Sequence Conservation of Pilus Subunits in Neisseria meningitidis Ana Cehovin1, Megan Winterbotham1, Jay Lucidarme2, Ray Borrow2, Christoph M. Tang1, Rachel M. Exley*1 and Vladimir Pelicic*1 1 The Centre for Molecular Microbiology and Infection, Flowers Building, Imperial College London, Armstrong Road, London, SW7 2AZ, UK 2 Vaccine Evaluation Unit, Health Protection Agency North West, PO Box 209, Clinical Sciences Building 2, Manchester Royal Infirmary, Manchester, M13 9WZ, UK *For correspondence: Tel: 44-20 7594 2080 FAX: 44-20 7594 3095 E-mail: v.pelicic@imperial.ac.uk; r.exley@imperial.ac.uk 1 Abstract The rapid onset and dramatic consequences of Neisseria meningitidis infections make the design of a broadly protective vaccine a priority for public health. There is an ongoing quest for meningococcal components that are surface exposed, widely conserved and can induce protective antibodies. Type IV pili (Tfp) are filamentous structures with a key role in pathogenesis that extend beyond the surface of the bacteria and have demonstrated vaccine potential. However, extensive antigenic variation of PilE, the major subunit of Tfp, means that they are currently considered to be unsuitable vaccine components. Recently it has been shown that Tfp also contain low abundance pilins ComP, PilV and PilX in addition to PilE. This prompted us to examine the prevalence and sequence diversity of these proteins in a panel of N. meningitidis disease isolates. We found that all minor pilins are highly conserved and, unexpectedly, the major pilin genes are also highly conserved within the ST-8 and ST-11 clonal complexes. These data have important implications for the re-consideration of pilus subunits as vaccine antigens. Key words: Neisseria, pilin, vaccine Running Title: Neisserial pilin subunit conservation 2 1. INTRODUCTION Infections with Neisseria meningitidis account for over 50,000 deaths globally each year. A large proportion of cases occur in developing countries which experience large seasonal epidemics [1]. Although endemic in developed countries, meningococcal disease is still a leading cause of disability and morbidity in childhood. The non specific early symptoms, rapid onset and traumatic consequences of meningococcal infections make these an important public health problem and this has led to the implementation of national vaccination campaigns in several countries [2]. Meningococci display an antigenically diverse polysaccharide capsule which forms the basis for grouping into serogroups. Most cases of meningococcal disease are attributable to serogroups A, B, C, Y and W-135 [3]. The recently developed multilocus sequence typing (MLST) also groups meningococci according to the allelic profile of seven housekeeping genes, thus assigning them into sequence type (ST) clonal complexes [4]. Meningococci from the same ST clonal complex may belong to different serogroups. In the USA and Europe, the majority of cases of invasive meningococcal disease are caused by isolates which belong to a limited number of clonal complexes known as the hyperinvasive lineages (including the ST-1, ST-4, ST5, ST-8, ST-11, ST-32, ST-41/44 and ST-269 clonal complexes) [3]. While anticapsular vaccines are available against serogroups A, C, Y and W135, an effective vaccine to prevent infection with serogroup B meningococci has yet to be produced [5]. 3 There is an ongoing quest for meningococcal components that are surface exposed, relatively conserved in sequence, widely prevalent among serogroups/clonal complexes and which induce antibodies that are either bactericidal or capable of interfering with pathogenesis or colonization of N. meningitidis. Several promising candidates have been identified. These include five antigens identified by reverse vaccinology which, along with an outer membrane vesicle, constitute an investigational 5 Component Vaccine against Meningococcus B (5CVMB) [6, 7]. Another investigational vaccine comprises two variants of the surface exposed antigen factor H binding protein (fHBP; previously LP2086 or GNA1870), which is also a major component of the 5CVMB [8]. However, the search for novel vaccine antigens is not over since additional meningococcal components may increase coverage and protection, whilst reducing the likelihood of vaccine escape due to mutation/recombination. The