Many carried meningococci lack the genes required for capsule synthesis and transport

Heike Claus1, Martin C. J. Maiden2, Rainer Maag1, Matthias Frosch1 and Ulrich Vogel1

Institute for Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Straße 2, 97080 Würzburg, Germany1
The Peter Medawar Building for Pathogen Research, University of Oxford, South Parks Road, Oxford OX1 3SY, UK2

Author for correspondence: Ulrich Vogel. Tel: +49 931 20146802. Fax: +49 931 20146445. e-mail: uvogel{at}hygiene.uni-wuerzburg.de


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Of 830 Neisseria meningitidis isolates obtained from healthy carriers in Bavaria, Germany, 136 (16·4%) lacked the operons necessary for the synthesis, lipid modification, and transport of capsular polysaccharide. These operons were replaced by a non-coding intergenic region either 113 or 114 bp in length, termed here the capsule null locus (cnl). Comparisons of the nucleotide sequence of this region in the meningococcus and its acapsulate relatives, Neisseria gonorrhoeae and Neisseria lactamica, revealed six distinct sequence variants (cnl-1 to cnl-6), with a total of 10 nucleotide substitutions and three indels. With the exception of one 4 bp insertion, which was unique to a gonococcal isolate, all of the individual sequence changes were present in the N. lactamica isolates examined. The meningococcal isolates with a cnl belonged to one of four otherwise genetically diverse genetic groupings: the ST-53 and ST-1117 complexes (75 isolates); the ST-845 complex (12 isolates); the ST-198 and 1136 complexes (46 isolates), and the ST-44 complex (one isolate). These data demonstrated that a substantial proportion of carried meningococci were incapable of capsule production, that the cnl circulated within Neisseria populations by horizontal genetic exchange, and that the expression of a polysaccharide capsule was not a requirement for person-to-person transmission of certain meningococcal lineages.

Keywords: Neisseria meningitidis, multilocus sequence typing, transmission, capsule null locus

Abbreviations: MLST, multilocus sequence typing; RM, restriction–modification; ST, sequence type

The GenBank accession number for the sequence of the cnl-1 allele is AJ308327.


   INTRODUCTION
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The causative organism of meningococcal disease, Neisseria meningitidis, is primarily a commensal bacterium which can be cultured from up to 40% of human nasopharyngeal swab samples (Broome, 1986 ). This figure, which is likely to be an underestimate of the actual carriage rate (Sim et al., 2000 ), is dependent on a number of factors particular to the individuals sampled, including age and social status, together with population factors such as geographical location and climatic conditions (Rosenstein et al., 2001 ). The meningococcus and the gonococcus are the only members of the genus Neisseria associated with human disease, the other species being harmless inhabitants of the mucosal surfaces of animals and humans (Morse & Knapp, 1992 ). Within the genus, the expression of a capsular polysaccharide is unique to N. meningitidis, and it is tempting to speculate that the acquisition of the genetic material necessary for the expression of a capsule was an important step in the evolution of the meningococcus from other Neisseria species.

Immunochemical differences among meningococcal capsules define the serogroups of the organism (Vedros, 1987 ). Five of the 13 recognized capsular polysaccharides, those conferring serogroups A, B, C, W-135 and Y, can be thought of as virulence determinants, as virtually all meningococcal disease is caused by organisms expressing one of these capsular antigens (Poolman et al., 1995 ). These capsules are capable of protecting the bacterium against opsonophagocytosis during disseminated infection (Kahler et al., 1998 ; Masson & Holbein, 1985 ; Vogel & Frosch, 1999 ; Vogel et al., 1997 ); however, as the majority of colonizations by organisms expressing these serogroups do not result in invasive disease, and as meningococcal disease is not a mechanism for person-to-person transmission, it is likely that the capsule genes have been acquired by, and maintained in, meningococcal populations because they have a biological role distinct from pathogenicity, for example the prevention of desiccation during transmission (Virji, 1996 ). This idea is supported by the observation that during colonization of the nasopharynx meningococci are frequently acapsulate as a consequence of phase variation of the capsule-synthesis genes (Hammerschmidt et al., 1996 ).

