Living in a changing environment: Insights into host adaptation in Neisseria meningitidis from comparative genomics

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International Journal of Medical Microbiology 297 (2007) 601–613 www.elsevier.de/ijmm

Living in a changing environment: Insights into host adaptation in Neisseria meningitidis from comparative genomics Christoph Schoena,, Biju Josepha, Heike Clausa, Ulrich Vogela,b, Matthias Froscha,b a

Institut fu¨r Hygiene und Mikrobiologie, Universita¨t Wu¨rzburg, Josef-Schneider-Str. 2, Bau E1, D-97080 Wu¨rzburg, Germany Nationales Referenzzentrum fu¨r Meningokokken, Institut fu¨r Hygiene und Mikrobiologie, Universita¨t Wu¨rzburg, Wu¨rzburg, Germany

b

Received 18 April 2007; received in revised form 25 April 2007; accepted 25 April 2007

Abstract Neisseria meningitidis (the meningococcus) colonizes the human nasopharynx of about 10% of the human population. However, for reasons that are still mostly unknown meningococci occasionally enter the cerebrospinal fluid leading to often fatal bacterial meningitis especially in children and young adults. The genetic basis for the observed differences in the pathogenic potential of different strains has only partially been unravelled so far. With the advent of whole genome sequencing technologies, complete genome sequences from three pathogenic meningococcal strains have become available and allow for a comprehensive analysis of the genomic and genetic differences occurring within this species. In this review, the general properties of the meningococcal genomes so far sequenced is given with an emphasis on the chromosomal rearrangements that have occurred, and the genomic islands and prophages that have been identified. The concomitant development of microarray technology for comparative genome hybridization studies of a large set of different meningococcal isolates as well as strains from other Neisseria species has extended our understanding of meningococcal population genetics on a genome-wide scale thus bridging the gap between meningococcal epidemiology and genomics. Finally, we briefly discuss the potential impact of meningococcal life style on its genome architecture and how in turn this genomic make-up might lead to a virulent phenotype making N. meningitidis an accidental pathogen. The overall properties of the meningococcal genome are characterized by genomic variability and instability, resulting in increased functional flexibility within this species. r 2007 Elsevier GmbH. All rights reserved. Keywords: Neisseria meningitidis; Comparative genomics; Microarray; Phase variation; Adaptation; Life-style

Introduction Neisseria meningitidis (the meningococcus) is a facultative commensal of the human nasopharynx and, as such, is carried by around 10% of the adult population (Claus et al., 2005; Cartwright et al., 1987). Corresponding author.

E-mail address: [email protected] (C. Schoen). 1438-4221/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2007.04.003

However, some strains are able to cross the mucosal barrier getting access to the bloodstream and subsequently the blood brain barrier causing septicaemia and meningitis, a major cause of disease worldwide (Rosenstein et al., 2001). Meningococcal meningitis is a lifethreatening disease, almost always fatal in the absence of prompt medical intervention and leading to long-term sequelae in many patients who survived thanks to timely antibiotic therapy. Interactions between N. meningitidis

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and its human host alternate between asymptomatic carriage, transmission from person to person via aerosols or secretions, and invasive infection. Strains of N. meningitidis have been classified into 12 or 13 serogroups based on the structure of the capsule (Frosch and Vogel, 2006), but invasive meningococcal infection is limited to the serogroups A, B, C, Y, and W-135. N. meningitidis serogroup A has caused major epidemics in the African meningitis belt in several subSaharan countries and in Asia, while in the industrialized countries 30–70% of the disease is caused by serogroup B strains. Serogroup C meningococci account for small-scale outbreaks worldwide. Cases of serogroup Y have been increasing in recent years which accounts for over 30% of the cases in USA (Rosenstein et al., 1999). Serogroup W-135 was associated with large outbreaks among Hajj pilgrims in Saudi Arabia in 2000 and 2001 and also in Burkina Faso in 2002 (Taha et al., 2000). The other serogroups of meningococci are rarely associated with disease with the exception of serogroup X being associated with an outbreak in Africa (for a recent review on the epidemiology of N. meningitidis see Maiden and Caugant, 2006). Currently, only vaccines against the serogroup A, C, W-135, and Y meningococci based on capsular polysaccharides are available, but no effective vaccine has been developed against serogroup B meningococci (Harrison, 2006). Capsule null locus meningococci lacking all genes necessary for capsule synthesis and transport have been encountered frequently in healthy carriage (Claus et al., 2002, 2005) and only rarely in invasive disease (Hoang et al., 2005; Vogel et al., 2004). However, a clear judgement of the pathogenicity of these strains cannot be made at the moment considering a recent report on three cases due to capsule null locus strains in patients from Burkina Faso (Findlow et al., 2007). Genomic typing methods such as multilocus enzyme electrophoresis typing (MLEE) and multilocus sequence typing (MLST) have shown that almost all cases of invasive disease are caused by a limited number of strains of genetically related bacteria which have been referred to as hyperinvasive lineages (Yazdankhah et al., 2004; Maiden et al., 1998; Caugant et al., 1986). In spite of extensive experimental studies with respect to putative virulence factors in N. meningitidis, the answer to the question why only some strains belonging to these hyperinvasive lineages cause the invasive and most often fatal meningitis in some individuals eludes us even today. In the recent years substantial information has been gathered on the susceptibility of the host, which is determined by a variety of genetic polymorphisms in genes influencing immune function and coagulation (Emonts et al., 2003; Tzeng and Stephens, 2000). From the reports available until now genetic predisposition appears to be a multifactorial one, with different factors

