meningococcal strain SD by polymerase chain reaction (PCR) and cloned into the inducible expression vector pQE30 to produce plasmid pQEApp. The gene encoded a predicted protein of 159 780 Da. The sequences of the corresponding genes in the clinical isolates Z4181 and Z3515 (Table 1) were obtained after PCR amplification and cloning of these genes. The predicted protein sequence of strain SD was . 98% identical to its counterpart in the recently published genome sequence of strain MC58 (B:15:P1.16) (Tettelin et al., 2000) and strain Z4181 and . 89% identical to its counterpart in strain Z2491 (Parkhill et al., 2000) and strain Z3515 at the amino acid level (Fig. 1). Most of the variation between strains was found in the N-terminus (Fig. 1). Induction of E. coli cells harbouring plasmid pQEApp with IPTG resulted in the expression of a major new protein migrating on SDS–polyacrylamide gels with an apparent molecular mass of 175 kDa (Fig. 2). In addition, three smaller new proteins with molecular masses of 120 kDa, 60 kDa and 40 kDa were expressed after induction (Fig. 2). We purified the recombinant App protein using Ni-NTA spin columns with the 6-histidine tag incorporated into the recombinant protein and used the purified protein to raise an App-specific rabbit polyclonal monospecific antiserum (RaApp). The specificity of the antiserum was demonstrated by probing a Western blot of purified recombinant App and a control preparation from the E. coli host not expressing App. A single band corresponding to purified recombinant App was detected, whereas no protein was detected in the control preparation (Fig. 3, lanes 1 and 2). Sequence analysis of the app gene and flanking DNA We analysed the DNA surrounding the app locus in the Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 611–623

Characterization of an autotransporter of N. meningitidis

613

Fig. 1. Identity of the App protein with selected homologues. The N-terminal passenger domains and C-terminal b-domains are compared separately in (A) and (B) respectively. The App protein of N meningitidis (Nm ) strain SD is compared with its counterparts in the meningococcal strains MC58, Z4181, Z2491 and Z3515, the App homologue in N. gonorrhoeae (Ng ), as well as the Hap protein of H. influenzae (Hi ) and the IgA proteases (Iga) of N. gonorrhoea, N. meningitidis and H. influenzae. Accession numbers are U11024(Hap), X04835(Iga; Ng ), X82474(Iga; Nm ), X64357(Iga; Hi ).

N. meningitidis strain Z2491 genomic sequence, and no genes were found that were likely to be functionally related (Fig. 4). An ORF encompassing nucleotides 22194 to 2850 with respect to the start codon of app was found in the opposite orientation to app. This putative gene displayed a high degree of homology to the thdF gene of E. coli. The thdF gene encodes the thiopene and furan oxidation protein, which belongs to the ERA/THDF family of GTP-binding proteins (Alam and Clark, 1991). A second ORF was predicted between 319 and 1429 nucleotides downstream of the app gene. This putative gene, which was also in the opposite orientation to app, was 93% identical to a previously described meningococcal gene encoding a putative transposase (Zhu et al., 1999). A ribosome binding site was predicted between nucleotides 212 and 27 with respect to the start codon of the app gene, and a promoter sequence was predicted spanning nucleotides 2171 and 2126. The predicted transcriptional start site was at nucleotide 2131. No inverted repeats were observed at the end of the app gene, which might have represented transcription stop signals. Furthermore, no runs of direct repeats (indicative of phase variation) were present upstream of or within the app gene. Finally, the first 42 amino acids of the predicted App precursor protein, which were identical in all five strains for which the sequence was available, were predicted to form a cleavable signal peptide. The arrangement of genes at the app locus of strain MC58 is essentially similar to that of strain Z2491 (not shown). Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 611–623

Mutagenesis of app We amplified a region of DNA containing < 1 kb of DNA both upstream and downstream of the start codon of app (Fig. 4). This fragment was cloned into plasmid pGEM-T Easy to yield plasmid pGTApp1. This construct was used as a substrate for inverse PCR, in which a region of DNA from nucleotides 26 to 124 with respect to the start codon was deleted and a new unique Bgl II site introduced to produce plasmid pGTApp2. A cassette encoding resistance to spectinomycin and streptomycin was cloned into the Bgl II site, and the resulting construct (pGTApp3) was used to mutate the meningococcal strain MC58 by natural

Fig. 2. Expression of recombinant App. Mid-logarithmic phase cultures of E. coli strain XL10GOLD harbouring plasmids pREP4 and pQE30 (lane 1) or pQEApp (lanes 2 – 5) were sampled, and proteins were separated on a 10% SDS – PAGE gel and stained with Coomassie blue. Cultures were sampled before (lanes 1 and 2) or 1 h (lane 3), 2 h (lane 4) or 3 h (lane 5) after the addition of 2 mM IPTG.

614 H. Abdel Hadi, K. G. Wooldridge, K. Robinson and D. A. A. Ala’Aldeen transformation and allelic exchange to yield MC58app. We probed whole-cell lysates of MC58 and MC58app with RaApp to confirm that App was not expressed in the latter. Strongly reactive proteins with apparent molecular weights of 175 kDa and 100 kDa were detected in lysates of MC58 (Fig. 3, lane 3). Minor (weakly reactive) protein bands with apparent molecular weights of < 140 kDa and < 160 kDa were detected in MC58 (Fig. 3, lane 3). All these bands were absent from lysates of MC58app.

App is surface localized, cleaved and secreted by N. meningitidis

Fig. 3. Detection of App in various preparations by immunoblotting using RaApp. Recombinant App was purified from E. coli lysates by affinity purification using Ni-NTA (lane 1). Similar preparations of control cultures of E. coli not expressing App did not contain any proteins that were recognized by RaApp (lane 2). Whole-cell lysates of strain MC58 (lane 3) were compared with those of MC58app (lane 4). Concentrated culture supernatants of strain MC58 (lanes 5 and 7) were compared with outer membrane-enriched fractions of MC58 cells (lane 6) and with concentrated supernatants of MC58app (lane 8). Arrows indicate the positions of prominent bands, and the apparent molecular weights are given in kDa.