The meningococcal capsule-synthesis (cps) gene cluster consists of five regions (Frosch et al., 1989 ): region A comprises the genes that are required for polysaccharide synthesis (Edwards et al., 1994 ; Swartley et al., 1998 ); region B contains genes responsible for lipid modification (Frosch & Müller, 1993 ); region C, containing the ctr genes, is required for polysaccharide transport (Frosch et al., 1991 , 1992 ); region D is involved in lipopolysaccharide synthesis (Hammerschmidt et al., 1994 ); region D' is a truncated duplication of the region D found in meningococci (Petering et al., 1996 ); and region E (the functions of the tex gene homologue present in region E are currently unknown). The order of the regions of the cps cluster in meningococcal isolates studied to date is B-D'-E-C-A-D or B-D-A-C-E-D' (reviewed by Vogel & Claus, 2000 ; Vogel et al., 2001 ). Because of the role of capsule expression in invasive disease, the ctrA gene (region C) is conserved in most meningococcal isolates from patients (Frosch et al., 1992 ) and is used as a target for the detection of meningococci in clinical specimens (Guiver & Borrow, 2001 ; Guiver et al., 2000 ). By contrast, region A contains genes which are serogroup specific with variants of the siaD gene required for synthesis of the sialic-acid-containing capsules (B, C, Y and W-135) and the myn genes necessary for the expression of a serogroup A capsule (Claus et al., 1997 ; Swartley et al., 1997 ). Regions E and D are present in Neisseria gonorrhoeae and Neisseria lactamica, which do not express a capsule (Petering et al., 1996 ).

Natural populations of N. meningitidis, i.e. those derived from carriers and not associated with invasive disease, are genetically diverse (Caugant et al., 1986a , b ), largely as a consequence of extensive inter- and intra-species horizontal genetic exchange (Feil et al., 1999 ; Smith et al., 1999 ; Zhou et al., 1997 ). Analyses of nucleotide sequence data acquired during multilocus sequence typing (MLST) studies (Maiden et al., 1998 ) have demonstrated that the meningococcus has a fundamentally non-clonal population structure (Holmes et al., 1999 ), which nevertheless contains a number of clonal complexes comprising genetically related organisms (Bygraves et al., 1999 ; Feavers et al., 1999 ). In contrast to the genetic diversity of carrier populations of meningococci (Jolley et al., 2000 ; Tzanakaki et al., 2001 ), most isolates obtained from patients with meningococcal disease belong to a small number of clonal complexes (Caugant et al., 1987 ; Maiden et al., 1998 ). The relationships of these ‘hyperinvasive’ meningococci to less-invasive isolates are potentially informative in the endeavour to understand meningococcal disease, especially with regard to the distribution of genes thought to be involved in pathogenesis. The present study established that a high proportion (16·4%) of the meningococci isolated from healthy people in Bavaria, Germany, were acapsulate as a consequence of the absence of regions A and C of the capsule gene cluster. These isolates belonged to four genetically distinct groups of meningococci.


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Isolate collection.
Meningococci were isolated from retropharyngeal swab samples collected from 8000 children and young adults in the German federal state of Bavaria between November 1999 and March 2000 (Alber et al., 2001 ). Briefly, the swabs were inoculated directly onto Martin–Lewis agar plates (Becton Dickinson), and Gram-negative, oxidase-positive colonies were tested for ß-galactosidase and {gamma}-glutamyltransferase activity. If necessary, biochemical confirmation of the identity of putative meningococci was achieved using the API NH system (BioMerieux). For comparison with the meningococcal isolates, the following were used: N. gonorrhoeae isolates MS11 and FA1090; N. lactamica reference strain DSM 4691 (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany); and N. lactamica isolates 149, 154, 159, 178, 205, 207, 208, 265, 267, 270 and 271 (obtained during the course of the Bavarian carriage study), all of which possessed distinct genotypes (Alber et al., 2001 ).

MLST.
All meningococcal isolates were characterized by MLST by following previously published procedures (Maiden et al., 1998 ; Jolley et al., 2000 ). An ABI Prism 3700 automated sequencer (Perkin-Elmer) was used to separate the sequence reaction products, and the resultant data were assembled with the Staden suite of computer programs (Staden, 1996 ). Sequence types (STs) and alleles were assigned from the MLST database (http://neisseria.mlst.net/) and STs were assigned to complexes with the aid of the program BURST (Day et al., 2001 ). In the present work, a clonal complex was defined as a central or ‘founder’ genotype and all isolates sharing alleles at four of the seven MLST loci with the central genotype.

