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Infection and Immunity, December 2000, p. 6685-6690, Vol. 68, No. 12
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Multiple Mechanisms of Phase Variation of PorA in Neisseria meningitidis

Arie van der Ende,* Carla T. P. Hopman, and Jacob Dankert

Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, and Reference Laboratory for Bacterial Meningitis, University of Amsterdam/RIVM, 1100 DE Amsterdam, The Netherlands

Received 6 July 2000/Returned for modification 14 August 2000/Accepted 25 September 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Previously, we reported that PorA expression in Neisseria meningitidis is modulated by variation in the length of the homopolymeric tract of guanidine residues between the -35 and -10 regions of the promoter or by deletion of porA. To reveal additional mechanisms of variation in PorA expression, the meningococcal isolates from 41 patients and 19 carriers were studied. In addition, at least 3 meningococcal isolates from different body parts of each of 11 patients were analyzed. Sequence analysis of the porA promoter showed that the spacer between the -35 and -10 regions varies in length between 14 and 24 bp. PorA expression was observed in strains with a porA promoter spacer of 16 to 24 bp. All but one strain with a porA promoter spacer of 16 to 20 bp and undetectable PorA expression have a homopolymeric tract of 8 or 6 instead of 7 adenine residues in the porA coding region. The other PorA-negative strain had a single-base-pair deletion in the coding region. The highest level of PorA expression was observed in strains with a promoter spacer of 17 or 18 bp. PorA expression was reduced twofold in strains with a porA promoter spacer of 16 or 19 bp. Strains with a 16-bp promoter spacer with substitutions in the polyguanidine tract displayed increased levels of PorA expression compared to strains with a homopolymeric tract of guanidine residues in the porA promoter. In conclusion, meningococci display multiple mechanisms for varying PorA expression.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Major outer membrane proteins of Neisseria meningitidis are of interest, since their antigenic variation is used for serological discrimination between isolates (1, 7). In addition, meningococcal outer membrane vesicles containing major outer membrane proteins are under investigation as experimental vaccines to prevent meningococcal disease (8, 12). Class 1 protein, or PorA, is of particular interest, since monoclonal antibodies directed against serosubtype-specific epitopes on PorA proved to exert bactericidal activity in serum and confer protection against N. meningitidis infection in an animal model (27, 28). In clinical trials with meningococcal outer membrane-based vaccines, the induced bactericidal activity in serum was predominantly attributed to the presence of antibodies directed against PorA (4, 22). Since PorA is exposed to antigenic variation, the newer PorA-based vaccines contain multiple antigenic variants of PorA (5, 35). Field trials with a hexavalent PorA-based vaccine have already been performed (24).

PorA can be expressed by most clinical isolates, but its levels of expression vary considerably (13, 33, 34). Since stable expression of PorA in meningococci during infection is a prerequisite for the PorA vaccine to be effective, knowledge of the genetic mechanism of the variable PorA expression is important. Recently, isolates from patients with meningococcal disease with the complete porA gene deleted have been described (34). Previously, we reported PorA phase variation at the transcriptional level, mediated by a variable homopolymeric tract of guanidine residues between the -35 and -10 domains of the porA promoter (33). For this report we studied additional mechanisms of PorA expression variation and porA promoter sequences among meningococcal isolates from 52 patients with meningococcal disease and 19 N. meningitidis carriers. From 11 of the 52 patients with meningococcal disease, three isolates, cultured from different sites and specimens, were studied.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Strains and culture conditions. From the collection of the Reference Laboratory for Bacterial Meningitis, University of Amsterdam, Amsterdam, The Netherlands, and Rijksinstituut voor Volkgezonheid en Milieu, Bilthoven, The Netherlands, 66 strains from 41 patients with meningococcal disease and 19 healthy carriers were selected (see Table 1). These 66 strains represent all 29 lineages present in the collection of the Reference Laboratory, including reference strains for serogrouping. A second group of isolates cultured from 11 patients was studied (see Table 2). Three isolates per patient were taken from different sites and specimens. All isolates were stored at -70°C within four passages.

From the frozen stock culture of each isolate, bacteria were grown on a chocolate agar plate (3% hemoglobin in GC agar base) (Oxoid CM 367; Unipath, Basingstoke, England) at 37°C in a humidified atmosphere of 5% CO2 in air for 18 h. One colony was selected from the culture plate and resuspended in 100 µl of deionized H2O. A loop of this suspension was subcultured on a chocolate agar plate. Part of this culture was stored at -70°C, and another part was used to inoculate 200 ml of tryptic soy broth (Difco Laboratories, Inc., Detroit, Mich.) for the preparation of the outer membrane protein fraction. The remaining suspension in deionized H2O was heated at 100°C for 10 min. After centrifugation for 2 min in an Eppendorf centrifuge at maximum speed, 10 µl of the supernatant was used in a PCR to amplify the porA promoter region.