triggering either higher susceptibility or severity of disease. The interpretation is even more difficult due to the fact that no systematic analyses are available on differences between clonal lineages of meningococci and their attack rates among individuals with varying genetic predispositions. As outlined above the meningococcal lineage must play a role, given the well-studied bias of invasive disease to only few lineages worldwide. For efficient survival in the blood stream, the role of the meningococcal capsule has been well documented (Vogel and Frosch, 1999), and iron acquisition systems have also been shown to enhance this capability (Perkins-Balding et al., 2004). Several proteins like the type IV pili and opacity proteins have been shown to be important for the interaction of meningococci with host cells which initiates the further downstream processes of meningococcal pathogenesis (Merz and So, 2000). Comparative genomics has already played a key role in the elucidation and understanding of factors that might contribute to the observed differences in the pathogenic potential between carrier strains and strains belonging to such hypervirulent lineages. With regard to complement activity, the molecular basis of increased killing of some strains has been well characterized recently. Strains with phospho-ethanolamine at the 6-position of the second heptose of the lipooligosaccharide bound more complement factor C4b and were more susceptible to serum activity than strains with this substitution at the 3-position (Ram et al., 2003). Furthermore, high expression of the outer membrane lipoprotein GNA1870 currently under evaluation as a broad-spectrum meningococcal vaccine candidate and expression of a GNA1870 allele permissive to regulatory factor H binding promoted serum resistance (Madico et al., 2006). This review will give an overview on the genome properties of the three pathogenic strains that have been fully sequenced so far and discuss possible evolutionary forces that might have shaped the meningococcal genomes.

The sequenced genomes of N. meningitidis To date, the genome sequences of N. meningitidis serogroup A strain Z2491 (Parkhill et al., 2000b), serogroup B strain MC58 (Tettelin et al., 2000) and serogroup C strain FAM18 (Bentley et al., 2007) have been published. In addition, the annotated genome of the other pathogenic Neisseria species, Neisseria gonorrhoeae has been made publicly available (GenBank accession number AE004969) as well as the finished genome sequence of the obligate commensal species Neisseria lactamica (genome sequence available from The Wellcome Trust Sanger Institute homepage via http://www.sanger. ac.uk/Projects/N_lactamica/). Our group has recently

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sequenced several isolates from healthy carriers, the annotation and analysis of the genome of a sequence type 53 isolate is almost finished and will be reported soon (Schoen et al., unpublished data). In this review we focus on the general features of the N. meningitidis serogroup A strain Z2491, the serogroup B strain MC58 and the serogroup C strain FAM18 genomes based in part on our own analyses of the primary sequences as well as the published data as given in the accompanying scientific publications (Bentley et al., 2007; Parkhill et al., 2000b; Tettelin et al., 2000). Table 1 gives a short overview, and Fig. 1 shows a multiple whole-genome alignment of these three genomes.

Comparison of meningococcal chromosome structure The size of the circular chromosome is about 2.2 Mb and similar for all three strains and all have an average Table 1.