Sequence analysis of the cps cluster.
The intergenic region between the region E and the region D of the cps cluster was amplified by PCR using primers GH26R (5'-GGTCGTCTGAAAGCTTGCCTTGCTC-3', GenBank accession no. Z21508, position 4863–4839) and HC287 (5'-CGCGCCATTTCTTCCGCC-3', position 4162–4179) or HC344 (5'-GGATTGGACGAGCGAGAC-3', position 4431–4448); in these PCR reactions, the annealing temperature was 53  °C and the extension time was 1 min. The nucleotide sequences of the amplified products were obtained for both strands with primers GH26R and HC287. Nucleotide sequence data were analysed with the LASERGENE sequence-analysis software (Dnastar). The sequence of the cnl-1 allele has been submitted to the EMBL database and given accession number AJ308327.

Dot-blot hybridization.
Chromosomal DNA was prepared with the QIAamp DNA Mini kit (Qiagen), and 200 ng samples were spotted onto nylon membranes (Macherey-Nagel). Dot-blot hybridizations were performed as described previously (Hilse et al., 1996 ; Claus et al., 2000a ). A probe specific to the meningococcal ctrA gene (GenBank accession no. M57677) was generated by PCR using the primers DM1 (5'-GTGTTTAAAGTGAAATTTTA-3', position 143–162) and DM2 (5'-CTTAATTACTCACATTAATT-3', position 1332–1313). A probe specific to the novel restriction–modification (RM) system nmeST1117 (EMBL database accession no. AJ311178, REBASE enzyme nos 5036 and 5038) was generated by PCR using the primers HC315 (5'-TGAAATGGTTCGTTCTGTTATC-3', position 514–535) and HC314 (5'-TCGCTTAACTGCTAATGTATTG-3', position 1893–1872). The oligonucleotide HC350 (5'-GAG CTG TTC CAT GCC TAC CGA ATG TAC CAC CTC AAC GTG ACC CAA ATT AAC GGC AAC TTC-3', GenBank accession no. Z13995, position 4452–4511) was used as a probe to region B of the cps cluster. A probe to the serogroup-A-specific mynB gene (GenBank accession no. AF019760) was amplified with the primers NT2 (5'-ATACTTAATAACAGAAAATGGCG-3', position 1605–1627) and NT4 (5'-ATGATGGTAATGGGAAAAGAGT-3', position 3222–3201). Probes specific to the siaD genes of serogroups B and C were amplified with the primer pairs UE11A/UE13 and HC2/HC4, respectively, which are as follows: UE11A, 5'-CTAATTCTCATTAATTT-3'; UE13, 5'-GGAGATCAGAAGTCATAGTA-3'; HC2, 5'-AAATCTATAAATTGACTC-3'; HC4, 5'-GGAGATTTGTTTAGCT-3' (Vogel et al., 2001 ). The oligonucleotide probes W1063–1130 and Y1063–1130 were used for specific detection of the siaD genes of serogroups W-135 (EMBL database Y13970, position 1063–1130) and Y (EMBL database Y13969, position 1063–1130).


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STs of carrier isolates lacking genes necessary for capsule synthesis and transport
A total of 830 meningococci were isolated from the 8000 samples – an overall carriage rate of 10·4%. Of these, 148 isolates (17·8%) did not hybridize to the ctrA, mynB and siaD gene probes. To determine whether the lack of the ctrA, mynB and siaD genes was due to a replacement with a small DNA fragment of the operons necessary for capsule synthesis (region A) and transport (region C), PCR amplifications using primers GH26R and HC344 specific to the tex gene (region E) and the galE gene (region D) were performed on all 148 isolates. A product of approximately 400 bp was obtained with 136 of the 148 isolates, suggesting a replacement of regions A and C by an approximately 100 bp nucleotide sequence. Lineage assignment showed that 57 of the 136 isolates belonged to the ST-53 complex, 18 to the ST-1117 complex, 44 to the ST-198 complex, 12 to the ST-845 complex, four to the ST-1136 complex, and one to the ST-44 complex (Table 1).