Fluorescence-based sequencing and analysis. The porA promoter region was amplified using primers PorA5 (34) and P21Rev (5'-ACGGCCGGCTTTGATTTCGCCGTACAG-3') or PorA72 (5'-GCACGAGGTCTGCGCTTGAATTG-3') and P21Rev. The DNA sequences of both strands of these amplicons, of 330 and 295 bp, respectively, were determined by a PCR-based sequence reaction with fluorescent dye-labeled dideoxynucleotide terminators using AmpliTaq DNA polymerase FS (Perkin-Elmer, Gouda, The Netherlands) and primers PorA5 and P21Rev or PorA72 and P21Rev according to the instructions supplied by Applied Biosystems Incorporated (Foster City, Calif.). The sequences were analyzed on an automatic sequenator (model 373-stretch; Applied Biosystems Incorporated).

PorA expression quantification. Outer membrane proteins were isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described by Poolman et al. (25). Ten micrograms of the outer membrane protein fraction of each isolate was loaded on a sodium dodecyl sulfate-11% polyacrylamide gel. After staining with 0.1% Coomassie brilliant blue R (Sigma, St. Louis, Mo.) in acetic acid-methanol (10%/25% in H2O) and destaining, protein patterns were visualized and an image was captured with a video camera. Analysis of the images was performed using Gelcompar software 3.1 (Applied Maths, Kortrijk, Belgium). The amount of PorA in each strain was expressed as the percentage (1% = 1 arbitrary unit) of the total amount of outer membrane protein of the strain visible in the gel.

Western and colony immunoblotting. PorA was identified by Western immunoblotting (32) using PorA-specific monoclonal antibodies (1). For colony blotting, colonies were transferred to nitrocellulose (pore size, 0.45 µm; Schleicher & Schluell, Dassel, Germany) and immunologically stained as described previously (13).

Statistics. Significance of differences was calculated by the chi-square test (Yates corrected) and the two-tailed Fischer exact test.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

PorA phase variation by length variation of a homopolymeric adenine tract in the porA coding region. First, 66 strains from 41 patients with meningococcal disease and 19 healthy carriers were studied (Table 1). The number of base pairs forming the spacer between the -35 and -10 domains of the porA promoter ranged from 14 to 24. PorA expression was observed in strains with a porA promoter spacer of 16 to 24 bp (Fig. 1; Table 1).

                              
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  		TABLE 1.   Serogroup, source, porA promoter spacer length and composition, and PorA expression of meningococcal isolates



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  		FIG. 1.   Outer membrane profiles of meningococci with different porA promoter spacer sequences. Strains YP1 and YP2 are from one carrier; strains 860183-I and 860183-II are the CSF and blood isolates from one patient.

Surprisingly, PorA expression was undetectable in four of the strains with a porA promoter spacer of 16 to 20 bp (Table 1; Fig. 1 and 2B). Strain 800377, with a porA promoter spacer of 17 bp, appeared to have a deletion of a single bp at position 39 (from the start codon) in the porA reading frame (Fig. 2B). Predicted translations of porA reveal that this deletion introduces a frame shift, which truncates the coding sequence at codon position 40. In strain 851267, with a porA promoter spacer of 20 bp, a 7-bp homopolymeric tract of adenine residues close behind the start codon was extended with 1 bp, also resulting in a frame shift. The primary culture plate YP of the throat swab of one carrier contained 91% PorA-positive and 9% PorA-negative expression variants as identified by colony immunoblotting. One PorA-negative expression variant (YP1) and a positive PorA expression variant (YP2) were selected for further analysis (Fig. 2B). The porA promoter spacer was 16 bp long, but the homopolyadenine tract in the porA coding region in these variants was 8 bp (strain YP1) and 7 bp (strain YP2) long, respectively. In addition, from the culture plate of strain YP1, a PorA-positive variant, which was present with a frequency of 10-3, was selected which had a polyadenine tract of 7 bp (not shown). Furthermore, the cerebrospinal fluid (CSF) isolate 860183-I, with a porA promoter spacer of 17 bp and a polyadenine tract in the porA coding region of 7 bp, displayed strong PorA expression, while the isogenic blood isolate 860183-II (from the same patient) with a polyadenine tract of 6 bp had undetectable PorA expression levels (Fig. 1 and 2B). In addition, the blood isolate contained variants in which the porA gene was completely deleted, as described previously (34).


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  		FIG. 2.   (A) Sequence variation in the promoter region of the outer membrane protein PorA among 66 strains of N. meningitidis. The putative -35 and -10 domains are underlined. Polymorphisms in the porA promoter spacer are in boldface. (B) Association between PorA expression and variation in the homopolymeric tract of adenine residues and a nonsense mutation in the porA coding region. The start codon of each sequence is in boldface.

