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Journal of Clinical Microbiology, December 1998, p. 3680-3682, Vol. 36, No. 12
Wellcome Trust Centre for the Epidemiology of
Infectious Disease,
Received 11 June 1998/Returned for modification 4 August
1998/Accepted 25 August 1998
The genetic diversity of porB genes from meningococcal
isolates characterized as serotype 22 was investigated by gene
sequencing. This procedure identified seven distinct porB
sequences, demonstrating variation in the PorB protein recognized by
the serotype 22 monoclonal antibody. This is consistent with the
genetic heterogeneity of serotype 22 meningococci reported previously.
Neisseria meningitidis
causes bacterial meningitis and septicemia worldwide (3).
For routine epidemiological surveillance, meningococci are classified
by immunological reagents into serogroups (by type of capsular
polysaccharide), serotypes (PorB, class 2 or 3 outer membrane
protein [OMP]), and serosubtypes (PorA, class 1 OMP)
(7). Many meningococcal isolates are nonserotypeable (NT),
or nonserosubtypeable (NST), as a result of antigenic variation in the
PorA and PorB proteins (5, 11, 14) and the assay-dependent reactivity of some of the monoclonal antibodies (MAbs) used (17, 20, 23).
A MAb identifying a new serotype, 22, was produced in the Czech
Republic in 1994 to combat the large proportion (50 to 80%) of NT
meningococci isolated there between 1973 and 1994 (9). Use
of this MAb in the National Reference Laboratory for Meningococcal Infections, Prague, Czech Republic, showed that 44% of the
meningococci previously characterized as B:NT during 1995, and 37% of
such isolates obtained between 1973 and 1994, were serotype 22 (10). Testing of meningococcal isolates in other European
countries gave the following rates of serotype 22 for isolates
previously classified as NT: Austria, 5.4%; Germany, 11.3%; and
Greece, 9.5% (19). Addition of this reagent to the
serotyping panel used at the Meningococcal Reference Unit for England
and Wales in 1995 identified serotype 22 organisms among invasive and
noninvasive serogroup B and C isolates of diverse serosubtype. A study
of 22 Czech serogroup B, serotype 22 (B:22), meningococci by
PCR-restriction endonuclease pattern analysis of the pilA
gene and multilocus enzyme electrophoresis concluded that these
organisms were highly heterogeneous, with 17 clonal complexes and 14 pilA alleles identified among serotype 22 isolates
(16).
In the present work, the antigenic heterogeneity of the PorB proteins
recognized by the serotype 22 MAb was examined by nucleotide sequence
determination of the porB genes of serotype 22 meningococci. The study included 10 Czech B:22 isolates (isolates 312204 to 312213)
and 3 United Kingdom (U.K.) isolates, 1 B:22 (isolate 312664) and 2 C:22 (isolates 312472 and 312597) (Table
1). The meningococcal template DNA
preparation was as described previously (20). Amplification
of porB genes by PCR (in 100-µl reaction mixtures) was
carried out with reaction buffer (Gibco BRL); 200 µM (each) dATP,
dCTP, dGTP, and dTTP; 1 µM concentrations of PCR primers PB1
(5'-TAAATGCAAAGCTAAGCGGCTTG-3') and PB2
(5'-TTTGTTGATACCAATCTTTTCAG); 0.5 U of Taq
polymerase (Gibco BRL); and 1 µl of template DNA (approximately 50 ng
µl Two class 2 OMP-encoding porB allele sequences were
identified among the 13 isolates (GenBank accession no. AF065125 and AF065126). The peptide sequences deduced from these alleles were
consistent with the porin model of PorB structure (22), with
eight surface exposed loop regions (loops I to VIII) in which the
serotype-specific peptide sequences reside (6). The new sequences were aligned with 92 meningococcal PorB protein sequences covering all known serotypes, including a distinct sequence from an
additional serotype 22 isolate (GenBank accession no. U92906 [17]) and many PorB sequences from isolates described
as NT, most of which had not been tested with the serotype 22 MAb
(2, 6, 17, 21).