Electron microscopy of immunogold-labelled whole meningococcal cells of strain MC58 demonstrated that App was localized to the cell surface (Table 2). We wished to determine whether App was cleaved and secreted by the meningococcus, as are some members of the autotransporter family of proteins. Meningococci were cultured overnight in DMEM, and the medium was concentrated by ultrafiltration after removal of meningococcal cells by centrifugation and filtration. Proteins were separated by SDS–PAGE and transferred to nitrocellulose for immunoblotting. Supernatants from strain MC58 grown in DMEM contained a strongly reacting band at

Fig. 4. The app locus and mutagenesis of app. The app locus is illustrated in (A). app is flanked by two ORFs in the reverse orientation. A putative promoter sequence (P) is indicated upstream of app, and uptake sequences (US) are indicated upstream and towards the 30 end of the gene. Primers used during this study are indicated. app1 and app2 were used to amplify the ORF for cloning into the expression vector pQE30. The primers app3 and app4 were used to amplify the region indicated, which, after cloning in pGEM-T Easy, was used as a substrate for inverse PCR using primers app5 and app6. The V cassette of plasmid pHP45V was inserted into the unique Bgl II site created after self-ligation of the inverse PCR product. Structural features of App are indicated in (B). The first 42 amino acid residues are predicted to form a cleavable signal peptide (pale grey). An asterisk indicates the conserved serine protease motif. Regions predicted to form amphipathic b-strands (black), including the C-terminal autotransporter consensus sequence, are indicated within the C-terminal b-portion of the protein (dark grey).

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 611–623


615

Table 2. Immunoelectron microscopy of N. meningitidis strain MC58 and MC58app. Strain MC58 MC58app MC58 MC58

Antiserum Preimmune RaApp RaApp RaTBPc

a

No. of cells counted

% of labelled cells

Mean no. of gold particles per cell (^ 1 SD)

26 26 25 26

80 92 100 100

3 (2.9) 9 (6.0) 31.5 (14.2)b 74.4 (41.8)

a . Preimmune serum from the rabbit used to raise the RaApp serum b . The numbers of gold particles detected on MC58 cells probed with RaApp were significantly greater than the numbers of MC58 cells treated with preimmune serum or MC58app cells probed with RaApp (P , 0.001). c . Rabbit antiserum raised against neisserial transferring-binding proteins.

< 100 kDa and a weakly reactive band at < 140 kDa, both of which were detected in an OMP-enriched fraction, in addition to the larger 175 kDa protein (Fig. 3, lanes 5 and 6). As a control for outer membrane contamination of supernatant fractions, we probed supernatant and outer membrane-enriched fractions in immunoblots with a monoclonal antibody to the outer membrane protein PorA. PorA was readily detected in OMP-enriched fractions but was not detected in supernatant preparations (not shown). No RaApp-reactive proteins were detected in fractions enriched for cytoplasmic membranes or pooled cytoplasmic and periplasmic fractions (data not shown). A similar pattern of secreted proteins was detected in supernatants of strain Z2491 and SD (data not shown). Culture supernatants of MC58app did not contain any bands that reacted with RaApp (Fig. 3, lane 8).

convalescing from meningococcal disease contained antibodies that recognized App. Recombinant App was probed by immunoblotting with sera from nine convalescent-phase patients, five acute-phase patients, one longterm carrier and one healthy donor with no history of meningococcal infection. Sera from all nine convalescing patients and the long-term carrier reacted with the recombinant protein. In contrast, sera from the one healthy donor and all five acute-phase sera did not react with the recombinant protein, confirming that the antibodies detected resulted from seroconversion after meningococcal infection or carriage. The results from paired acute-phase and convalescent-phase sera are shown in Fig. 7.

App is a strong T-cell stimulant App is conserved in N. meningitidis and is expressed in vivo We wished to determine whether App is expressed in a range of meningococcal clinical isolates. RaApp-reactive proteins were detected by immunoblotting in whole-cell lysates of all 18 strains that we tested (Table 1). These included 12 meningococcal isolates representing the clonal groups responsible for the majority of meningococcal infections worldwide. In addition, strains MC58, Z2491, SD and laboratory isolates of serogroup 29E, W135 and Y contained immunoreactive proteins. In all isolates, proteins with apparent molecular weights of < 175 and < 140 kDa were found. The relative proportions of the two forms were not consistent and varied from strain to strain and, in some cases, only one of the bands was apparent after photography. Typical examples are shown in Fig. 5. Supernatant preparations of the same panel of strains were also probed with RaApp. Proteins with apparent molecular weights of < 140 kDa and < 100 kDa were observed in all strains, although the relative amount of each protein was strain dependent and, in some strains, additional proteins with apparent molecular weights of between 88 and 134 were detected (Fig. 6). Next, we investigated whether sera taken from patients Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 611–623

To determine whether the App protein was capable of inducing responses in human T cells, we used recombinant App purified from E. coli lysates by elution from a preparative SDS–polyacrylamide gel to test its ability to induce T-cell proliferation in vitro. Peripheral blood mononuclear cells (PBMCs) were initially isolated from four healthy donors (HD1 –4). PBMCs were cultured with either purified App or with whole-cell meningococcal extracts prepared from strain SD for 7 and 10 days to detect a secondary memory response or in vitro priming

Fig. 5. Immunoblots demonstrating the expression of App in a panel of meningococcal isolates. Whole-cell lysates of meningococcal strains SD (lane 1), Z1035 (lane 2), Z1001 (lane 3), Z5010 (lane 4), Z6414 (lane 5), Z5035 (lane 6) and Z4181 (lane 7) were probed with RaApp. Arrows to the right indicate the positions of the major protein bands.

616 H. Abdel Hadi, K. G. Wooldridge, K. Robinson and D. A. A. Ala’Aldeen between 0.65 and 1.3; Table 3B). Although the patient cells did respond to the control protein preparation with a SI of 3.2, this was not as large as the SI for these cells after stimulation with purified App (8.1). Taken together, App resulted in significantly higher SI than the negative control (P , 0.05). Antibodies to App are bactericidal

Fig. 6. Immunoblots demonstrating secretion of App in a panel of meningococcal isolates. Concentrated cell-free supernatant of mid-log phase cultures of meningococcal strains MC58 (lane 1), Z5035 (lane 2), Z1035 (lane 3) and Z4181 (lane 4) were probed with RaApp. Arrows to the right indicate the positions of the major protein bands.