Predicted translations of porA show that full coding integrity is maintained with a repeat of seven adenine residues, while expansion or reduction by one residue introduces a frame shift, which truncates the coding sequence.

PorA expression associated with the length and composition of the spacer between the -35 and -10 domains of its promoter. The porA promoter contained a variable homopolymeric tract of 8 to 18 guanidine residues in the spacer between the putative -35 and -10 domains (Fig. 2A). Nine strains had one or more mutations in the porA promoter spacer, replacing one or two of the residues in the homopolymeric tract of guanidine residues with a thymidine or adenine (Fig. 2). In addition, for isolate 980218 and isolates YP1 and YP2, the homopolymeric tract of thymidine residues was extended by 1 or 2 bp, respectively, compensated for by a reduction of an equal number of base pairs of the interrupted polyguanidine tract.

PorA expression of the strains ranged widely (Fig. 1 and 3). The PorA expression was strongest in 33 strains with a spacer region of 17 or 18 bp (Fig. 3). The PorA expression level of the majority of strains with promoter spacer regions of 16 and 19 bp was significantly lower. The average PorA expression level in 6 strains with a mutated porA promoter spacer of 16 bp was 1.6 times higher than that of the strains with a corresponding porA promoter spacer with a homopolymeric tract of guanidine residues, although this difference did not reach a significant level (Fig. 3). Expression of PorA was undetectable in strains with a promoter spacer region shorter than 16 bp (Fig. 1 and 3). The PorA expression of the 7 strains with a polyguanidine tract in the porA promoter spacer length of >= 21 bp was only weak or not detectable. For those 4 strains with a weak PorA expression level and of subtypes for which a monoclonal antibody was available, Western immunoblotting also showed a low level of PorA expression.


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  		FIG. 3.   Association between the mean PorA expression levels and the length of the porA promoter spacer region with a contiguous polyguanidine tract () or with an interrupted polyguanidine tract (). The whiskers indicate the standard errors of means. The amount of PorA is expressed as a percentage of the outer membrane protein fraction. Isolates with a mutation in the porA coding region were excluded. ns, not significant.

PorA expression of meningococcal isolates from different specimens of a single patient. The distributions of the porA promoter spacer lengths among isolates from carriers and among isolates from patients were different. The isolates from 8 of the 19 (42%) carriers had a promoter spacer of less than 17 bp, but the isolates from only 3 of the 41 (7%) patients had such a low number of base pairs in the porA promoter spacer (P < 0.05). These results might indicate that the porA promoter spacers of carrier isolates were shorter than those of patient isolates. Therefore, three isolates from different sites and specimens from 11 patients were analyzed for their PorA expression, porA promoter spacer sequence, and homopolymeric adenine tract in the porA coding region (Table 2). The multiple meningococcal isolates from different specimens from the same patient had identical porA promoter sequences, except for patient 901168. The throat isolate had a porA promoter spacer of 18 bp and high PorA expression, while the isolates from blood and CSF had a porA promoter spacer of 16 bp and low PorA expression. The PorA expression in the isolates from the skin biopsy specimen, CSF, and blood of patient 930470 was negative, due to a homopolymeric adenine tract of 6 bp instead of 7 bp in the porA coding region.

                              
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  		TABLE 2.   Meningococcal strains from three different specimens of patients


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

It was reported earlier that the spacer between the putative -35 and -10 domains of the porA promoter varies in length due to a variable homopolymeric tract of guanidine residues in this spacer (2, 29, 33). Meningococcal isolates with the porA gene deleted have been described (34). In addition, meningococcal isolates with PorA expression levels that were not anticipated based on their promoter sequence were recently encountered. To reveal additional mechanisms of PorA expression variation, the meningococcal isolates from 41 patients and 19 carriers were studied.

The porA promoter spacer ranged from 14 to 24 bp in length due to variation in the length of the homopolymeric guanidine tract. In addition, the homopolymeric tract of thymidine residues of the porA promoter spacer may also vary. One strain was found in which the polythymidine tract was increased by one thymidine residue, and in the two variants (YP1 and YP2) from the primary culture plate of a throat swab the polythymidine tract was increased by two thymidine residues.

Carrier isolates had shorter porA promoter sequences than patient isolates. However, the mean PorA expression levels of the isolates with a porA promoter spacer of <17 bp were higher in carrier isolates than in isolates from patients, due to mutations in the polyguanidine tract in four out of seven carrier isolates with a porA promoter spacer of 16 bp. In addition, PorA expression did not differ among meningococci isolated from different sites of the body, indicating that the level of PorA expression of meningococci at different locations in the host had no selective advantage during infection. However, porA-negative meningococci, which may appear after selection, being resistant to bactericidal antibodies against PorA were isolated from a patient's blood (34).

