ARTICLE IN PRESS

International Journal of Medical Microbiology 294 (2004) 75–82

www.elsevier.de/ijmm

REVIEW
Genetic lineages and their traits in Neisseria meningitidis
Ulrich Vogel, Heike Claus, Matthias Frosch
Institute for Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Strabe 2, D-97080 Würzburg, Germany

Abstract
Neisseria meningitidis is a model organism for the study of bacterial population biology, for genome sequencing and
pathogenicity research. In the recent years, our group has identified a variety of markers for hypervirulent lineages of
meningococci, which in part could be validated for typing purposes. Furthermore, carrier strain collections of
meningococci and N. lactamica were studied by multilocus sequence typing, and elucidated the impressive genetic
variability of those species. Characterisation of meningococcal carrier strains allowed to define the capsule null locus
(cnl) of meningococci, which frequently occurs among carrier isolates and renders strains constitutively
unencapsulated. This finding poses the question about the yet unclear role of the meningococcal polysaccharide
capsule in transmission and carriage. O-acetylation of the meningococcal polysaccharides is another variably expressed
trait in meningococci. We identified the genes responsible for O-acetylation of the serogroup C, W-135 and Y capsules,
and provided the genetic basis for understanding the variability of O-acetylation patterns in meningococci. The oatC
and oatWY genes proved to be the first genes identified to be responsible for O-acetylation of polysialic acid.
r 2004 Elsevier GmbH. All rights reserved.

Keywords: Neisseria meningitidis; Population structure; Differential distribution; Carriage; Capsule null locus; Capsule
O-acetylation

Content

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Markers of hypervirulent lineages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

The Bavarian N. meningitidis and N. lactamica strain collections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Capsule genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Capsular O-acetylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Corresponding author. Tel.: +49-931-2014-6802; Fax: +49-931-2014-6445.

E-mail address: uvogel@hygiene.uni-wuerzburg.de (U. Vogel).