respectively. PBMCs cultured for 7 days with the T-cell mitogen phytohaemagglutinin (PHA) served as a positive control, whereas cells cultured with no added antigen served as negative controls. PHA stimulated PBMCs from all the donors tested with stimulation indices (SI) of between 12.5 and 542.9 with respect to cells cultured with no added antigen (Table 3A). Responses to whole-cell meningococcal extracts were observed with SI of between 2 and 8.5 after 7 days and between 13.7 and 33.1 after 10 days (not shown). Finally, PBMCs from all four donors responded to purified App with SI of between 2.8 and 9.5 after 7 days and between 11.4 and 36.9 after 10 days (Table 3A). After these experiments, we determined that the App preparation contained a minor co-migrating protein derived from the E. coli host cells. In order to ensure that App, and not the contaminating protein, was responsible for the observed induction of T-cell proliferation, a second preparation of App was obtained. This time, App was affinity purified using Ni-NTA, followed by a second purification step involving SDS –PAGE and elution from the gel. A control preparation was obtained from the same number of E. coli cells not expressing App, which were treated identically to those expressing App. This preparation contained no detectable protein on Coomassie-stained SDS –PAGE gels (not shown). Both preparations were tested as described above with PBMCs obtained from HD1 and three new donors known to have memory response to meningococcal whole cells (HD5 –7). PBMCs from a patient who had recovered from group C meningococcal meningitis 1 month previously were also examined. Again, cells from all the individuals tested responded strongly to PHA with SI of between 122 and 2998 (Table 3B). All four healthy donors responded to the purified App with SI of between 2.1 and 17.4, whereas none responded significantly to the control preparation (SI

It was important to examine the ability of the antiserum raised against App to kill meningococci in the presence of a complement source. The RaApp antiserum killed 100% of the homologous strain (SD, ET-5) at a concentration of . 1:128. This experiment was repeated six times with reproducible results. In contrast, the corresponding preimmune serum failed to produce statistically significant bactericidal activity even at a dilution of 1:2. In order to determine the ability of RaApp to kill heterologous serogroup B of other clonal lineages, the antiserum was tested for activity against isolates representing clonal groups ET-37 (Z4701), A4 (Z4671) and lineage 3 (Z6426). Only the last strain was killed at dilutions greater than 1:16 at which the antiserum did not kill the other two strains. Discussion We have identified a meningococcal gene encoding a large polypeptide with homology to the autotransporter family of proteins. These proteins are a divergent family with a variety of biological functions that are thought to be secreted by a common defining mechanism (Jose et al., 1995; Henderson et al., 1998). This mechanism of secretion was first described for the IgA1 protease of Neisseria gonorrhoeae (Pohlner et al., 1987; Jose et al., 1995). Secretion across the cytoplasmic membrane appears to be, at least in the majority of cases, via the sec-dependent, type II secretion pathway, in which a

Fig. 7. Detection of App-reactive antibodies in convalescent serum. Purified recombinant App was probed with convalescent-phase antiserum from a patient after recovery from meningococcal septicaemia (lane 1) or with acute-phase serum from the same patient (lane 2). The meningococcal strain isolated from this patient was group C, subgroup 2a.



617

Table 3A. Proliferation of PBMCs in response to recombinant App protein purified from lysates of E. coli by elution from SDS – polyacrylamide gels. Mean c.p.m. ^ SDa (SIb) in response to Donor

Time

APPc

PHA (10 mg ml21)

Negative controld

HD1

7 days 10 days 7 days 10 days 7 days 7 days

1762.8 ^ 340.2 (9.5) 2816.9 ^ 793 (36.9) 1387.3 ^ 376.9 (2.8) 1210.8 ^ 192.3 (11.4) 1227.1 ^ 596 (3.7) 1822.5 ^ 574.1 (5.4)

42 409.1 ^ 3947.5 (228) 41 988 ^ 923.1 (55) 9657.2 ^ 1671.4 (19.3) 1332.8 ^ 123.4 (12.5) 1814 ^ 31 (543) 744 ^ 33.8 (220)

186.2 ^ 43.2 76.3 ^ 5.2 501 ^ 296 106.5 ^ 27 334.2 ^ 104 338.6 ^ 251.1

HD2 HD3 HD4

a . SD, 1 standard deviation. b . Stimulation index calculated as mean counts per minute (c.p.m.) for cells with the antigen divided by mean c.p.m. for the negative control. c . App was added at final concentrations of 0.125, 0.25, 0.5 and 1.0 mg ml21. The mean c.p.m. for the concentration that gave the optimal SI is reported. d . Cells cultured without antigen.

Table 3B. Proliferation of PBMCs in response to recombinant App protein purified by affinity purification followed by elution from SDS – polyacrylamide gels. Mean c.p.m. ^ SDa (SIb) in response to Donor

Time

APPc

HD5 HD6 HD7 HD1 Patient

7 days 7 days 7 days 7 days 7 days

4353.6 504 165.4 236.6 3920.5

^ 1814 (17.4) ^ 340 (5.6) ^ 74.6 (2.08) ^ 162.8 (2.15) ^ 3717.8 (8.1)

Negative controld

PHA (10 mg ml21)

Unstimulated controle

220.6 125 89.9 71.9 1574.1

93518.4 127251 238683 187580 58993.1

250.3 90 79.6 109.9 479.9

^ 81.1 (0.88) ^ 31.5 (1.3) ^ 27.4 (1.13) ^ 19.7 (0.65) ^ 1015.2 (3.2)

^ 10224.1 (374) ^ 11129 (1413.9) ^ 500021.5 (2998.5) ^ 41419 (1707.6) ^ 6493.9 (122.9)

^ 89.3 ^ 26.7 ^ 37.5 ^ 47.3 ^ 346

a . SD, 1 standard deviation. b . Stimulation index calculated as mean counts per minute (c.p.m.) for cells with the antigen divided by mean c.p.m. for the negative control. c . App was added at final concentrations of 0.125, 0.25, 0.5 and 1.0 mg ml21. The mean c.p.m. for the concentration that gave the optimal SI is reported. d . Cells cultured with control preparation of XL10GOLD containing pQE30 and pREP4 without cloned app. e . Cells cultured without antigen.

cleavable N-terminal signal peptide mediates secretion across the cytoplasmic membrane. The C-terminal bdomain is then inserted into the outer membrane, where it is thought to form a pore through which the functional passenger domain is passed. The passenger domain may then remain covalently attached to the b-domain, it may be cleaved but remain associated with the b-domain, or it may be cleaved and released from the cell surface (Henderson et al., 1998). Most, if not all, autotransporter proteins studied to date have been found in pathogenic bacteria and are associated with virulence. The biological roles of autotransporter proteins include adhesion to cell surfaces, proteolytic degradation of host molecules, iron (haem) acquisition, cytotoxicity, host cell invasion, intracellular motility and serum resistance. We initially identified App as a homologue of the haemoglobin protease of a pathogenic E. coli strain isolated from a wound infection (Otto et al., 1998). However, the App protein was much more closely related to other members of the autotransporter family of proteins. This family includes the IgA proteases of N. meningitidis, N. gonorrhoea and H. influenzae as well as the recently Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 611–623

described Hap protein of H. influenzae. The latter protein has been implicated in attachment and invasion of this pathogen to host cells (St Geme et al., 1994; Hendrixson and St Geme, 1998). Analysis of the DNA sequence at the app locus indicated that the app gene was downstream of a gene encoding the housekeeping enzyme thdF and upstream of a transposase gene. As both these genes were in the opposite orientation with respect to app and a promoter was predicted with high probability upstream of app, it appears that it is transcribed independently of other genes. No homopolymeric runs or runs of direct repeats were present either within or upstream of app. Many phase-variable antigens, particularly in Neisseria species, are encoded by genes in which such sequences serve to allow for phase switching. App is therefore unlikely to be phase variable. Although there is an insertion sequence downstream of the app gene, App does not appear to be part of a composite transposon. There are, however, two neisserial DNA uptake signals, one of which is part of an incomplete inverted repeat. The first 42 amino acids of App were predicted to form a