contribution of type IV pili (Tfp) to pathogenesis and their surface location made these organelles attractive candidates in the early 1980s for inclusion in vaccines [9, 10]. Tfp are polymers predominantly of PilE, the major pilin, and are important virulence factors that contribute to the survival of meningococci in the human host during both colonization and disease [11]. Notably they have a key role in adhesion to human cells, either by inducing microcolony formation on cells and/or promoting twitching motility [12-14]. Furthermore, Tfp promote the rapid emergence of variants with novel, heritable characteristics by mediating natural competence for DNA transformation [15]. Tfp in Neisseria have been grouped into two immunotypes, according to their reaction (class I) or lack of reaction (class II) with the monoclonal antibody SM1 [16, 17]. Several studies on the closely related pathogen Neisseria 4 gonorrhoeae showed that a vaccine based on pilus preparations is safe and induces the production of specific antibodies that enhance phagocytosis of bacteria by peripheral blood leukocytes and inhibit the attachment of gonococci to epithelial cells [9, 10]. Furthermore, PilE has been shown to be immunogenic during the course of infection as anti-pilin antibodies can be found in patients infected with N. gonorrhoeae or N. meningitidis [18, 19]. However, the development of a pilus-based vaccine has been halted because PilE undergoes extensive antigenic variation in Neisseria species [20]. Single strains of meningococcus or gonococcus can rapidly and efficiently evolve to express PilE with different amino acid (aa) sequence [21], which results from recombination of promoterless pilS cassettes into the pilE gene in a process known as gene conversion [22-24]. The resulting major pilin subunits have conserved N-termini but widely variable C-termini. Consequently, anti-Tfp vaccines provide limited cross protection and field trials were largely unsuccessful [19]. It was recently reported that Tfp in Neisseria species also contain low abundance proteins that share important sequence and structural features with PilE and are hence known as minor pilins [25]. In brief, they have a specific prepilin leader peptide that is cleaved by a dedicated prepilin peptidase and they co-purify with Tfp. As shown for PilX [26] minor pilins are expected to have 3D structure characteristic of type IV pilins, including a conserved hydrophobic N-terminal α-helix important for assembly in the pilus and a variable C-terminal region, which folds into a globular domain. Within the C-terminal region, two conserved cysteine residues define a hypervariable domain known as the D-region that is exposed on the surface of the filaments [26, 27]. Minor pilins have important roles in Tfp biology. PilX, PilV and ComP are dispensable for Tfp biogenesis, but they are essential for pilus-mediated bacterial 5 aggregation, adherence to human cells and competence for natural transformation [2830]. Therefore, in the present study, we investigated the prevalence and sequence variation of the genes encoding the minor pilins among disease-causing isolates of N. meningitidis and compared them to that of the gene encoding the major pilin. This has led to two important findings: 1) we unexpectedly show that although major pilins have been shown to be extremely variable, the class major class II pilins within the ST-8 and ST-11 clonal complexes are highly conserved and 2) that the minor pilins, PilX, PilV and ComP, have highly conserved nucleotide and aa sequences. These data have important implications for the possible use of the pilus subunits as vaccine antigens. 2. MATERIALS AND METHODS 2.1 Bacterial isolates and preparation of boilates N. meningitidis isolates (n=102) were from the Meningococcal Reference Unit (Manchester, UK). All isolate information including year of isolation, sequence type (ST), clonal complex, serogroup and site of origin is shown in supplementary data (Tables S1 and S2). Isolates were grown overnight on blood agar at 37°C, in the presence of 5% CO2 and suspensions prepared as follows. Several colonies were resuspended in 250 μL of 0.9% (w/v) saline, heated to 106°C for 20 min and snap frozen at -20°C for 5 min. The suspensions were then centrifuged at 12000 x g for 5 min and 100 μL of supernatants retained as