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GC content of about 52%. Strain MC58 has the largest chromosome which is almost 100 kb larger than the other two mainly due to a large duplication of about 30 kb comprising 36 coding sequences (CDS) (NMB1124NMB1159 duplicated in NMB1162-NMB1197) and the acquisition of two additional islands of horizontally transferred DNA (IHT-B and -C) 17.1 and 32.6 kb in size, respectively, both being absent in the genomes of strains Z2491 and FAM18 (Fig. 1). Although Neisseria is considered to be an haploid organism like all other prokaryotes, a recent publication has challenged this common notion in providing evidence that N. gonorrhoeae is in fact homo-polyploid with about 4–10 genome equivalents per diplococcal cell (Tobiason and Seifert, 2006). Being closely related to N. gonorrhoeae it would therefore not come as surprise if future studies might prove this also to be true for N. meningitidis. All three genomes are largely collinear to each other with three reciprocal inversions around the origin of replication (Bentley et al., 2007) (Fig. 1). The first inversion event with respect to strain Z2491 is closest to

Comparative overview of the sequenced meningococcal genomes

Strain

Z2491a

MC58a

FAM18a

Reference Serogroup Sequence type Clonal complex

Parkhill et al. (2000b) A 4 ST-4

Tettelin et al. (2000) B 74 ST-32

Bentley et al. (2007) C 11 ST-11

General information Genome size (bp) G+C content (%) GenBank accession

2184406 51.8 AL157959

2272351 51.5 AE002098

2194961 51.6 AM421808.3

Functional RNAs Number of tRNAs Number of rRNA operons

58 4

59 4

59 4

Coding sequences Putative number Average CDS length (bp) Coding area (%) Putative pseudogenes

1993 902 78.9 84

2063 871 79.1 92

1975 918 80.2 58

Repeat elementsb DNA uptake sequences dRS3 CREE IS elements

1892 772 286 56

1935 756 262 52

1888 718 274 60

Mobile DNA GEI Prophages (total)c Restriction/modification systemsd

0 X9 25

1 X9 22

0 X10 22

a Unless stated otherwise, the numbers given for the strains Z2491, MC58, and FAM18 are based on the GenBank accession numbers AL157959, AE002098, and AM421808.3, respectively. b Numbers for repeat elements were taken from Bentley et al. (2007). c Numbers are lower estimates and include also defective forms. d Numbers are based on the number given in the REBASE database (Roberts et al., 2007).

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Fig. 1. Multiple alignment of the three meningococcal genomes so far fully sequenced using the programme Mauve (Darling et al., 2004). Locally collinear blocks of DNA are depicted in the same colours. Equally coloured blocks on different sides of black lines corresponding to the respective genome sequence indicate chromosomal inversions. The chromosomal locations of some prophages and islands of horizontally transferred DNA are depicted by black bars. Larger chromosomal regions being collinear between the genomes are depicted by arrows with identical colour. Due to space limitations, not all prophages and islands of horizontal transfer could be shown. For details see text.

the origin of replication and may be caused by a recombination event between repeat arrays in Z2491 with one array flanking the pilin gene pilC2 while the other is adjacent to genes involved in pilus retraction (pilTU). As pilC2 is thus adjacent to pilTU in MC58 and in FAM18 and both genes may be co-transcribed this rearrangement may have an effect on pilus phenotype (Bentley et al., 2007). The insertion of a copy of the neisserial filamentous phage Nf1 (Kawai et al., 2005) in the repeat array directly upstream of pilC2 in strain FAM18 (Nf1-C1 in Fig. 1) represents another level of variability at this locus. As this phage has been associated with strains that cause disease (Bille et al., 2005) (see below) this inversion is also likely to alter the fitness of the bacterium with respect to its interaction with the human host or with other strains of N. meningitidis. A second inversion is probably caused by a recombination event between copies of IS1106 in FAM18 and is associated with the insertion of another IHT containing a putative type II restriction–modification system in FAM18 (IHT-D in Fig. 1). The third is the most complex of the three inversion events and comprises almost half of the chromosome being inverted in strain MC58 with respect to the other two genomes. It has been proposed that a circular plasmid containing IHT-B as well as IHT-C together with the Nf2-B3 prophage has been integrated into the ancestor of strain MC58. Compared to other more complex scenarios it would require only two events of homologous recombination and would explain elegantly not only the origin of this large inversion but also the origin of IHT-B and -C which are specific only to MC58 but absent from the other two genomes (for details see Kawai et al., 2006). The presence of three short sequence homologies

(NMB1752-1755 in IHT-C; Fig. 1) with the gonococcal cryptic plasmid (Snyder et al., 2005) adds further observational evidence to this explanation. In addition to these inversions, a thorough computational analysis by Kawai et al. (2006) revealed a number of other genome rearrangements and stressed the role of mobile genetic elements in the formation of complex genome polymorphism in N. meningitidis. For example, an IS-mediated replicative inversion of a 29-kb region comprising a putative composite transposon (Tn in Fig. 1) was found to be translocated in the genome of strain MC58 relative to the other two genomes.