618 H. Abdel Hadi, K. G. Wooldridge, K. Robinson and D. A. A. Ala’Aldeen signal peptide. Similar, unusually large signal sequences have been found in a number of autotransporter proteins, but the significance of this is not known (Henderson et al., 1998). Autotransporters described to date have few cysteine residues, and App contains only three, one of which is located in the presumed signal peptide. It is thought that a high cysteine content is not tolerated because the formation of disulphide bonds in the periplasm would block translocation through the outer membrane (Jose et al., 1996). The terminal 20 amino acids of App conform to a proposed signature based on the established autotransporter proteins (Henderson et al., 1998). This motif is assumed to form the last of an even number of b-strands of a b-barrel structure with which the b-domain traverses the outer membrane. Computer modelling of the C-terminus of App predicts a number of regions of amphipathic b-strands, which could possibly span the outer membrane. These data are consistent with a model that has been proposed for other autotransporter b-proteins, in which up to 14 trans-membrane b-strands make up a b-barrel structure (Jose et al., 1995; Hendrixson et al., 1997; Henderson et al., 1998). Several autotransporter proteins contain RGD (Arg – Gly –Asp) motifs, which are implicated in attachment to the host cell via b-integrin molecules. Although one such motif is present in the sequence of the App protein of the group A strains Z3515 and Z2491, it was not conserved in the sequences of the group B strains SD or MC58 or in the group C strain Z4181. Furthermore these motifs, when present in autotransporter proteins, are usually present in pairs. It is likely, therefore, that the occurrence of a single RGD motif within the App sequence of strain Z2491 was fortuitous. Another motif found in several autotransporter proteins is the serine protease motif GDSGSP (Henderson et al., 1998). This motif, which is found in all members of the IgA protease subfamily, is also present and conserved in the App proteins from all five meningococcal App sequences. This sequence forms the active site of these proteases and is responsible for cleavage of human IgA by the IgA proteases and for autocatalytic cleavage of the Hap protein (Hendrixson et al., 1997) as well as the IgA proteases. It is likely, therefore, that App is also autocatalytically cleaved. The salient features of App are shown in Fig. 4B. When we expressed recombinant App from strain MC58 in E. coli, we observed four new protein bands on SDS – PAGE gels with apparent molecular weights of 175 kDa, 120 kDa, 60 kDa and 40 kDa. It is likely that the latter three proteins are cleavage products. Only the full-length protein would be expected to contain the N-terminal 6-histidine tag and, indeed, this was the only form retained by Ni-NTA affinity purification. As the recombinant protein is expressed in a foreign host and, in addition, incorporates a non-native, 10-amino-acid N-terminal extension, we

cannot draw any conclusions about the normal processing of App from these observations. When whole-cell meningococcal lysates of strain MC58 were probed with the RaApp antiserum, we detected strong bands with apparent molecular weights of 175 kDa and 100 kDa and weaker bands with apparent molecular weights of < 160 kDa and < 140 kDa. Although the precursor protein, including the 42-amino-acid presumed signal sequence, has a calculated molecular weight of 159 789 Da, we presume that the < 175 kDa band corresponds to this form of the protein. After processing and removal of the signal sequence, the protein would have a predicted molecular weight of 156 119 Da, which would agree well with the 160 kDa band observed. The identities of the 140 kDa and 100 kDa bands are presumed to be the products of autocatalytic cleavage or cleavage by an unidentified protease. The presence of the conserved serine protease motif in App supports the likelihood of autoproteolytic cleavage of a passenger domain. Both these forms were also detected in the supernatant of MC58 cultures, whereas these preparations did not contain the 175 kDa band. By probing the supernatant preparations with a monoclonal antibody to the outer membrane protein PorA, we excluded the possibility that these preparations were significantly contaminated with other subcellular fractions, such as membrane blebs, which are known to be shed by growing meningococci. This antibody did not detect any protein in supernatant preparations, whereas PorA was readily detected in outer membrane-enriched fractions. The Hap protein has been shown to be cleaved at several sites (Hendrixson et al., 1997). It is likely, therefore, that the App-derived products observed in meningococcal supernatants also result from autocatalytic cleavage at alternative sites. The precise cleavage sites within App have not been determined because of difficulties in obtaining sufficient quantities of the b-protein. It is interesting to note, however, that there is a hydrophilic, proline-rich region of App that forms the beginning of a region of strong homology with the neisserial IgA proteases. The corresponding region in the IgA proteases is proximal to the known cleavage site of these proteins. It is possible, therefore, that App is cleaved in this region, which would result in a b-protein of 35 kDa. The relative amounts of the two main secreted forms of the protein originally detected in supernatants of strain MC58 varied considerably between strains, and other minor bands were also observed in supernatants of some strains. The heterogeneity of secretion profiles between strains might reflect sequence variation at the cleavage sites. The sequences of the MC58 and Z4181 strains, however, are virtually identical despite these strains having markedly different profiles of secreted proteins, suggesting that these observed differences do not result from differences at the primary sequence level. We would predict that Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 611–623