1438-4221/$ - see front matter r 2004 Elsevier GmbH. All rights reserved.
doi:10.1016/j.ijmm.2004.06.027
 ARTICLE IN PRESS
76 U. Vogel et al. / International Journal of Medical Microbiology 294 (2004) 75–82
Introduction
Neisseria meningitidis, a Gram-negative b-proteobac-
terium commonly known as the meningococcus, colo-
nises the human nasopharynx in a significant proportion
of the population. Colonisation is age-dependent with
low carriage rates in infants and toddlers and increasing
rates in teenagers and young adults (Cartwright, 1995).
Carriage of pathogenic isolates infrequently leads to
systemic disease with peak incidence in infants and
teenagers. Although the total incidence of disease in
industrialised countries is low, meningococcal disease
has become a leading cause of septicaemia and
meningitis in the affected age groups in countries having
introduced Haemophilus influenzae b conjugate vaccine
(Schuchat et al., 1997). Despite the availability of potent
antimicrobial agents, meningococcal disease is consid-
ered a major burden for public health, because it is
communicable, and because the time course of disease
may be extraordinarily rapid leading to fatal outcome in
a substantial proportion of affected individuals even
after initiation of effective therapy. Furthermore,
sequelae of disease are frequently observed such as
hearing and limb loss, as well as kidney failure (Steven
and Wood, 1995). Epidemics of meningococcal disease
are observed in the meningitis belt in Africa, whereas in
industrialised countries most cases are sporadic, and
secondary cases are rare. Hyperendemic disease has
resulted in vaccination campaigns against serogroup C
disease (Miller et al., 2001). However, public health
systems are still awaiting the development of a generally
available vaccine preventing serogroup B disease.
Two obvious discrepancies stimulated research in the
field of meningococcal population structure in the last
three decades (for reviews see Vogel, 2001; Vogel and
Claus, 2003; Vogel and Frosch, 2002). One is the
observation of high meningococcal carriage rates con-
trasting low incidences of disease. The other is the
observation of extensive horizontal gene transfer con-
trasting clonal expansion of successful lineages with a
limited degree of genetic and antigenic variation during
spread. The key to those questions has been the
application of phylogenetically valuable typing systems
to unbiased international strain collections comprising
either disease or carriage isolates. In the 1980s, multi-
locus enzyme electrophoresis was used (Caugant et al.,
1986a, b, 1987, 1988; Selander et al., 1986). In the late
1990s, multilocus sequence typing (MLST) was devel-
oped using meningococci as the model organism
(Maiden et al., 1998). The latter approach has proven
to be extremely valuable and flexible resulting in its use
for different bacterial and fungal species (see
www.mlst.net). Both approaches revealed that patho-
genic meningococcal lineages continuously spread at a
global level. The frequency of carriage of hypervirulent
lineages correlates to their proportion among the total
number of cases (Jolley et al., 2000); however, hyper-
virulent lineages account only for a minority of strains
found in healthy carriers (Caugant et al., 1988). Despite
of global spread over decades, hypervirulent lineages
display a remarkable degree of genetic and antigenic
stability (Caugant et al., 1986b). Although variations in
outer membrane proteins are observed (Achtman, 1994;
Linz et al., 2000; Morelli et al., 1997; Zhou et al., 2001),
and although the capsular antigen may switch due to
clonal replacement or horizontal exchange of capsule
synthesis genes (Alcala et al., 2002; Swartley et al., 1997;
Vogel et al., 2000a), the typing patterns of genetic
lineages remain remarkably stable. Microevolution-
induced changes may be purified constantly by
bottlenecking and maintenance of genotypes represent-
ing the fitness peak of a lineage. Our group there-
fore attempted to define stable markers for hyperviru-
lent lineages. We furthermore investigated the hypoth-
esis that restriction-modification systems contribute
to the stability of lineages. We investigated the popula-
tion structure of N. meningitidis and a closely
related organism, N. lactamica, which induces protec-
tive immunity against meningococcal disease in
children (Coen et al., 2000; Kremastinou et al., 1999),
by the application of sequence typing to strain
collections gathered during an extensive swabbing
campaign.
A traditional focus of our laboratory is the genetics of
the meningococcal capsular polysaccharide. Thirteen
different capsular polysaccharides have been described
for N. meningitidis, five of which are associated with
most cases of meningococcal disease, i.e. the serogroups
A, B, C, W-135, and Y. Serogroup B, C, W-135, and Y
capsules contain sialic acid and share the genes
necessary for its biosynthesis. Those genes are organised
in the sia operon (region A of the cps locus) (Edwards et
al., 1994; Frosch et al., 1989) (reviewed in Vogel et al.,
2001; Vogel and Claus, 2000). Serogroups are associated
to hypervirulent lineages. The emergence of serogroup
variants, e.g. within the ST-11 complex has been
observed especially after prolonged clonal spread in a
population or following vaccination campaigns (Alcala
et al., 2002). It is unclear whether those capsule variants
are the result of clonal replacement or switching due to
horizontal gene transfer. We recently described the first
scenario in which the latter hypothesis was the most
likely one, because there was a direct epidemiological
linkage of a case and a carrier with clonally identical
isolates exhibiting different serogroups (Vogel et al.,
2000a).
In serogroups C, W-135, and Y meningococci, sialic
acid may be modified by O-acetylation (Bhattacharjee et