Comparison of meningococcal gene content The number of CDS based on the primary annotation as deposited in GenBank in N. meningitidis serogroup A strain Z2491 is 1993, 2063 in serogroup B strain MC58 and 1975 in serogroup C strain FAM18, respectively (Table 1). However, not at least due to the different computational approaches applied, these numbers might be subject to future changes in ongoing semiautomated re-annotations and should be taken with a grain of salt. Based on reciprocal BLASTP best-hits, around 61% of the CDS represent the neisserial core genome and around 9% of the CDS are specific to the respective strains (Fig. 2). Within this putative core genome, around 90% of the proteins have been assigned to approximately 1200 different clusters of orthologous groups (COGs) with 10% of these having no specified function. Genes involved in basic biological processes like energy production, amino acid synthesis,

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Fig. 2. Venn diagram of the encoded proteomes in the three meningococcal genomes based on the primary annotation and bi-directional best-hit BLASTP analysis (Altschul et al., 1990) with cut-offs of at least 50% sequence identity over at least 50% sequence length.

translation, and DNA replication and repair form the largest family of COGs with a functional assignment. The fact that meningococci are fastidious bacteria being able to grow only on a restricted range of carbon sources finds its reflection also in the metabolic blueprint deduced from the meningococcal genome sequences. The encoded metabolome suggests that N. meningitidis is able to degrade glucose, the amino acids serine, proline, and glycine, and the organic acids acetate, gluconate, glutamate, lactate, malate, oxaloacetate, and pyruvate. Genome analysis also revealed that N. meningitidis has a large number of ABC transporters as well as systems for scavenging iron, including previously recognized haemoglobin, transferrin, and lactoferrin-binding proteins, as well as additional systems for iron acquisition, including siderophore acceptor and utilization homologues. Regarding those categories that are potentially important for the pathogenesis of meningococcal disease, 9% of all functionally assigned CDS of the core genome might be involved in cell wall/membrane biosynthesis, and 5–6% for proteins involved in transport and binding functions. In contrast to the meningococcal core genome, almost two third of the CDS unique to each strain do not belong to any COG, and about 10% code for proteins involved in replication, recombination and repair, indicating that they might belong to mobile genetic elements such as IS elements and prophages. Besides these CDS, the genome of N. meningitidis also harbours a substantial number of pseudogenes. In particular, in a recent comprehensive analysis of pseudogenes in prokaryotes among the 64 genomes compared including 38 genomes from pathogenic species, the meningococcal genomes had the second and third highest proportion of pseudogenes, respectively (Liu et al., 2004). Due to their size, many large gene families were also among the top pseudogene families like the divergent family of ABC transporters. However, in the two strains MC58 and Z2491 the analysis by Liu et al. (2004) revealed 26 and 22 copies of

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transposase pseudogenes, respectively, while both strains contain only 11 and 5 copies of functional transposase genes. In addition, in strain MC58 transposase pseudogenes have been found in most of the 29 remnant insertion sequences thus indicating that N. meningitidis strains probably undergo high selection pressure against transposases. Together with the high number of IS elements ranking among the highest found for a bacterial species with a free-living replicative stage, this is indicative of an organism undergoing adaptive evolution towards a host-restricted life-style (Moran and Plague, 2004). Most IS elements are mainly members of at least four specific families (Mahillon and Chandler, 1998).

Repetitive DNA One of the most obvious characteristics of the neisserial genomes is the abundance and diversity of repetitive DNA which contributes to genome fluidity and phenotypic variability (see below). Altogether, approximately 20% of the meningococcal chromosome has been found to be included in repeats, a number that again ranks among the highest given for an eubacterial species (Achaz et al., 2002). Among the functionally most important examples are homo- or heteropolymeric repeat tracts leading to phase variable gene expression, the neisserial DNA uptake sequence (DUS) as well as the so-called dRS3, Correia and REP2 repeats. Loci termed as ‘‘simple sequence contingency loci’’ which contain short tandem sequence repeats are abundantly present either within the coding region or in the promoter region (Bayliss et al., 2001). The numbers of these tandem repeat motifs are modified during replication through slipped-strand mispairing which in turn influences transcription or translation resulting in reversible on-off switching of gene expression (phase variation) or an altered function of the encoded proteins (Lovett, 2004). N. meningitidis has the largest repertoire of phase variable genes described for any species so far (Bentley et al., 2007; Martin et al., 2003; Snyder et al., 2001; Saunders et al., 2000). The genes, which are found to be phase variable in meningococci include those involved in biosynthesis and modification of pili, capsular polysaccharide, lipopolysaccharide, opacity proteins, haemoglobin receptors, PorA outer membrane protein, Opc outer membrane protein, ferric receptor, and the putative adhesin NadA. In addition, N. meningitidis also contains potentially phase variable type III restriction modification systems. Here, genes may come under the influence of the methyltransferases by the introduction of point mutations resulting in transcriptional alterations (Srikhanta et al., 2005). This system thus controls