Characterization of an autotransporter of N. meningitidis the b-domain would be present in the outer membrane, but we were not able to detect a protein in either whole cells or outer membrane-enriched fractions. It is possible that this domain did not contain immunodominant epitopes and thus was not recognized by RaApp. The biological role of App is currently unknown, but its close homology to the Hap protein of H. influenzae suggests that it may mediate meningococcal –host cell interactions. The Hap protein has been shown to mediate attachment to human cells in vitro and subsequent invasion into these cells (St Geme et al., 1994). Interestingly, a host protein, secretory leucocyte protease inhibitor, is implicated in preventing autoproteolysis of Hap and facilitating adhesion by the intact surfaceexposed protein (Hendrixson and St Geme, 1998). We are currently investigating the role App in interaction with host cells. The protein sequences of strains SD and MC58 were virtually identical. This was not surprising, as these isolates belong to the same clonal subgroup (group B, subgroup ET-5). The sequence of App from strain Z4181 (group C, subgroup ET-37) is also more than 98% identical to App from the group B strains. More variation was observed between these strains and the serogroup A strains Z2491 (subgroup IV-1) and Z3515 (subgroup III), which were . 99% identical to each other and 85% identical to the groupB/C strains. It is notable that the variation is mainly in the N-terminal putative passenger domain of the protein. Proteins of the size expected for App, including the secreted form, were specifically detected by the RaApp antiserum in a range of meningococcal isolates. The isolates tested included strains representative of the major clonal groups responsible for the majority of invasive meningococcal disease worldwide, which have been identified using multilocus electropherotyping (MLEE) and confirmed by multilocus sequence typing (MLST) (Maiden et al., 1998). These data indicate that App is a conserved antigen among meningococci. The high degree of identity between App protein sequences of the group B and group C strains is very encouraging with regard to vaccine potential but, in order to evaluate fully the vaccine potential of App, it will be necessary to determine the App sequences from a greater number isolates representing hypervirulent lineages and especially those lineages associated with serogroup B meninogococci. Antiserum raised against denatured recombinant App was capable of killing meningococci of the homologous strain (SD; ET-5) in the presence of complement. In addition, strain Z6426 (representing lineage 3) was also sensitive to this antiserum. In contrast, strains Z4701 and Z4671 (representing the ET-37 and A4 clonal subgroups) were insensitive to killing. Insensitivity of strain Z4701 was unlikely to result from sequence variation, because the Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 611–623

619

App sequence of this strain is 99% identical to that of the sensitive SD strain. This anomaly may result from differences in processing and surface exposure of the App protein in these strains. It is likely that antiserum raised against a preparation of the protein in native form will be more effective in binding to surface-exposed epitopes of App, which may include conformational epitopes not present in denatured preparations of the protein. We have demonstrated that App is a strong T-cell stimulant in several unrelated individuals, inducing proliferative responses of the same order of magnitude as those elicited by an outer membrane vesicle vaccine in human volunteers (van der Voort et al., 1997). Although it cannot be excluded that lipopolysaccharide (LPS) present in the App preparation may have had some modulatory effect upon T-cell proliferative responses via induction of cytokines such as tumour necrosis factor (TNF)a and interleukin (IL)-10 (Lorenzen et al., 1999), recent evidence has shown that LPS actually downregulates T-cell proliferative responses in a manner dependent upon IL10 (Hessle et al., 2000). That sera from patients convalescing from invasive meningococcal disease contained antibodies that recognized recombinant App provides evidence that App is both expressed during infection and a B-cell immunogen. Thus, the App protein provides a promising candidate vaccine component. The pertactin molecule, which is an autotransporter protein of Bordetella pertussis, provides a precedent for an autotransporter protein that is used as a component of a successful multicomponent acellular vaccine (Pichichero et al., 1997). App is conserved, expressed during infection and carriage, stimulates B cells and T cells, and antibodies to App are bactericidal. In addition, App may be a primary target for neutralization of a potential role in pathogenesis. We propose that App is a worthy candidate for further study as a potential component of a future vaccine.

Experimental procedures Bacterial strains and growth conditions Escherichia coli strains XL10GOLD (Stratagene) and JM109 (Promega) were used in all recombinant work described here. N. meningitidis strains MC58, Z2491 and SD, laboratory isolates of serogroups 29E, W135 and Y and strains representing the major hypervirulent lineages used in this study are described in Table 1. N. meningitidis were routinely cultured on chocolate agar at 378C in 5% CO2. Liquid cultures were grown in Mueller –Hinton broth supplemented with Vitox (Oxoid) at 378C with shaking. E. coli were grown on Luria agar at 378C or in Luria broth at 378C with shaking. Antibiotics were used at the following concentrations: ampicillin, 50 mg ml21; kanamycin, 25 mg ml21; streptomycin, 150 mg ml21; spectinomycin, 150 mg ml21.

620 H. Abdel Hadi, K. G. Wooldridge, K. Robinson and D. A. A. Ala’Aldeen Preparation of culture supernatant fractions

DNA manipulations

Neisseria meningitidis cells grown overnight in DMEM at 378C in an atmosphere of 5% CO2 were pelleted by centrifugation for 10 min at 11 000 g. The supernatant was filtered through a 0.2 mm filter and concentrated 100-fold by ultrafiltration using a Vivaspin-2 protein concentrator (50 000 molecular weight cut-off; Vivascience) according to the manufacturer’s instructions. Proteins were then separated by SDS –PAGE and transferred to nitrocellulose for detection with anti-App antiserum (RaApp).

Plasmid DNA was extracted and purified using Qiagen spin columns according to the manufacturer’s instructions. Chromosomal DNA was prepared as described previously (Chen and Kuo, 1993). Restriction enzymes were purchased from Kramel Biotech, New England Biolabs or Fermentas and used according to the directions of the manufacturer. T4 DNA ligase was purchased from Boehringer Mannheim. Taq DNA polymerase was purchased from Qiagen. The app gene was amplified from strain SD using the primers app1 (50 -CGCG GATCCATGAAAACAACCGACAAACGGACAACCG-30 ) and app2 (50 -CGCGTCGACCTTTCGGCATATCCGGCGGTTA CC-30 ). The resulting amplicon was purified using PCR purification spin columns (Boehringer Mannheim), digested overnight at 258C with Bam HI and Sal I and ligated to pQE30 DNA (Qiagen) digested with the same enzymes. The insert was sequenced by primer walking at the Protein and Nucleic Acid Chemistry Laboratory at the University of Leicester. All other DNA manipulations were carried out according to standard protocols (Sambrook and Maniatis, 1989).

Subcellular fractionation Meningococccal cells grown on a chocolate agar plate overnight at 378C in an atmosphere of 5% CO2 were suspended in phosphate-buffered saline (PBS) and harvested by centrifugation for 20 min at 11 000 g. Proteins were recovered from the supernatant by trichloroacetic acid (TCA) precipitation as described previously (Brunder et al., 1996). Cells were resuspended in PBS and disrupted by sonication. After removal of unbroken cells by centrifugation at 250 g, the supernatant was centrifuged at 11 000 g for 40 min. The supernatant fraction containing cytoplasmic and periplasmic fractions was concentrated by TCA precipitation as above. The pellet was resuspended by sonication in PBS, followed by the addition of an equal volume of PBS containing 4% Triton. After incubation at 378C for 30 min, insoluble material enriched for OMPs was removed by centrifugation at 11 000 g for 40 min, and the final supernatant containing cytoplasmic membrane proteins was collected.