al., 1975, 1976), which in the case of serogroup C
meningococci alters the immunogenicity of the poly-
saccharide (Richmond et al., 2001). Although O-
acetylation of meningococcal capsules has been known
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U. Vogel et al. / International Journal of Medical Microbiology 294 (2004) 75–82
since the seventies, its genetics and population biology
remained obscure until it was resolved recently by our
group (Claus et al., 2004).
Another aspect of capsule genetics we addressed was
the earlier observation of transiently unencapsulated
strains among carrier isolates (Dolan-Livengood et al.,
2003; Sadler et al., 2003). The assembly of a compre-
hensively typed carrier strain collection comprising 830
strains allowed us to determine the mechanisms leading
to loss of capsule expression in a population-based
approach. Furthermore, we identified constitutively
unencapsulated meningococci harbouring the capsule
null locus (cnl). A significant proportion of carried
meningococci belong to this group of strains. Their
existence stimulates questions concerning the role of the
capsular polysaccharide during transmission of the
bacteria.
Markers of hypervirulent lineages
Hypervirulent lineages of meningococci display the
capability to expand clonally. Despite evidence for
microevolution and horizontal gene transfer, founder
genotypes remain remarkably stable over time. By using
DNA-sequence analysis, representational difference
analysis, and isolation of plasmids, we were able to
identify and characterise several markers for hyperviru-
lent lineages. The results of these experiments are
summarised in Table 1. A variety of restriction-
modification systems, plasmids, and other markers
could be identified which are linked to lineages of
meningococci (Claus et al., 2000a, b; Hilse et al., 2000;
Vogel et al., 2000b). A more detailed overview of
meningococcal plasmids is provided in a thesis available
at http://opus.bibliothek.uni-wuerzburg.de/opus/voll-
texte/2003/701/. One of the markers identified by our
group, a single nucleotide polymorphism at position 640
of the fumC gene, is now widely used for the
identification of the ET-15 clone, a derivative of the
ST-11 complex responsible for a variety of outbreaks
and hyperendemic situation in several countries world-
wide (Vogel et al., 1999). We also generated monoclonal
antibodies against the restriction-modification system
NmeDI which exhibits a 100% negative predictive value
for the ST-8 and ST-11 complexes (Claus et al., 2003),
which are responsible for most of the cases of serogroup
C disease world-wide. The identification of differentially
distributed restriction-modification systems was a no-
table finding (Claus et al., 2000a, b). In other model
systems such as Helicobacter sp., DNA restriction
contributes to ecological separation of clones (Ando
et al., 2000; Aras et al., 2002). We therefore also
attempted to identify an effect of NmeBI on DNA
transfer in a co-cultivation system. The effect was rather
77
weak and the presence of NmeBI did not abrogate DNA
transfer (Claus et al., 2000a). We therefore assume that
DNA restriction is only a minor contributor to the
separation of meningococcal lineages, which seems to be
mainly an effect of clonal expansion, fitness peaks and
selection.
The Bavarian N. meningitidis and N. lactamica
strain collections
Local cross section carrier isolate collections of N.
meningitidis are valuable for several reasons: (i) they
provide a snapshot view on the population structure of
meningococci in comparison to strains recovered from
invasive disease, (ii) typing data can be used for
geographical and longitudinal comparisons, and serve
for international surveillance of pathogenic lineages, (iii)
well-characterised strain collections of a genetically
diverse organism are invaluable resources for hypoth-
esis-driven research. In collaboration with the Bavarian
government and the German Federal Armed Forces we
assembled a strain collection of 830 isolates recovered
from 8000 volunteers during a 5-months swabbing
campaign conducted throughout Bavaria. MLST of all
isolates was performed in collaboration with the group
of Martin Maiden in Oxford (manuscript in prepara-
tion). The results of the MLST study have not been
published yet, but the data could already be used to
identify the cnl (Claus et al., 2002) (see below), and to
validate the monoclonal antibody 3/9-2 specific to the
ST-11/8 complexes (Claus et al., 2003). Furthermore,
our data allowed to select strains for genome sequencing
efforts within the frame of the competence network
PathoGenoMik funded by the German Federal Ministry
of Education and Research. The first genome of an
apathogenic meningococcal isolate (ST-53 complex) is
currently annotated.
The swabbing campaign also provided the possibility
to set up a collection of N. lactamica isolates. N.
lactamica is related to N. meningitidis. The apathogenic
organism colonises the nasopharynx in age groups not
harbouring meningococci extensively and is thought to
elicit protective immunity to meningococcal disease. N.
lactamica has been considered a potential vaccine
candidate (Oliver et al., 2002). Therefore, knowledge
of the population structure of this organism is highly
desirable. We performed sequence typing of a selection
of isolates and demonstrated a striking genetic diversity
driven by recombination (Alber et al., 2001). In 26
isolates collected in three Bavarian towns, we found 17
different genotypes. Twelve of the 17 genotypes were
observed only once. In most cases, for which an identical
genotype was observed, an epidemiological linkage
could be postulated. This finding suggests rapid
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78 U. Vogel et al. / International Journal of Medical Microbiology 294 (2004) 75–82