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the coordinated random switching of a whole set of genes in a bacterial pathogen and has been termed phasevarion. The neisserial DUS comprises 12 base pairs and occurs alone or in inverted repeats forming transcriptional terminators (Ambur et al., 2007; Goodman and Scocca, 1988). These sequences present in around 2000 copies are involved in the recognition and uptake of DNA from the environment. They are not equally distributed in the neisserial genome, but are rather scattered with a higher density within gene loci involved in DNA repair, recombination, restriction modification, and replication (Davidsen et al., 2004). The presence of these DUS in such large numbers coupled with the natural competence of Neisseriae facilitate the incorporation of foreign DNA from the same or related species via homologous recombination (Davis et al., 2001; Linz et al., 2000; Holmes et al., 1999; Kroll et al., 1998; Zhou et al., 1997; Feil et al., 1996; Bowler et al., 1994; Hobbs et al., 1994; Frosch and Meyer, 1992; Spratt et al., 1992; Zhou and Spratt, 1992). Further repetitive sequence elements in the N. meningitidis genome are concentrated within intergenic repeat arrays between 200 and 2700 bp in length. These repeat arrays are composed of several different repeat types, the most abundant of which are the so-called ‘neisserial intergenic mosaic elements’ (NIMEs), which comprise repeat units of approximately 50–150 bp (RS elements), each flanked by 20-bp inverted repeats (dRS3 elements) (Bentley et al., 2007; Parkhill et al., 2000b). Within each array, there are between 1 and 60 copies of RS elements belonging to 117 different families. dRS3 elements are present in around 750 copies in each genome and have been found to serve as the target sites for the integration of the neisserial filamentous phage Nf1 (see below) (Kawai et al., 2005). Also present in the repeat arrays are larger units, which are also found in isolation, including the ‘Correia repeat-enclosed elements’ (CREEs, 156-bp sequences bounded by 26-bp inverted repeats). CREEs are sequence indels like small insertion sequences 100–155 bp in length with long terminal inverted repeats consisting of the 26 bp Correia sequence, but without coding for a transposase (Buisine et al., 2002; Liu et al., 2002). They carry transcription initiation signals and functional integration host factor binding sites and hence may play a role in modulating expression of various virulence-related genes (Rouquette-Loughlin et al., 2004). The last kind of repeat structures which were shown to have a biological role in transcriptional regulation of some genes are the repetitive extragenic palindromic sequences called REP2. These elements contain promoter and ribosome-binding sites (Parkhill et al., 2000b) and were found to influence expression of virulence genes such as pilC1 being necessary for the interaction of meningococci with host cells (Morelle et al., 2003).

Prophages and islands of horizontal transfer Sequencing of the neisserial genomes revealed the presence of an unexpected number and diversity of prophages, some of them are members of the Mu-related family of phages, the phage l-related group of phages of enteric hosts and the family of filamentous M13-like phages (Bentley et al., 2007; Morgan et al., 2002; Parkhill et al., 2000b; Tettelin et al., 2000) (Fig. 1). For example, three partially defective mosaic relatives of the Mu-like group of prophages were found in the genome of N. meningitidis strain Z2491, called Pnm1, Pnm2, and Pnm3, respectively. The MC58 genome contains similar prophages at the Pnm2 and Pnm3 sites called NeisMu1 and NeisMu2, respectively, but none at the Pnm1 integration site. The proteins encoded by these prophages code for membrane-associated antigenic proteins which contribute to the variability in the surface structure and thereby influence virulence and pathogenicity (Masignani et al., 2001). Strain FAM18 lacks Pnm1 and Pnm2 but is the only one among the sequenced strains to harbour a putatively intact copy of a l-like prophage on IHT-E (Fig. 1) (see below). During an in silico genome comparison Kawai et al. (2005) have further identified filamentous prophages in Neisseria called neisserial filamentous phages (Nf) which belong to a group that includes the Escherichia coli phage M13 as well as the Vibrio cholerae phage carrying the cholera toxin (Kawai et al., 2005). Comparison of the three genomes of N. meningitidis and one of N. gonorrhoeae revealed four subtypes Nf1–Nf4. Eleven intact copies are located at different loci in the four genomes, some of them are depicted in Fig. 1. Subtype Nf1 is present in one copy in the Z2491 genome, in two copies in the MC58 genome and in four copies in the FAM18 genome, respectively, and was found to be associated with hypervirulent meningococcal lineages (see below) (Bille et al., 2005). However, by now it is still unclear how this island contributes to the pathogenicity of the meningococci since it seems not to contain genes coding for known virulence factors. In support of these in silico findings it was also experimentally shown that Nf1 is able to excise from the chromosome and is secreted from the bacteria via type IV pilin secretin (PilQ) in the form of a circular plasmid (Bille et al., 2005). It has been hypothesized that the presence of this phage might enhance transmission rates and thereby attack rates in certain age groups (Moxon and Jansen, 2005) However, there is no experimental proof or epidemiological confirmation available for this elegant hypothesis until now. The meningococcal genomes all contain a different number and repertoire of islands of horizontally transferred DNA that are characterized by atypical DNA composition but otherwise lack the typical hallmarks of canonical genomic islands (GEIs) (Dobrindt