Immunogold labelling and electron microscopy Cells of strain MC58 or MC58app were grown in Mueller – Hinton broth, prepared for immunogold staining, and electron microscopy was performed as described previously (Ala’Aldeen et al., 1993). RaApp was used as the primary antibody, and preimmune serum from the same rabbit was used as the negative control. Both sera were preadsorbed overnight with MC58app cells. The secondary antiserum was anti-rabbit IgG (whole molecule) conjugated to 5 nm gold particles (Sigma). The numbers of gold particles associated with at least 25 randomly selected organisms from each strain –antiserum combination were counted.

Construction of the MC58app mutant A 2029 bp region of DNA flanking the start codon of app and containing an incomplete neisserial uptake sequence was amplified by PCR using the primers app3 (50 -GCTGTCG ATTGCCTGACCG-30 ) and app4 (50 -GGTTTCTGTTACCG TACCCG-30 ) and DNA from strain Z2491. This fragment was cloned into the vector pGEM-T Easy (Promega) according to the manufacturer’s instructions. The resulting plasmid (pGTApp1) was used as a template for inverse PCR using the primers app5 (5-CGCAGATCTACCGAAACACACCGCA AAGC-30 ) and app6 (5-CGCAGATCTCCTTATCTGACGGGA TTCGG-30 ), and the resulting product was digested overnight at 258C with Bgl II before being self-ligated. The resulting plasmid (pGTApp2) was digested with Bgl II and ligated to the 2 kb V element of plasmid pHP45V (Prentki and Krisch, 1984) excised using Bam HI. The resulting plasmid (pGTApp3) was used to mutate N. meningitidis strain MC58 in a biphasic system. MC58 was grown to an optical density of 0.2 in Mueller –Hinton broth and added to a well of a 24-well plate containing 1.5 ml of GC agar supplemented with Vitox. After incubation for 4 h at 378C in 5% CO2, 250 ng of the mutagenic plasmid in 2.5 ml of TE was added to the well, and incubation was continued for 16 h. The cells were then harvested and plated onto GC plates supplemented with Vitox at the concentration suggested by the manufacturer and spectinomycin and streptomycin, both at 150 mg ml21. Colonies were observed after a further 48 h and selected for analysis. The mutation was confirmed by PCR.

Screening a genomic library of N. meningitidis strain SD A lZapII genomic library of strain SD has been described previously (Palmer et al., 1993; Ala’Aldeen et al., 1996). The library was screened with antisera raised in a mouse against a detergent-insoluble meningococcal extract prepared essentially as described previously (Kizil et al., 1999), except that the proteins were size selected between 15 and 25 kDa. The library was screened, and plasmid DNA was derived from positive plaques as described previously (Ala’Aldeen et al., 1996).

SDS–PAGE, immunoblotting and protein purification Proteins were separated by SDS– PAGE using 8% or 10% acrylamide gels as described previously (Sambrook and Maniatis, 1989). Immunoblotting was carried out after SDS – PAGE by transferring proteins onto nitrocellulose sheets as described previously (Ala’Aldeen et al., 1994). After immunoblotting, nitrocellulose filters were probed with rabbit polyclonal antiserum to purified recombinant App or with Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 611–623

Characterization of an autotransporter of N. meningitidis convalescent patients’ serum as described previously (Ala’Aldeen et al., 1994). Recombinant App was purified from inclusion bodies after the induction of mid-log phase cultures of E. coli cells harbouring the recombinant gene with 2 mM IPTG for 4 h in Luria broth. The cells were harvested by centrifugation and sonicated. Insoluble material was harvested by centrifugation and solubilized in 6 M GuHCl, 0.1 M NaH2PO4, 0.01 M Tris-HCl (pH 8.0). His-tagged recombinant protein was then purified using Ni-NTA spin columns (Qiagen) under denaturing conditions according to the manufacturer’s recommendations. To obtain protein of higher purity for T-cell proliferation assays, recombinant App was also separated by SDS– PAGE and eluted from the gel using the Bio-Rad Mini Whole Gel Eluter according to the manufacturer’s recommendations.

Antiserum and bactericidal assay Polyclonal antiserum against App (RaApp) was produced by immunizing a New Zealand White rabbit once with 30 mg of purified recombinant App mixed with Freund’s complete adjuvant and twice with the same amount of antigen mixed with Freund’s incomplete adjuvant at 2 week intervals. The animal was test bled 7 days after the third inoculation and boosted once more with 30 mg of App in Freund’s incomplete adjuvant. The animal was sacrificed after a further 10 days, and the serum was stored at 2208C. Before use in immunoblots and in immunogold electron microscopy, RaApp was preadsorbed overnight at 48C with a 10% (v/v) suspension of MC58app cells before being cleared by centrifugation and filtered through a 0.2 mm membrane filter. Antiserum against meningococcal transferrin-binding proteins has been described previously (Ala’Aldeen et al., 1993). Human sera were collected from patients recovering from invasive meningococcal disease. The bactericidal assay was performed as described previously (Borrow et al., 2001). Briefly, 20 ml of RaApp serum (preadsorbed with MC58app ), 10 ml of a suspension of bacteria (grown to exponential phase and diluted to < 800 cfu per well) and 10 ml of undiluted sterile baby rabbit serum (PelFreeze) were added sequentially to a sterile 96-well tissue culture plate. Plates were covered and incubated for 60 min at 378C on a shaker (Stuart Scientific) at 150 r.p.m. Aliquots (10 ml) were plated on blood agar plates at time zero and after 60 min incubation, and serial dilutions were performed to determine the number of organisms in each microtitre well. Colony-forming units (cfu) were determined at time zero and at 60 min, and the serum bactericidal titres were reported as the reciprocal of the serum dilution yielding $ 50% killing. An antiserum to serogroup A and the preimmune serum corresponding to RaApp were included on each plate to provide positive and negative controls respectively. Other control wells included a complement control containing PBS – BSA, complement and bacteria (to determine that the organisms were viable in complement in the absence of antibody); an inactive complement control containing RaApp serum, heat-inactivated complement (568C, 30 min) and bacteria; and an antibody control containing PBS –BSA, the RaApp and the bacteria (to determine that the organisms were viable in antibody in the absence of complement). Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 611–623