boilates. Isolates were from individuals with invasive meningococcal disease in the UK between 1975 and 2001. The samples 6 were from blood (n=73), cerebrospinal fluid (n=8) and nose/throat/other (n=16); information was not available for five isolates. The isolates were from serogroup B (n=47), C (n=46), W135 (n=6), Y (n=1) and non groupable (n=2) and from the following clonal complexes ST11 (n=29), ST269 (n=23), ST41/44 (n=13), ST22 (n=4), ST-32 (n=2), ST-8 (n=29) and one for both ST167 and an unassigned ST (UA). Also included in the analyses were the sequences of the pilin genes from meningococcal strains for which whole genome sequences are available: serogroup B MC58, serogroup A Z2491 and serogroup C FAM18, 053442 and 8013 (Table S3). 2.2 PCR amplification and sequencing of pilin genes The major pilin gene, pilE, was amplified using primers designed for amplification of class I or class II alleles either designed in this study or described previously [31]. These primers hybridize to sequences overlapping the stop and start codons (for class II) or in the regions upstream and downstream (for class I) of the distinct pilE coding sequences and are based on the sequences of meningococcal strains MC58 and FAM18 (Figure 1A). The minor pilin genes, pilX, pilV and comP were amplified using specific primers designed in this study (Figure 1B). All primers are shown in Table 1. PCR was performed using the following conditions. Reaction mixes contained either 1.7 U of Expand High Fidelity polymerase (Roche) for amplification of pilE or Pfu Ultra Fusion polymerase (Agilent Technologies) for amplification of minor pilin genes. A volume of 2-4 μL of boilates was added to make a total reaction volume of 25-50 μL. Standard PCR cycling conditions were as follows: 94°C for 2 min, 30 cycles of (94°C for 30 sec, 52°C for 45 sec and 72°C for 3 min), followed by 3 min at 72°C for the amplification of pilE, and 95°C for 5 min, 30 cycles of (95°C for 30 sec, 50°C for 45 sec and 72°C for 1 min), followed by 10 min at 72°C for the 7 amplification of the minor pilins. The reactions were cooled and stored at 4°C. Where no pilE gene product was obtained using NG1680 and NG1681, hybridization temperatures were adjusted to 50°C and the PCR was repeated with primers 5’pilE and 3’pilE. Following PCR amplification, the DNA fragments were purified using PCR Clean-up kit (Qiagen). The fragments encoding pilE were cloned into plasmid pGEMTEasy (Promega). The nucleotide sequence of the forward and reverse strand of each pilE gene was determined by sequencing with primers M13F and M13R (Table 1) which hybridize to sequences within the pGEMTeasy vector. Sequencing reactions were performed by MRC Sequencing Service, Hammersmith Hospital. Where forward and reverse strands showed sequence discrepancies, sequence reactions were repeated. 2.3 Sequence alignment and analysis The nucleotide sequence of each gene was determined for each DNA strand. The coding sequences of the pilE, pilX, pilV and comP genes were identified by homology searches using NCBI nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and by manual alignment with either MC58 or FAM18 for pilE sequences or strain 8013 for pilV, pilX and comP sequences using the Serial Cloner 1.3-11 and DNA Strider 1.4 programs. Nucleotide sequences were translated using Serial Cloner 1.3-11 and DNA Strider 1.4. Protein-coding nucleotide sequences and the deduced aa sequences were aligned using the ClustalW2 program available at EBI (http://www.ebi.ac.uk/Tools/clustalw2/index.html) [32] by Neighbour-joining method and presented using Boxshade (http://mobyle.pasteur.fr). Phylogenetic analysis of nucleotide and deduced aa sequences was performed by MEGA 4 (http://www.megasoftware.net/) [33] using the Nei-Gojobori and Kimura 2-parameter 8 models [34]. Amino acid identity was determined as percentage of identical residues common to all sequences. Phylograms were constructed using the ClustalW2 and visualized with Treeview version 0.5.0. Secondary structure predictions were made using ESyPred3D Web Server 1.0 [35], with predictions for class I and class II pilins based on PilE from N. gonorrhoeae and predictions for minor pilins based on N. meningitidis PilX. 3. RESULTS 3.1 Prevalence, distribution and diversity of gene encoding major pilin (PilE) in N. meningitidis The initial strain