Table 1. Markers of lineages of N. meningitidis

Lineage Predominant Genetic element Category Comment Reference

serogroup

ST-11 complex, ST- C nmeDI R-Ma Insertion site: pheS/ Claus et al.
8 complex pheT intergenic region. (2000a)
Monoclonal antibodies
available for typing
nmeAI R-M Also present in Claus et al.
serogroup A (2000a)
meningococci
37/1–7 putative R-M IS1655 insertion in orf 2 Claus et al.
specific to ST-11 (2000b)
complex
37/1–26 Prophage fragment 6,7 kb were sequenced in Claus et al.
the original paper (2000b)
(GenBank accession
No. AJ278707)
pJS-B Plasmid Plasmid integration into Claus et al.
chromosome via (2000b)
inverted repeat region
IR1/IR2
ST-11 complex C IS1655 in orf 2 of Insertion sequence Claus et al.
37/1-7 (2000b)
ST-32 complex, ST- B nmeBI R-M Insertion site: pheS/ Claus et al.
41/44 complex pheT intergenic region (2000a)
ST-1-7 A nmeAI R-M Claus et al.
(2000a)
Subgroup VI A pJS-A Plasmid 2-kb plasmid of Hilse et al.
(isolated in former unknown function (2000)
GDR)b
ST-11 complex C Position 640 of Housekeeping gene Single nucleotide Vogel et al.
clone ET-15 fumC gene polymorphism specific (2000b)
to the ET-15 clone in
comparison to other
strains of the ST-11
complex
ST-53, ST-1117, Unencapsulated Capsule null locus — Strains lack the genes Claus et al.
ST-198, ST-1136, (cnl) necessary for capsule (2002)
ST-845 and related synthesis, modification,
sequence types and transport
a
R-M: restriction-modification system.
b
Sequence types not available.