621

Preparation of PBMCs and T-cell proliferation assay PBMCs isolated from healthy donors and one patient were used as a source of T cells for use in T-cell proliferation assays. PBMCs were isolated from blood by liquid gradient centrifugation over Histopaque 1077 (Sigma-Aldrich) as described previously (Kizil et al., 1999) and suspended in RPMI-1640 medium (Life Technologies) supplemented with 10 mM HEPES, 2 mM L -glutamine, 100 U ml21 penicillin, 100 mg ml 21 streptomycin, 50% human AB serum (RPMI250% HS) and 10% dimethyl sulphoxide (DMSO) and stored in liquid nitrogen. Resuscitated cells were washed and resuspended in RPMI210% HS medium at 1 107 cells ml21 and rested overnight on ice. A previously described protocol was used for proliferation assays. Briefly, aliquots of 200 ml of PBMCs at 1 106 ml21 were placed into the wells of a flat-bottomed 96-well culture plate (Nunc). Purified App was added to wells in quadruplicate at final concentrations of 0.125 mg ml21, 0.25 mg ml21, 0.5 mg ml21 or 1.0 mg ml21. Equivalent volumes of the negative control preparation were added, and PHA (Sigma) was added at 10 mg ml21 as a control. The plates were incubated at 378C in an atmosphere of 5% CO2 for 7 and 10 days. [3H]-thymidine (1 mCi; Amersham) was added to each well for the final 18 h of incubation. The cells were harvested and proliferation measured by uptake of [3H]-thymidine. Proliferation was defined by a SI (ratio of c.p.m. from stimulated cells to unstimulated cells) of . 2.0.

Statistical analysis T-cell proliferation data were analysed using the Wilcoxon signed rank test for paired data. The means of the numbers of gold particles associated with organisms in immunogold experiments were compared using a one-tailed Student t-test for means with unequal variance.

Protein and nucleic acid sequence analysis and EMBL accession number Public databases of published protein and DNA sequences were searched using the BLAST programs available at http:// www.ncbi.nlm.nih.gov/Blast/index.html. The genome databases of strains Z2491 and MC58 were interrogated using the BLAST and GRASTA servers available at http://www.sanger.ac. uk/Projects/N_meningitidis/blast_server.shtml and http://tigr. org/tdb/CMR/gnm/htmls/SeqSearch.html respectively. The DNA region flanking the app gene in the genome database of strain Z2491 was searched for probable genes using the GENEMARK HMM algorithm available at http://genemark. biology.gatech.edu/GeneMark/hmmchoice.html. The promoter prediction program available at http://www.fruitfly.org/ seq_tools/promoter.htm was used to identify the probable promoter upstream of app. The SIGNALP program, available at http://www.cbs.dtu.dk/services//SignalP/#submission, was used to predict the presence of a signal peptide at the N-terminus of App. Prediction of protein secondary structure was performed using the LASERGENE suit of programs (DNAstar). Other DNA and protein sequence analysis was carried out using the DNAMAN package of programs (Lynnon BioSoft). The nucleotide sequence of the app genes of

622 H. Abdel Hadi, K. G. Wooldridge, K. Robinson and D. A. A. Ala’Aldeen N. meningitidis strains SD, Z3515 and Z4181 have been deposited with the EMBL database and assigned the accession numbers AJ242535, AJ296276 and AJ296277 respectively.

Acknowledgements We thank Dr Dominique Caugaunt for kindly providing N. meningitidis stains representing hypervirulent lineages. We also thank Dr Ray Borrow for kindly providing the baby rabbit complement. This work was funded in part by the Ralf Sutcliff Foundation for Meningitis Research, Remedy and the Meningitis Research Foundation.

References Achtman, M. (1995) Global epidemiology of meningococcal disease. In Meningococcal Disease. Cartwright, K. (ed.). Chichester: John Wiley & Sons, pp. 159– 175. Ait-Tahar, K., Wooldridge, K.G., Turner, D., Atta, M., Todd, I., and Ala’Aldeen, D.A.A. (2000) Auto-transporter A (AutA) protein of Neisseria meningitidis: a potent CD41 T-cell and B-cell stimulating antigen detected by expression cloning. Mol Microbiol 37: 1094 –1105. Ala’Aldeen, D.A.A. (1997) Neisseria meningitidis. In Principles and Practice of Clinical Bacteriology. Emmerson, A.M., Hawkey, P., and Gilaspy, S. (eds). Chichester: John Wiley and Sons, pp. 277–304. Ala’Aldeen, D.A.A., and Griffiths, E. (1995) Vaccines against meningococcal diseases. In Molecular and Clinical Aspects of Bacterial Vaccine Development. Ala’Aldeen, D.A.A. (ed.). Chichester: John Wiley & Sons, pp. 1 –39. Ala’Aldeen, D.A., Powell, N.B., Wall, R.A., and Borriello, S.P. (1993) Localization of the meningococcal receptors for human transferrin. Infect Immun 61: 751 –759. Ala’Aldeen, D.A., Stevenson, P., Griffiths, E., Gorringe, A.R., Irons, L.I., Robinson, A., et al. (1994) Immune responses in humans and animals to meningococcal transferrin-binding proteins: implications for vaccine design. Infect Immun 62: 2984– 2900. Ala’Aldeen, D.A., Westphal, A.H., De Kok, A., Weston, V., Atta, M.S., Baldwin, T.J., et al. (1996) Cloning, sequencing, characterisation and implications for vaccine design of the novel dihydrolipoyl acetyltransferase of Neisseria meningitidis. J Med Microbiol 45: 419– 432. Alam, K.Y., and Clark, D.P. (1991) Molecular cloning and sequence of the thdF gene, which is involved in thiophene and furan oxidation by Escherichia coli. J Bacteriol 173: 6018– 6024. Borrow, R., Andrews, N., Goldblatt, D., and Miller, E. (2001) Serological basis for use of meningococcal serogroup C conjugate vaccines in the United Kingdom: reevaluation of correlates of protection. Infect Immun 69: 1568 –1573. Brunder, W., Schmidt, H., and Karch, H. (1996) KatP, a novel catalase-peroxidase encoded by the large plasmid of enterohaemorrhagic Escherichia coli O157:H7. Microbiology 142: 3305 –3315. Campagne, G., Garba, A., Fabre, P., Schuchat, A., Ryall, R., Boulanger, D., et al. (2000) Safety and immunogenicity of three doses of a Neisseria meningitidis A 1 C diphtheria