collection consisted of 56 clinical isolates (Table S1) which were analysed by PCR for the presence of all the pilin genes: pilE, pilX, pilV and comP. The pilE gene was successfully amplified from total of 48 of these isolates with primers specific for either class I or class II alleles (Figure 1A). Results from this primary analysis demonstrated that the class I pilE allele was present in 31/56 isolates belonging to five different clonal complexes, while the class II pilE was present in all isolates belonging to either the ST-11 or ST-8 clonal complexes (17/56). No pilE product was detectable in a total of eight isolates from ST-32, ST-269 or ST-41/44 clonal complexes using the PCR conditions described in Materials and Methods, possibly due to sequence variation at the pilE locus rather than absence of the pilE gene. The amplified pilE genes were sequenced and the nucleotide and deduced aa sequences of the corresponding mature pilins (excluding the leader peptides) were aligned. The sequence of class I pilins from sequenced strains of N. meningitidis MC58 [36], 8013 [37] and Z2491 [38] and class II pilin from meningococcal strain 9 FAM18 [39] were also included for comparison. The extent of variation of class I and class II pilE genes was calculated as the mean distance between nucleotide sequences which refers to the number of base differences per nucleotide site from averaging all sequence pairs using the Kimura’s 2-parameter model, as described in [40]. As expected on the basis of previous observations [41], in the strains harbouring class I pilins there was considerable variation in pilE, shown by the substantial mean distance between nucleotide sequences (0.379) (Table 2). Analysis of the mature pilin sequences revealed that two sequences encoded pilins that were truncated by premature stop codons (isolates 55 and 66, not shown). The remaining full length pilins exhibited significant variation, in particular over the hypervariable D-region in the C-terminus of the protein (Figure 2A). Even strains within the same clonal complex contained distinct PilE sequences. In striking contrast, when the class II PilE sequences, found only within the ST-8 and ST-11 clonal complexes, were aligned they were found to be highly conserved. We therefore amplified pilE from an additional set of 46 isolates; 41 belonging to these two clonal complexes and five further isolates from other clonal complexes as controls (Table S2). We obtained class I pilE sequences from these five isolates and these are included in Table 1 and Figure 2A. Overall, class II pilE could be amplified from 29 N. meningitidis isolates from the ST-11 clonal complex and 29 from the ST-8 clonal complex. Pilins within either the ST-8 and ST-11 clonal complexes were almost identical, as indicated by the very small mean distances between nucleotide sequences (0.099 and 0.004 respectively) in these strains (Table 2). The majority (26/29) of ST-8 isolates were found to encode a PilE that was identical apart from a 10 single amino acid change at position 48. The remaining isolates, which are all of the same sequence type (ST-153) within the ST-8 clonal complex (Table S2), encode a slightly different PilE in comparison, but which has 99% identity amongst these three strains (Figure 2B). The mature pilins from the isolates belonging to the ST-11 clonal complex differed merely by one or two aa at positions 48, 79, 88, 116 and 123 (Figure 2C). The level of conservation of class II pilins was further highlighted by the results of phylogenetic analysis of all the PilE aa sequences (Figure 3), which identified only nine aa sequence types among all the clinical isolates expressing class II pilins (n=59) whereas as many as 37 different aa sequence types were found for the class I pilins (n=37). Most interestingly, the D-region which is usually ‘hypervariable’ is highly conserved in the class II pilins (Figure 2). 