microevolution in N. lactamica. In light of the tremen-
dous genetic diversity of N. lactamica it will be
challenging to find conserved antigens in this species,
which elicit protective immunity against N. meningitidis.
Furthermore, the results suggest that selection of an N.
lactamica strain for a vaccine development project has
to be handled with great care.
We provided the group of Martin Maiden with 48
isolates for their MLST approach on N. lactamica,
which used primers identical to the ones of the
meningococcal MLST scheme (http://pubmlst.org/Neis-
seria/). The results of this approach will provide
evidence for the suitability of MLST datasets with
regard to taxonomy. Furthermore, MLST data from
several countries including Germany allowed to select a
representative Neisseria lactamica strain for a genome
sequencing project (Martin Maiden, personal commu-
nication).
Capsule genetics
The capsule of meningococci is the most important
pathogenicity factor. The antigenic diversity of capsular
polysaccharides is determined by the biosynthesis region
within the chromosomal cps locus, the region A.
Exchange of genes of the region A by horizontal gene
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U. Vogel et al. / International Journal of Medical Microbiology 294 (2004) 75–82
transfer results in capsule switching, which probably is a
rare event in comparison to capsule phase variation.
Capsule phase variation has been demonstrated in vitro
for serogroup B meningococci (Hammerschmidt et al.,
1996; Hilse et al., 1996). The mechanisms behind
phase variation are reversible insertion of the insertion
sequence IS1301 and slipped strand mispairing in
the region A. Reversible loss of encapsulation
allows for tight adhesion to epithelial cells. There is
no experimental or ecological evidence for the function
of the capsule during transmission. However, the
capability to produce capsules of most meningococcal
lineages irrespective of their pathogenic potential
suggests that the capsule is a prerequisite during
some steps of the life cycle of the bacterium. Genotypic
and phenotypic characterisation of the Bavarian
meningococcal carrier strain collection revealed that
30% (n=166) of all strains, which harbour a polysialyl-
transferase gene necessary for the synthesis of either of
the serogroup B, C, W-135, or Y polysaccharides,
were unencapsulated (Claus et al. unpublished). We
have now finished a comprehensive population-based
survey on 115 of 166 strains to characterise the
mechanisms leading to loss of the capsule (Weber et
al. manuscript in preparation). Interestingly, 34% of the
115 strains analysed so far displayed an irreversible
phenotype with regard to their lack of encapsulation.
This finding raises the question whether irreversibly
unencapsulated strains are readily eliminated or main-
tained in the population.
The analysis of the Bavarian meningococcal carrier
strain collection demonstrated that 16.4% of all isolates
lacked all regions of the cps locus with the exception of
region D (lipopolysaccharide biosynthesis) and the
region E (tex gene), both of which are also present in
commensal Neisseria sp. and N. gonorrhoeae (Claus et
al., 2002). Between region E and region D, encapsulated
meningococci harbour region A (polysaccharide synth-
esis) and region C (capsule transport). Instead, consti-
tutively unencapsulated meningococcal lineages
exhibited a 113- or 114-bp intergenic region occurring
in three different alleles. This so-called cnl was highly
related to the homologue regions in N. lactamica and N.
gonorrhoeae. We analysed the DNA sequence of the tex
gene in an ST-53 isolate, in order to determine the
source of cnl, because horizontal gene transfer resulting
in replacement of the cps should involve also the
adjacent tex gene. In the ST-53 isolate, however, the
tex gene was most related to meningococcal tex genes.
There are two possible interpretations of this finding: (i)
the tex gene sequences are phylogenetically uninforma-
tive because consecutive events of horizontal gene
transfer blurred the original event, and (ii) cnl lineages
represent ancient unencapsulated genotypes, which have
been selected against after the emergence of encapsu-
lated variants.
79
There were five founder sequence types and 25 related
sequence types with the cnl. The finding that cnl was not
randomly distributed, although there was evidence for at
least infrequently occurring horizontal gene transfer of
cnl into encapsulated genetic backgrounds, suggested
that cnl provided for a fitness benefit only in some
genotypes. This benefit may arise from specific antigenic
and functional combinations of components of the outer
membrane. Again this finding points to the unresolved
question of the ecological role of the meningococcal
capsular polysaccharide, which seems to be dispensable
for the transmission of some meningococcal lineages as
well as of N. lactamica. It might be discussed that