conjugate vaccine in infants from Niger (in process citation). Pediatr Infect Dis 19: 144 –150. Chen, W.P., and Kuo, T.T. (1993) A simple and rapid method for the preparation of gram-negative bacterial genomic DNA. Nucleic Acids Res 21: 2260. Finne, J., Bitter-Suermann, D., Goridis, C., and Finne, U. (1987) An IgG monoclonal antibody to group B meningococci cross-reacts with developmentally regulated polysialic acid units of glycoproteins in neural and extraneural tissues. J Immunol 138: 4402 – 4407. Frasch, C.E., and Chapman, S.S. (1973) Classification of Neisseria meningitidis group B into distinct serotypes. 3. Application of a new bactericidal-inhibition technique to distribution of serotypes among cases and carriers. J Infect Dis 127: 149– 154. Henderson, I.R., Navarro-Garcia, F., and Nataro, J.P. (1998) The great escape: structure and function of the autotransporter proteins. Trends Microbiol 6: 370 –378. Hendrixson, D.R., and St Geme, J.W., III (1998) The Haemophilus influenzae Hap serine protease promotes adherence and microcolony formation, potentiated by a soluble host protein. Mol Cell 2: 841 –850. Hendrixson, D.R., de la Morena, M.L., Stathopoulos, C., and St Geme, J.W., III. (1997) Structural determinants of processing and secretion of the Haemophilus influenzae hap protein. Mol Microbiol 26: 505– 518. Hessle, C., Hanson, L.A., and Wold, A.E. (2000) Interleukin10 produced by the innate immune system masks in vitro evidence of acquired T-cell immunity to E. coli. Scand J Immunol 52: 13 –20. Jones, D.M., and Eldridge, J. (1979) Development of antibodies to meningococcal protein and lipopolysaccharide serotype antigens in healthy-carriers. J Med Microbiol 12: 107– 111. Jose, J., Jahnig, F., and Meyer, T.F. (1995) Common structural features of IgA1 protease-like outer membrane protein autotransporters (letter). Mol Microbiol 18: 378– 380. Jose, J., Kramer, J., Klauser, T., Pohlner, J., and Meyer, T.F. (1996) Absence of periplasmic DsbA oxidoreductase facilitates export of cysteine-containing passenger proteins to the Escherichia coli cell surface via the Iga beta autotransporter pathway. Gene 178: 107– 110. Kizil, G., Todd, I., Atta, M., Borriello, S.P., Ait-Tahar, K., and Ala’Aldeen, D.A. (1999) Identification and characterization of TspA, a major CD4(1) T-cell- and B-cell-stimulating Neisseria-specific antigen (in process citation). Infect Immun 67: 3533– 3541. Lomholt, H., Poulsen, K., and Kilian, M. (1995) Comparative characterization of the iga gene encoding IgA1 protease in Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae. Mol Microbiol 15: 495– 506. Lorenzen, D.R., Dux, F., Wolk, U., Tsirpouchtsidis, A., Haas, G., and Meyer, T.F. (1999) Immunoglobulin A1 protease, an exoenzyme of pathogenic Neisseriae, is a potent inducer of proinflammatory cytokines. J Exp Med 190: 1049 –1058. Maiden, M.C., and Spratt, B.G. (1999) Meningococcal conjugate vaccines: new opportunities and new challenges. Lancet 354: 615–616. Maiden, M.C., Bygraves, J.A., Feil, E., Morelli, G., Russell, J.E., Urwin, R., et al. (1998) Multilocus sequence typing: a Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 611–623

Characterization of an autotransporter of N. meningitidis portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 95: 3140 –3145. Otto, B.R., van Dooren, S.J., Nuijens, J.H., Luirink, J., and Oudega, B. (1998) Characterization of a hemoglobin protease secreted by the pathogenic Escherichia coli strain EB1. J Exp Med 188: 1091 –1103. Palmer, H.M., Powell, N.B., Ala’Aldeen, D.A., Wilton, J., and Borriello, S.P. (1993) Neisseria meningitidis transferrinbinding protein 1 expressed in Escherichia coli is surface exposed and binds human transferrin. FEMS Microbiol Lett 110: 139– 145. Parkhill, J., Achtman, M., James, K.D., Bentley, S.D., Churcher, C., Klee, S.R., et al. (2000) Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491 (see comments). Nature 404: 502– 506. Pichichero, M.E., Deloria, M.A., Rennels, M.B., Anderson, E.L., Edwards, K.M., Decker, M.D., et al. (1997) A safety and immunogenicity comparison of 12 acellular pertussis vaccines and one whole-cell pertussis vaccine given as a fourth dose in 15- to 20-month-old children. Pediatrics 100: 772– 788. Pohlner, J., Halter, R., Beyreuther, K., and Meyer, T.F. (1987) Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease. Nature 325: 458– 462. Prentki, P., and Krisch, H.M. (1984) In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29: 303– 313. Richmond, P., Borrow, R., Miller, E., Clark, S., Sadler, F., Fox, A., et al. (1999) Meningococcal serogroup C conjugate


623

vaccine is immunogenic in infancy and primes for memory. J Infect Dis 179: 1569 –1572. Richmond, P., Goldblatt, D., Fusco, P.C., Fusco, J.D., Heron, I., Clark, S., et al. (2000) Safety and immunogenicity of a new Neisseria meningitidis serogroup C-tetanus toxoid conjugate vaccine in healthy adults. Vaccine 18: 641 –646. Rumke, H.C., Labadie, J., van den Dobblesteen, G., and van Alphen, L. (1998) Clinical studies with RIMV hexavalent meningococcal PorA vesicle vaccine. In Proceedings of the Eleventh International Pathogenic Neisseria Conference, Nice, p. 58 Sambrook, J.F.E., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. St Geme, J.W., III, de la Morena, M.L., and Falkow, S. (1994) A Haemophilus influenzae IgA protease-like protein promotes intimate interaction with human epithelial cells. Mol Microbiol 14: 217–233. Tettelin, H., Saunders, N.J., Heidelberg, J., Jeffries, A.C., Nelson, K.E., Eisen, J.A., et al. (2000) Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287: 1809 –1815. van der Voort, E.R., van Dijken, H., Kuipers, B., van der Biezen, J., van der Ley, P., Meylis, J., et al. (1997) Human B- and T-cell responses after immunization with a hexavalent PorA meningococcal outer membrane vesicle vaccine. Infect Immun 65: 5184 – 5190. Zhu, P., Morelli, G., and Achtman, M. (1999) The opcA and (psi) opcB regions in Neisseria: genes, pseudogenes, deletions, insertion elements and DNA islands. Mol Microbiol 33: 635 –650.