Comparison of the alignment showed that the only peptide sequence
unique to the serotype 22 PorB sequences was AKNNDGTANQGKKH, located in
putative loop VI, all other loop sequences being diverse among serotype
22 isolates or shared with PorB sequences from isolates with different
serotypes (Fig. 1 and data not shown). This loop VI sequence was also encoded by the porB genes
from isolates 315/85, EG 011, NG H38, and 528 (GenBank no. AF065127, AF065128, AF065129, and AF065130, respectively) (Fig. 1). These
isolates had not been typed with a reagent panel including the serotype
22 MAb and were classified as NT. To test the hypothesis that the loop
VI peptide sequence was required for serotype 22 MAb recognition, the
isolates were reserotyped, in a blinded fashion, with a MAb panel
including the serotype 22 MAb. All four meningococci were characterized
as serotype 22 (Table 1), strengthening the evidence that loop VI is
the critical loop for serotype 22 recognition.
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Heterogeneity of the PorB Protein in Serotype 22 Neisseria meningitidis
ABSTRACT
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1). Reaction conditions were 30 cycles of 94°C for
1 min, 60°C for 1 min, and 72°C for 2 min, followed by incubation
at 72°C for a further 2 min. Purification and direct nucleotide
sequence determination of amplified porB genes were done as
described previously (20).
TABLE 1.
Meningococcal serotype 22 strains examined and GenBank
accession numbers of their porB gene sequences
View larger version (30K):
[in a new window]
FIG. 1.
Alignment of seven meningococcal PorB protein sequences
obtained by translation of the nucleotide sequences of each of the
porB alleles identified among serotype 22 meningococci. The
locations of the putative surface loops (I to VIII) of the porin are
indicated. The boxed area defines the putative surface loop VI that is
likely to be important for recognition of the PorB proteins by the
serotype 22 MAb. Sequences AF065126 and U92906 were different at the
nucleotide sequence level but possessed identical peptide sequences.
The relationships among the seven porB allele sequences identified in serotype 22 meningococci were represented graphically by the split-decomposition method (1) (Fig. 2). The split graph obtained illustrates a network of possible pathways linking the porB allele sequences obtained from serotype 22 meningococci, suggesting that genetic recombination, which occurs within and between Neisseria species (12, 15, 18), has resulted in the circulation of a sequence encoding the serotype 22 epitope among the porB alleles present in populations of N. meningitidis.
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In conclusion, these data demonstrate that while meningococcal strains that react with the serotype 22 MAb possess an identical peptide sequence in variable surface loop VI of the PorB protein, the PorB proteins may be encoded by mosaic gene structures that are highly diverse in one or more of the other variable surface loops. Furthermore, the serotype 22 epitope is encoded by only a small part of the meningococcal genome, the remainder of which has been shown previously to be highly heterogeneous among serotype 22 meningococci (16). Positive reactions with the serotype 22 MAb therefore do not provide a robust indication of the genetic relatedness of meningococcal isolates unless they are supported by additional epidemiological information, obtained, for example, from multilocus sequence typing (13) or multilocus enzyme electrophoresis analyses (4).
Nucleotide sequence accession numbers. Nucleotide sequences have been deposited in the GenBank database under accession no. AF065125 to AF065130.
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ACKNOWLEDGMENTS |
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M.C.J.M. is a Wellcome Senior Research Fellow in Biodiversity, and R.U. and M.C.J.M. are grateful to the Wellcome Trust for financial support. P.K. and M.M. acknowledge the support of research grant 310/96/K102 from the Grant Agency of the Czech Republic and research grant 3982-3 from the Internal Grant Agency of the Ministry of Health of the Czech Republic.
We thank Steve Gray at the Meningococcal Reference Unit for England and Wales for serological characterization of meningococcal isolates.
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FOOTNOTES |
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* Corresponding author. Mailing address: Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, South Parks Rd., Oxford OX1 3PS, United Kingdom. Phone and fax: 44 1865 271284. E-mail: martin.maiden{at}zoo.ox.ac.uk.
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Journal of Clinical Microbiology, December 1998, p. 3680-3682, Vol. 36, No. 12
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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