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et al., 2004). Some of them code for proteins that are involved in meningococcal pathogenesis. For example, IHT-A contains the genes of the capsulation cluster, and IHT-B and -C appear to encode two-partner, or type V, secretion systems and show a similarity in sequence and genetic arrangement to those of Bordetella species where fhaC and fhaB, respectively, encode secretion accessory protein and a filamentous haemagglutinin important in virulence. The only region described in N. meningitidis so far that in contrast to IHTs shows all features of a typical GEI is MGI1 in the genome of strain MC58 (Fig. 1) (Hotopp et al., 2006). With the exception of the GC content being identical to the rest of the MC58 chromosome, MGI1 displays all features of a classical GEI (Dobrindt et al., 2004) like a direct repeat at the 50 and 30 end, localization at a tRNA locus and the accumulation of genes involved in DNA metabolism and transposition such as an integrase (pseudo-) gene and two transposases. MGI1 might represent a defective form of the l-like prophage present on IHT-E in FAM18 that has lost the genes for replication, head maturation and tail structure assembly and would thus be a case in point for the hypothesis that GEIs have evolutionarily originated from integrated prophages (Dobrindt et al., 2004). For detailed comparative analyses of the prophage content in the meningococcal genomes the reader is referred to (Bille et al., 2005; Kawai et al., 2005; Casjens, 2003; Morgan et al., 2002; Masignani et al., 2001).

Neisserial genome comparisons based on microarray hybridization It was the advent of whole genome sequences and in its wake the design of DNA macro- and microarrays that allowed for high-throughput comparative genome hybridization (mCGH) studies of a large range of isolates including invasive as well as carriage strains of N. meningitidis, different N. gonorrhoeae strains and a number of commensal species of Neisseria (Hotopp et al., 2006; Snyder and Saunders, 2006; Bille et al., 2005; Stabler et al., 2005; Perrin et al., 2002) (for a recent review see Claus et al., 2007). These studies mainly focussed on genomic typing of different meningococcal strains and the search for virulence gene candidates being restricted only to invasive isolates. The genome sequence of the meningococcal strain Z2491 enabled the design of a first DNA macroarray which was used for the analysis of the genomic differences between N. meningitidis and other Neisseria species based on the comparison of eight different N. meningitidis, three different N. gonorrhoeae and two different N. lactamica strains (Perrin et al., 2002). With