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Table 1. Genotypes of the meningococcal clonal complexes harbouring cnl

 
Nucleotide sequences present in the cps gene cluster
The nucleotide sequence of the genome between regions E and D of the capsule locus was determined for the following: the ST-44-complex isolate, two examples each for the remaining clonal complexes shown to have the 100 bp replacement; two isolates of N. gonorrhoeae; and 12 N. lactamica isolates. A total of six distinct sequences were identified between region E and region D of the cps cluster, with 10 nucleotide substitutions (base positions 10, 11, 12, 14, 21, 22, 23, 25, 33, 44 and 90; Fig. 1) and three indels, comprising two single-site indels (base positions 12 and 14; Fig. 1) and one 4 bp indel present in one of the gonococcal isolates (base positions 110–113; Fig. 1). Each unique sequence was assigned an arbitrary locus name [cnl-1 to cnl-6 (cnl, capsule null locus); Fig. 1]. Three variants (cnl-1, cnl-2 and cnl-5) were 114 bp in length, two were 113 bp in length (cnl-3 and cnl-4), and one (cnl-6) was 118 bp in length. With the exception of the 4 bp indel in the cnl-6 sequence, all of the sequence polymorphisms were present in N. lactamica isolates. In this sample of isolates, the cnl-1 sequence was only found in meningococci, differing from the cnl-4 sequence present in N. lactamica by a single indel at base position 12, cnl-5 was unique to N. lactamica, differing from cnl-2 by a substitution at base position 44, and cnl-6 was unique to N. gonorrhoeae, differing from cnl-2 by the 4 bp indel (Fig. 1).



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Fig. 1. Nucleotide sequences of the capsule null locus (cnl) sequences present in N. meningitidis, N. lactamica and N. gonorrhoeae. The sequences are shown aligned and compared with the sequence from meningococcal isolate {alpha}14 (ST-53 complex; EMBL database accession no. AJ308327). The vertical numbers at the top of the figure indicate base positions, a period (.) indicates an identical base, and a hyphen (-) indicates a deletion. Nucleotide changes are indicated by the appropriate letter.

 
Southern hybridization experiments demonstrated the absence of region B of the capsule gene cluster in all of the meningococcal isolates harbouring the capsule null locus, except for the ST-43 isolate (ST-44 complex). None of the N. gonorrhoeae and N. lactamica isolates tested reacted with the probe derived from region B.

Inter-isolate relationships with other loci
Uniquely in the collection of 830 carried meningococci, all members of the ST-53 and ST-1117 complexes possessed a previously undescribed RM system, nmeST1117, located between the pheS and pheT genes. The isolates belonging to the ST-198, ST-1136 and ST-845 complexes possessed the RM system nmeBI at this position (Table 1). The nmeST1117 RM system (EMBL accession no. AJ311178) comprised two ORFs: ORF1 was 1386 bp in length, with an A+T content of 67·8 mol%, and ORF2 was 930 bp long, with an A+T content of 72·3 mol%. The deduced amino acid sequences of these ORFs were 40 and 42% identical, respectively, to the two ORFs of a probable type II RM system of Helicobacter pylori strain J99 (PIR accession nos C71907 and D71907). The relationship between complexes ST-53 and ST-1117 harbouring nmeST1117 was supported by the MLST data; four of seven loci were identical (Table 1). The ST-53, ST-1117, ST-198 and ST-1136 complexes shared the nmeAI RM system and prophage 37/1-26 (Claus et al., 2000a , b ), both of which were absent in the ST-845 complex. The ST-1136 and ST-198 complexes shared three alleles of the seven loci used in MLST. One isolate harbouring the capsule null locus belonged to the ST-43 (ST-44) complex; this isolate shared three MLST loci and nmeBI with ST-845 but possessed a distinct cnl sequence.


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The biological role of the meningococcal capsule remains unclear, but it is almost certain that, despite its importance in disease, this antigen did not evolve as a virulence determinant. Although there is little direct evidence that hydrophilic capsules promote person-to-person transmission, it has been suggested that they improve meningococcal survival in aerosols by reducing desiccation (Virji, 1996 ). This view is consistent with the biology of the gonococcus, which is acapsulate, and implies that some form of direct person-to-person contact may be important in the transmission of other acapsulate Neisseria spp. such as N. lactamica. The evidence presented here established that a substantial proportion of meningococci (16%) can persist in nasopharyngeal transmission systems without the genetic material necessary for capsule synthesis.