Although PorA expression was observed in strains with a promoter spacer of 16 to 23 bp, the highest level of PorA expression was observed in strains with a promoter spacer of 18 or 17 bp. These results are in concordance with our earlier results (33) and with those from Arhin and colleagues (2). The results are also consistent with the recently published study of Sawaya and colleagues (29). In their study, PorA expression gradually decreased when the length of the promoter spacer region was decreased from 18 to 16 bp by in vitro mutagenesis. However, in that study, porA promoters with spacers of >18 bp were not studied.

In Escherichia coli, the length of the promoter spacer region is constrained to 17 ± 1 bp (10, 30). The data from our study indicate that the length of the porA promoter spacer in N. meningitidis might be less restricted. The -10 domain of the porA promoter is a complete match with the consensus sequence of -10 domains of promoters in other prokaryotes, while the putative -35 domain of the porA promoter is ill defined. The putative -35 domain ATGGTT matches the E. coli consensus -35 domain (TTGACA) only in two out of six nucleotides (10). Absence of an identifiable -35 region or poor homology with the -35 consensus sequence may be indicative of involvement of an auxiliary protein binding upstream of the -10 domain and in this way substituting for the role of the -35 region (6). However, the sequence TGGTTT, which is shifted 1 bp downstream compared to the ATGGTT sequence, also has two matches with the consensus -35 sequence. The broad range of lengths of the porA promoter spacer region allowing PorA expression may indicate that both -35 domains are being recognized by RNA polymerase and may be used alternatively at the initiation of porA transcription.

An alternative explanation may be that the homopolymeric tract of guanidine residues in the porA promoter adopts the A-helical structure instead of the B-helical conformation (6, 29, 36). The A-helical DNA structure has a shorter physical distance between base pairs as well as a decreased net twist compared to the B-helical DNA structure. In promoters with an A-helical structure, deletion of 1 bp from the optimal 17-bp spacer would have a greater impact on protein expression than an insertion of 1 bp (6, 36). Our results are consistent with this model. The average PorA expression levels of strains with a porA promoter with a spacer of 16 bp is 1.9 times lower than that of strains that have a promoter with a spacer of 17 bp. Conversely, strains with a porA promoter spacer of 17 or 18 bp expressed similar amounts of PorA. In addition, PorA expression is 1.6 times higher (although this difference is not significant) in strains with a porA promoter with a 16-bp spacer containing an interrupted polyguanidine tract than in strains with a homopolymeric tract of guanidine residues in the porA promoter, which is consistent with conversion of the A-helical DNA structure into the B-helical DNA conformation of the porA promoter spacer (6, 36).

In addition to the variation in PorA expression due to sequence variation in the porA promoter region, PorA expression variation may also be affected by other mechanisms. In our study, porA expression also was altered due to extension or shortening of a homopolymeric tract of adenine residues in the coding region of porA of isogenic meningococcal strains. So the DNA strand slippage mechanism causes PorA phase variation at the transcriptional level and at the translational level. Phase variation by the DNA strand slippage mechanism has been described for a variety of microorganisms (11). Translational control by DNA slipped-strand mispairing in Neisseria has been described for the opacity proteins (18), lipopolysaccharide immunotypes (14), hemoglobin receptor HmbR (16, 26), capsular polysaccharide production (9), and fimbrial expression (15) and seems to be a common mechanism for varying the expression of genes that are important in the virulence of pathogenic microorganisms.

PorA is the important component of group B meningococcal protein-based vaccines, since capsule polysaccharides of group B meningococci are poorly immunogenic. However, meningococci avoid the humoral host immune response to this protein by antigenic variation (3, 17, 19, 20, 31) within PorA as well as by variation of its expression (2, 13, 33). This study shows that PorA expression can be varied in multiple ways. Slipped-strand mispairing during replication in the homopolymeric tract of guanidine residues and/or thymidine residues in the porA promoter as well as the homopolymeric tract of adenine residues in the porA coding region yields progenitors with a PorA expression different from that of their parental bacterial cells. In addition, point mutations in the coding region may result in meningococci without PorA expression. Also, PorA expression may be absent due to deletion of the complete porA gene as previously described (34). In addition, insertion of an IS element in the porA coding region in isolates without PorA expression has also been observed (23).

In summary, PorA expression can be varied by multiple mechanisms, which in concert with antigenic variation of PorA might be indicative of a limited efficacy of PorA-based vaccines.


    FOOTNOTES

* Corresponding author. Mailing address: Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. Phone: (31-20) 5664862. Fax: (31-20) 6979271. E-mail: a.vanderende{at}amc.uva.nl.

Editor:   E. I. Tuomanen


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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Infection and Immunity, December 2000, p. 6685-6690, Vol. 68, No. 12
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