3.2 Prevalence and diversity of genes encoding minor pilins (PilX, PilV and ComP) in N. meningitidis The three genes encoding minor pilins were amplified from 56 clinical isolates described in Table S1. The pilX, pilV and comP genes were found to be widely distributed as they were present in 100% of isolates tested. These 168 minor pilin genes were sequenced and the nucleotide and deduced aa sequences of the corresponding mature proteins were aligned (Figures 4-6). The corresponding genes from sequenced strains (Table S3) were also included for comparison. Minor pilins were all found to be highly conserved with mean distances between nucleotide sequences ranging from only 0.005 for comP to 0.071 for pilV. A ratio between the rate of non-synonymous (dN) and synonymous (dS) substitutions per (non)synonymous nucleotide site was used to estimate whether the genes encoding minor pilins are under selection pressure, as described before [42]. In brief, dN/dS ratios of 11 <1, >1 or 1 suggests that mutations are either under negative or positive selection pressure or are neutral. The dN/dS ratios calculated for these proteins (Table 3) suggest that none of the minor pilin genes is under positive selection pressure. Consequently, the corresponding mature proteins display 73% (PilX), 64% (PilV) and 97% (ComP) aa identity. The most conserved protein, ComP, shows an unexpectedly limited variation for a surface-exposed protein. Only six aa sequence types were observed (Table 3), differing only at aa positions 29, 36, 83, 142 and 143 (Figure 4). For PilX, we identified 23 aa sequence types (Table 3), while for PilV there were 20 aa sequence types (Table 3). In PilX or PilV, the N-terminal extended -helix, which is necessary for assembly into a pilus fibre [26], was conserved as expected, while variability was restricted to the C-terminal domain, specifically to the two regions predicted to be exposed on the surface of the pilus (Figures 5 and 6). Due to the extremely low variation for ComP, phylogenetic analysis of the aa sequences was performed only for PilX and PilV. Phylograms revealed that the deduced aa sequences of these two minor pilins are not randomly distributed but rather that sequences obtained from the same clonal complexes cluster together. Only three major protein variants could be identified for both PilX (Figure 5) and PilV (Figure 6). Variant 1 was present mostly in ST-41/44 and ST-22 clonal complexes, variant 2 in ST-11 and variant 3 in ST-269, consistent with the very low variability of minor pilins within clonal complexes. 4. DISCUSSION 12 Subunits of Tfp, one of the most widespread virulence factors in the bacterial world [11] display many properties which have made them popular candidates for inclusion in vaccines. Consequently, several studies have so far evaluated their efficacy. For example, antibodies against the TcpA pilin from Vibrio cholerae inhibit microcolony formation and attachment of bacteria to epithelial cells in vitro and are protective in challenge experiments in vivo [43, 44]. Similarly, antibodies directed against N. gonorrhoeae Tfp were shown to inhibit attachment of gonococci to epithelial cells and prevent gonococcal infection [9, 10]. In Neisseria the extreme antigenic variability of PilE provides the bacteria with a mechanism of immune escape and results in limited cross protection, which has proven the major stumbling block to development of pilus-based vaccines [19]. Over recent years however, it has become evident that in addition to the major pilin subunit, minor pilins are also associated with the Tfp of Neisseria [25, 29]. These proteins influence pilus functions and dynamics and three of them PilX, PilV and ComP are believed to be components of the pilus fibre, although only PilX has been shown to co-localize with the assembled pili [26]. In the present study, as a preliminary evaluation of their potential as vaccine antigens, we determined sequence conservation of the genes encoding the three minor pilins PilX, PilV and ComP and sought to compare it to the sequence variability of the major pilin PilE. The pilE, pilX, pilV and comP genes were originally amplified from 56 isolates of N. meningitidis. These were clinical invasive isolates from patients with septicaemia and/or meningitis collected in the UK from 1975-2001. The majority of isolates belong to hyperinvasive clonal complexes which are the predominant causes of invasive meningococcal disease [4]. These include ST-11 isolates which were 13 responsible for the majority of serogroup C meningococcal infections in the UK prior to the introduction of the serogroup C conjugate vaccination programme [45]. Furthermore the collection includes ST-269 and ST-41/44 clonal complex strains which are currently responsible