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this macroarray it was shown that the meningococcusspecific regions are scattered as small islands around the chromosome and that dramatic differences in the pathogenic potential of the different Neisseria species might result from only small genetic changes. In addition, many differences were strain and not species specific. By extending these analyses to a greater collection of 29 invasive and 20 non-invasive meningococcal isolates the filamentous phage Nf1 (see preceding chapter) was found to be specific to hyperinvasive meningococcal complexes (Bille et al., 2005). As strains carrying this genetic island were significantly more likely to cause disease it was also termed ‘‘meningococcal disease-associated (MDA) island’’. However, in another mCGH study of one N. cinerea, two N. lactamica, two N. gonorrhoeae and 48 N. meningitidis isolates, this phage was present only in 60% of the virulent strains and in 42% of the carriage strains, suggesting that this phage may be a marker more for certain hypervirulent clonal groups than for virulent strains in general and it was not a characteristic of all invasive meningococcal strains (Hotopp et al., 2006). Besides this phage, another six of these islands (NeisMu1/PNM2, PNM1, IHT-B, IHT-C, IHT-D, IHT-E) were found to be differently distributed among the 49 meningococcal isolates tested and differentiated N. meningitidis into five groups. Surprisingly, in spite of the high degree of lateral gene transfer within N. meningitidis (Smith et al., 1999), isolates with the same sequence type had similar genomic content profiles and demonstrated congruence between the DNA array technique and the clonal complexes defined by MLST (Stabler et al., 2005). In a study comprising 13 unrelated strains of N. lactamica and focussing on the presence of meningococcal genes also in this commensal species, the vast majority of genes present in pathogenic N. meningitidis and including those designated as virulence genes were found also present in N. lactamica, indicating that the differences that make a particular species pathogenic or not are not as great as might be supposed on the basis of their distinct behaviours and previous lists of virulence genes (Snyder and Saunders, 2006). In accordance with this finding, with the exception of the capsule gene cluster also all other studies failed to identify a gene set that was clearly restricted to pathogenic strains and absent from all the commensal strains and species (Bille et al., 2005; Stabler et al., 2005). Therefore, it might be more the ‘genetic personality’ of a particular Neisseria sp., deriving from the combinations of genes and/or differences in their regulation, rather than the mere presence or absence of genes that underlies their different pathogenic potentials (Snyder et al., 2005). In summary, these mCGH analyses have already shed new light on meningococcal genome composition and dynamics on a population level and provided meningococcal epidemiology with a genomic foundation.

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Impact of life-style on the genome structure in N. meningitidis Organisms such as N. meningitidis living in complex environments like the human mucosal surface with its local immune defence mechanisms and the presence of competing members of the local microbiota would be expected to have sophisticated systems of environmental sensing. However, in a recent survey of signal transduction proteins encoded in 167 bacterial and archaeal genomes N. meningitidis was found to be among the genomes harbouring the smallest number of signal transduction proteins (Fig. 3) (Galperin, 2005; Ashby, 2004). Thus, what are the mechanisms that allow N. meningitidis to adapt to and grow in such a challenging environment and how might its life-style as a respiratory commensal restricted to human hosts have influenced its genomic make-up? A putative key for the understanding of the interconnection of life-style and genome architecture in N. meningitidis might be a selective advantage of a fast growth rate and the concomitantly selective pressure towards a small genome size. A short generation time might be a positively selected trait in N. meningitidis given the fact that direct person-to-person spread occurs via airborne transmission resulting in only a small number of bacteria being transmitted (Bayliss et al., 2000). Upon contact with the mucosa of the respiratory tract these founder organisms have to multiply rapidly to form microcolonies (Lappann et al., 2006; Sim et al., 2000) and thus to successfully compete with the residential microflora as well as the local immune responses. This in turn might have several impacts on genome architecture as well (Fig. 4). First, it has already been shown that the total number of regulatory proteins encoded by each given organism’s genome positively correlates with the genome size and the total number of encoded proteins (Galperin, 2005). Therefore, as the genome size of N. meningitidis is relatively small compared to other free-living microorganisms this might in part explain the observed small numbers of regulatory elements with the putative adverse effects on its ability to adapt to a changing environment (Fig. 4). On the other hand, a small genome also enables a short generation time. As phase variation due to slipped-strand mispairing requires DNA replication (Henderson et al., 1999) a short generation time might thus promote phase-variable gene expression as an evolutionary strategy to adapt to environmental changes. In support of this notion, a recent mathematical analysis has shown that if cells are unlikely to sense environmental transitions and if different environmental states select for different phenotypes, then variability among potential hosts or time-varying immune selection within a single host selects for phase varying phenotype

Fig. 3. Percentage of signal transduction proteins and putative phase variable genes due to slipped-strand mispairing in the encoded proteomes of N. meningitidis (Nm) (Saunders et al., 2000), Helicobacter pylori (Hp) (Saunders et al., 1998), Campylobacter jejuni (Cj) (Parkhill et al., 2000a), Streptococcus pneumoniae (Sp) (Tettelin et al., 2001), Haemophilus influenzae (Hi) (Fleischmann et al., 1995), and E. coli (Ec) (Torres-Cruz and van der Woude, 2003). The numbers for the signal transduction proteins were taken from Galperin (2005). Compared to the other genomes, the numbers for the phase variable genes in H. influenzae depicted here are the lower estimates as only tetrameric repeat sequences were considered. The figure is not meant to represent a comprehensive survey of phase variable genes in all eubacterial genomes, but to illustrate the loose inverse correlation between the numbers of phase variable genes and the numbers of two-component systems in the selected organism for which numbers on putative phase variable genes were available. In addition, the numbers for the genome size as well as the microbiological classification of each species according to NCBI are shown. In contrast to E. coli, all organisms are more or less restricted to the human host with the exception of C. jejuni frequently found also in poultry.