At least four genetically distinct groups of meningococci possessed a cnl sequence. One of these groups was represented by the members of the ST-53 and ST-1117 complexes. These meningococci contained the cnl-1 sequence, most shared identical alleles at three MLST loci (abcZ-16, adk-2 and pdhC-25), all had the nmeST1117 and nmeAI RM systems, none possessed the nmeBI RM system, and all possessed phage 37/1-26. A shared acapsulate common ancestor could be invoked to explain the relationships among these meningococci and their possession of a cnl sequence. Similarly, two of the clonal complexes with the cnl-2 sequence, ST-198 and ST-1136, shared many characteristics, suggesting that they might have shared a common acapsulate ancestor distinct from the putative ancestor of the ST-53 and ST-1117 complexes. Conversely, although the ST-845-complex isolates (cnl-2) and the ST-43 isolate (cnl-3) shared a number of MLST loci and other genetic characteristics, the presence of different cnl sequences suggested that these acapsulate isolates did not share a common origin with each other or with any of the other complexes described here. This was supported by the observations that ST-43 was part of the normally capsulate ST-44 complex (lineage 3) and, uniquely among the acapsulate meningococci described here, this isolate possessed region B of the cps gene cluster.

With the exception of the 4 bp insertion relative to the other cnl sequences found in gonococcal isolate FA1090 (base positions 110–113; Fig. 1), all of the nucleotide polymorphisms in the six cnl sequences were present in at least one of 12 N. lactamica isolates examined, two sequence changes being unique to this species (base positions 14 and 44; Fig. 1). Although the cnl-1 sequence was observed only in the meningococcus, it was a single nucleotide different from cnl-4, which was found in N. lactamica. Taken together, these observations suggested that either the cnl sequences have arisen in meningococci as a consequence of horizontal genetic exchange from N. lactamica, or several cnl sequences have been retained independently in the meningococcal population since speciation. The lack of regions A, B and C of the cps gene cluster in most of the cnl-containing meningococci implied that these organisms may have a genome segment of approximately 2·3 kb (region E) in place of the approximately 21 kb genome segment (region A through region B) present in capsulate meningococci.

Inspection of the MLST database showed that the clonal complexes harbouring the cnl sequences that were isolated in Bavaria have been identified elsewhere. Carried non-groupable ST-198-complex isolates were recovered from a population of children in the Gambia (J. M. MacLennan & R. Urwin, personal communication). A number of non-groupable ST-53-complex isolates were identified in a population of meningococci isolated from asymptomatic meningococcal carriers in the Czech Republic (Jolley et al., 2000 ). Furthermore, a serogroup C ST-53 variant caused a case of invasive disease in the UK in 1997 (M. C. J. Maiden, unpublished), indicating that meningococci of the ST-53 complex may be capable of reverting to capsule expression. These observations, together with the presence of cnl in a high proportion of the Bavarian carrier isolates and in at least four distinct genetic types, suggest that cnl can be a stable feature of at least some meningococcal genotypes.

We propose that cnl represents an alternative ‘allele’ for regions C and A of the meningococcal cps gene cluster which circulates among clonal complexes by horizontal genetic exchange. While the cnl sequences may resemble an ancestral state for meningococci, they may have been acquired from N. lactamica on at least four separate occasions post-speciation. The occurrence of this sequence mostly in particular clonal complexes suggests that the lineages represented by these complexes may be less dependent on the expression of a capsule for transmission than other meningococci. It is possible that the mode of spread of these lineages is similar to that of N. lactamica, but further investigation is required to test this idea.


   ACKNOWLEDGEMENTS
 
This work was supported by Deutsche Forschungsgemeinschaft grant VO718/3 (SPP 1047), the German Federal Ministry of Education and Research (project III/2 of the network ‘Pathogenomik’) and by Deutsche Gesellschaft für Hygiene und Mikrobiologie. M.C.J.M. is a Wellcome Trust Senior Research Fellow in Biodiversity. This publication made use of the MLST website located at http://neisseria.mlst.net/, developed by Man-Suen Chan, University of Oxford, and funded by the Wellcome Trust. We thank Rachel Urwin and Keith Jolley for their advice and assistance with this work. The Bavarian Government and the German Armed Forces are gratefully acknowledged for help during the collection of isolates.


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METHODS
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DISCUSSION
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Received 18 December 2001; accepted 11 February 2002.