for the majority of disease in Europe and North America [46, 47]. Analysis of the distribution of the class I or class II pilE genes revealed that the class II pilE allele is present in relevant clinical isolates, consistent with previous reports that invasive meningococci express pili of both classes [48]. The class I pilE gene was found in strains belonging to six different clonal complexes, and there was considerable sequence diversity in the pilE alleles amplified. Of note, eight strains did not yield a PCR product with primers either for class I or class II pilE. All these strains belonged to clonal complexes from which class I pilE alleles had been amplified and the failure to obtain products is consistent with sequence diversity in the class I pilE locus. In contrast, class II pilE genes were amplified only from isolates in the related ST-8 and ST-11 clonal complexes [49, 50]. Sequencing of the class I genes confirmed the well known diversity of the major pilin subunit genes since we identified a different sequence in every isolate. In contrast, class II pilE genes exhibited a high degree of sequence conservation, which prompted us to sequence an additional 41 genes from strains belonging to ST-8 or ST-11 clonal complexes. Only nine different class II pilins were identified from a total of 59 sequences amplified from meningococci that belong to either the ST-8 or ST-11 clonal complex, but which are from various clinical sites, express different capsular types, are of different sequence types and were isolated from patients in different 14 years. Of particular interest, the small degree of variation in the class II pilin proteins from either clonal complex corresponds to aa changes which do not localize to the characterised surface-exposed regions of hypervariability previously described [51]. This absence of variability is remarkable, since it suggests that there is no gene conversion of pilE in these strains, consistent with previous suggestions [52]. It remains to be determined why the major pilin subunit in these clonal complexes is so conserved but these findings suggest that a PilE-based vaccine (a previously abandoned idea) might be an interesting approach. This is particularly appealing for ST-11 strains, which are notably associated with higher bacterial loads [53] and increased morbidity [54-56] and continue to be responsible for outbreaks worldwide [57, 58] including the epidemics associated with the Hajj pilgrimage [59]. These facts, together with the recent evidence of possible capsule switching in ST-11 strains [6062] emphasize that it is worth investigating the immunogenicity of these major pilin subunits and the level of protection they might confer. Our data indicate that the minor pilins may also represent promising vaccine candidates. Their prevalence was extremely high: 100% of the isolates tested harboured all three genes. In addition, the nucleotide and aa sequences of all three minor pilins were found to be surprisingly conserved (between 64% and 97% identity) across different serogroups and clonal complexes. The prevalence and conservation of the minor pilins in N. meninigitidis appear to be comparable to those of the antigens in the investigational 5CVMB vaccine [6, 7]. As could be predicted, the variability observed for PilX and PilV (ComP is almost identical in all strains) was restricted to regions which are proposed to be exposed on the surface of the fibre [26]. However, the level of variation is very low when compared to the hypervariable class I major 15 pilins. Therefore, since it has been shown that PilX mediates bacterial aggregation [29], ComP is necessary for natural transformation [28] and PilV impacts on adhesion [63], this could suggest that sequence conservation of minor pilins may be necessary for preservation of the Tfp-linked functions they mediate. The surface exposure of minor pilins and their high conservation across clonal complexes suggests that these proteins may be potential vaccine candidates for inclusion in a broadly protective vaccine against N. meningitidis. The observed clustering of PilV and PilX from strains belonging to the same clonal complex suggests that a vaccine based on only up to three protein variants of each minor pilin may cover most hyperinvasive clonal complexes. The most important assessment to follow is the evaluation of immunogenicity of minor pilins and of their potentially dual vaccine potential. Indeed, they might protect not only by inducing complementmediated bactericidal activity, which is a generally accepted correlate of protection [64-66], but also by interfering with Tfp-linked functions, most notably adhesion to human cells and thereby increasing the protection against N. meningitidis. 