expression (Wolf et al., 2005). In fact, as can be seen in Fig. 3 there is a loose inverse correlation between the numbers of phase variable genes and the numbers of two-component systems in the organisms investigated. In N. meningitidis, the negative effect of the reduced numbers of these regulatory molecules might thus be compensated for by the excessively higher numbers of phase variable genes described above. In addition, a higher number of replicative cycles within a short time also increases the probability for the observed chromosomal inversions around the replication origin to occur (Fig. 1) as this kind of genome rearrangement has also been shown to be replicationdirected (Eisen et al., 2000; Tillier and Collins, 2000). Finally, slipped-strand mispairing has also been shown to be one of the major mechanisms for the

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Fig. 4. Schematic drawing illustrating the proposed interconnection of life-style and genome organization in N. meningitidis and the selective forces shaping the meningococcal genome. Being dependent on humans as natural host and directly transmitted between two individuals via aerosols Neisseria meningitidis might face three major challenges when colonizing a new host: the small number of founder organisms, the presence of the residential microflora and the local immune responses. Therefore, a fast replicative cycle might be of selective advantage to grow to large enough numbers for forming microcolonies (Lappann et al., 2006; Sim et al., 2000) and to meet these environmental challenges. A small genome might promote a short replication time at the cost of a lower coding capacity and consecutively fewer signal transduction proteins. The latter compromises the organism’s ability to adapt to a complex environment such as the human nasopharyngeal mucosa. However, many rounds of DNA replication within a short time firstly increase the chance to generate slipped-strand mispairing mutations and therefore to generate phenotypic diversity due to phase variation of simple sequence contingency loci. Second, slipped-strand mispairing mutations also lead to generation of further repeated sequences in the genome increasing its propensity to rearrangements due to recombination events between homologous repeated sequences within the chromosome or with stretches of homologous DNA taken up from the environment via horizontal gene transfer. Finally, it has been shown that the observed chromosomal inversion around the origin of replication might also be replication-directed which might result in alterations of the expression of genes located at the chromosomal breakpoints. Therefore, the latter three mechanisms being driven by a fast replication cycle might compensate for the low number of signal transduction proteins and the concomitantly less sophisticated systems of environmental sensing.

generation of simple DNA repeats that abound in the meningococcal genome (Levinson and Gutman, 1987). Amongst other mechanisms like the acquisition of IS elements via horizontal gene transfer, the selective advantage of a short generation time might thus also lead to the accumulation of repeated sequences (Achaz et al., 2002) which in turn might further increase the genomic flexibility serving as targets for intrachromosomal rearrangements (Shapiro, 2002).

Conclusion The key messages from meningococcal genome comparisons are ones of change and flexibility (Snyder et al., 2005). In a challenging environment like the mucosa of the human nasopharynx, a fast replication rate resulting in an outgrowth of other competitors upon colonization of a new host might constitute a selective advantage. Accordingly, many of the peculiarities of meningococcal genome organization like the small genome size compared to other free-living extra-

cellular bacteria, the corresponding low number of twocomponent systems, the high amount of repeated sequences, the large number of phase variable genes, the observed rearrangements around the origin of replication, and the high rate of horizontal gene transfer due to natural competence might be best understood in the light of an adaptation of N. meningitidis to a frequently changing environment. As an unwanted byproduct of phase variable phenotype expression, virulence of this bacterium might be an inadvertent consequence of short-sighted within-host evolution, which is exasperated by the increased mutation rates associated with phase variation, making N. meningitidis more an accidental than a facultative human pathogen (Meyers et al., 2003). Therefore, sequencing of the genomes from carrier strains and comparison with the described genomes of the invasive isolates might be a test for this hypothesis and will reveal whether only subtle differences between the genomes of diseasecausing and carrier isolates might exist. In the future, with a broader availability of affordable sequencing technology, genome sequencing will also help to address

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pressing questions of molecular epidemiology, such as the molecular basis for the recent emergence of virulent serogroup Y strains in the US (Rosenstein et al., 1999; Racoosin et al., 1998), or of serogroup X strains in Niger (Boisier et al., 2007).

Acknowledgements The authors thank Dr. Gabriele Gerlach for critical reading of the manuscript and two anonymous reviewers for their helpful comments. The work on the meningococcal genomes was supported by the BMBF in the context of the PathoGenoMik and PathoGenoMikPlus funding initiative.

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