unencapsulated meningococci, N. gonorrhoeae, and N.
lactamica are transmitted via direct person-to-person
contact, whereas the aerosol transmission of meningo-
cocci requires protection from desiccation by the
capsular polysaccaride. However, experimental evidence
for this suggestion is lacking.
Capsular O-acetylation
It is known from structural studies that the polysialic
acid structures of serogroup C, W-135, and Y meningo-
cocci are O-acetylated (Bhattacharjee et al., 1975, 1976).
Serogroup B meningococci lack this property, although
Escherichia coli K1, which similarly expresses a-2,8-
linked polysialic acid, is O-acetylated (Orskov et al.,
1979). O-acetylation alters the physico-chemical proper-
ties of the polysaccharide (Klein and Roussel, 1998). O-
acetylation in meningococci is interesting from a
population biological point of view, because O-acetyla-
tion occurs only in subsets of strains (Longworth et al.,
2002). It is furthermore of interest, because O-acetyla-
tion of polysialic acids is of general biological relevance
also in mammalian systems. Genes encoding sialic acid
O-acetyltransferases have not been described until now.
This lack of information might be due to the fact that
functional purification of those O-acetyltransferases is
very difficult. We were able to identify the O-acetyl-
transferase genes of serogroup C, W-135, and Y
meningococci, termed oatC and oatWY, respectively
(Claus et al., 2004). The major features of those genes
are summarised in Table 2. Our data will allow to
determine the role of meningococcal O-acetyltrans-
ferases for pathogenicity. They will help to stimulate
the search for other bacterial or mammalian polysialic
acid acetyltransferases. Purification of those enzymes
will provide tools for in vitro modification of polysialic
acids. It is unclear why O-acetylation of meningococci is
either a matter of phase variation (in the case of oatC)
or linked to certain genotypes (in the case of oatWY).
It will be therefore challenging to rule out the eco-
logical role of this biochemical modification in
 ARTICLE IN PRESS
80 U. Vogel et al. / International Journal of Medical Microbiology 294 (2004) 75–82
Table 2. Properties of O-acetyltransferase genes in serogroup C, W-135, and Y meningococci
OatC
Serogroup C W-135, Y
Gene length (bp) 1383
Deduced protein length (aa) 461
A+T content 71%
Operon Transcribed with sia operon (synthesis of
polysialic acid)
Consensus motif None
Phase variation Yes, homopolymeric tracts
Deletions, insertions Not observed
person-to-person transmission and meningococcal
spread within human populations.
Conclusion
This review summarised the results obtained by our
group and collaborators during the course of the
priority programme 1047 (Ecology of Bacterial Patho-
gens: Molecular and Evolutionary Aspects). Despite of
extensive intra- and interspecific horizontal gene trans-
fer, several clonal groupings of meningococci displayed
a sufficient degree of clonality for the establishment of
stable and epidemiologically useful markers. N. lacta-
mica was shown to be more panmictic than N.
meningitidis. The reasons for this attribute are unclear,
but the finding has implications for future work on the
induction by N. lactamica of immunity against menin-
gococci. Several clonal lineages of meningococci were
shown to be constitutively unencapsulated, and might be
transmitted by direct person-to-person contact. How-
ever, experimental evidence for the role of the poly-
saccharide capsule in droplet transmission and for its
benefit in specific clonal lineages is still lacking.
O-acetylation of meningococcal capsules was shown to
be driven by genes physically linked to the polysacchar-
ide synthesis genes. Different mechanisms of variation
of O-acetylation were disclosed, which determine the
O-acetylation phenotype during meningococcal circula-
tion. After the cloning of the respective genes, the
structure of the O-acetyltransferases and their relevance
for meningococcal life-style will be subject to further
studies.
Acknowledgements
We thank the German Research Foundation and
Jürgen Heesemann for initiating and maintaining the
OatWY
636
212
68%
Transcribed with sia operon (synthesis of
polysialic acid)
NodL–LacA–CysE family of O-
acetyltransferases
Not observed
Gene deletions and insertion of IS1301
observed in defined lineages
priority programme 1047 (Ecology of Bacterial Patho-
gens: Molecular and Evolutionary Aspects) over a 6-
years period. We are indebted to all our co-workers,
students, and technicians, who were involved in the
programme. The following collaborators are thanked
for their invaluable contributions: Martin Maiden,
Keith Jolley and Rachel Urwin, Oxford; Mark Acht-
man, and Giovanna Morelli, Berlin; Sebastian Suer-
baum, Hannover (formerly Würzburg); Dominique
Caugant, Oslo; Ray Borrow and Emma Longworth,
Manchester.
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