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Gene pilE (class I) pilE (class II) pilX Primer Sequence (5' to 3') Reference NG1680 CCTTGCAAACCTTTAAAAAGG This study NG1681 TAATACACAGGTATCGCAACA This study 5’ pilE CCGATGGTCAAATACATTGC This study 3’ pilE GCGCCTGTCAGATAAACCACG This study NG1705 GTCAAACCCGGTCATTGTCC [31] NG1706 CAGGAGTCATCCAAATGAAAGC [31] pilX-F TTATCGGGTTACTGCCAAGG This study pilX-F2 GTCGTCCTTCAATCTTATGT This study pilX-R CCTGTCGGTTTCGGTTTTT This study pilX-R2 TGTTAATCCACTATACCCATAAA This study pilV-F TGCTTTCCCGAAAATTTCAA This study pilV-F2 GTTTACATATTCCGCCATTT This study pilV-R TAAGCATACGCTTCCGCATC This study pilV-R2 TCCGATATAAGGGTAAGCAT This study comP-F TGTTGAATAATCTGTTCTTATTGGAAG This study comP-R CGGAAAACCGCACAAATACT This study cloned in M13F GTAAAACGACGGCCAG Invitrogen pGEMTeasy M13R CAGGAAACAGCTATGAC Invitrogen pilV comP 26 Table 2. Class I and class II pilE genes. Length, number of isolates from which genes were sequenced, corresponding number of alleles identified and overall mean distance between nucleotide sequences. Length No. of No. of Mean distance between (bp) isolates alleles nucleotide sequencesa Class I 177-522 39b 39 0.379 Class II (total) 444-459 59c 12 0.204 Class II (ST-8) 444-459 29 4 0.099 Class II (ST-11) 447 30 8 0.004 pilE a The number of base differences per nucleotide site from averaging all sequence pairs using the Kimura’s 2-parameter model. b 39 isolates corresponds to 31 isolates described in Table S1, 5 isolates described in Table S2 and 3 sequenced strains of N. meningitidis c 59 isolates corresponds to 17 isolates described in Table S1, 41 isolates described in Table S2 and 1 sequenced strain of N. meningitidis 27 Table 3. Minor pilin genes in 56 meningococcal isolates and five sequenced genomes. Length and number of alleles, length and number of deduced aa sequence types of mature pilins. The overall mean distances, numbers of synonymous and nonsynonymous substitutions and dN/dS ratios are also indicated for each gene. Mean No. of sites distance No. of aa No. of Gene Length (bp) dN/dS between alleles Non- nucleotide Length (aa) sequence ratioa Synonymous synonymous types sequences pilX 489-513 26 0.033 107 375 0.683 152-160 23 pilV 387-390 23 0.071 94 291 0.53 121-122 20 comP 450 6 0.005 96 350 0.06 143 6 adN/dS: A measure for estimating whether a particular sequence is under selection pressure. dN/dS < 1: non-synonymous mutations are under negative selection; dN/dS = 1: all changes are neutral; dN/dS > 1: substitutions are driven by positive selection. 28 FIGURE LEGENDS Figure. 1. Schematic representation of the genomic loci encoding major and minor pilins. (A) Representative loci encoding class I and class II pilE genes in MC58 and FAM18 genomes, respectively. The positions of primers used to amplify the pilE genes are shown (arrows). Sma/Cla [67] and G4 sequences [68] implicated in pilin antigenic variation through gene conversion are shown as black and grey bars respectively. lpxC: UDP-3-0-(s-hydroxymyristoyl) N-acetylglucosamine deacetylase; fbp: putative FKBP-type peptidyl-prolyl cis-trans isomerase; kat: catalase. (B) Representative loci encoding pilX, pilV and comP in MC58. Genetic environment surrounding the genes is similar in other sequenced genomes of N. meningitidis. The positions of primers used to amplify each minor pilin gene are shown (arrows). All the genes are drawn to scale. dut; deoxyuridine 5’- triphosphte nucleotidohydrolase; adh; alcohol dehydrogenase; mfp; membrane fusion protein. Figure 2. Alignment of major pilin PilE sequences. Isolate identification number or name and clonal complex (ST) are shown for each sequence. Conserved residues are black, non-identical residues are white on black background, similar residues are grey and gaps are indicated with hyphens. (A) Class I pilins. The mature full length pilin sequences deduced from 34 N. meningitidis isolates and three sequenced strains MC58, Z2491 and 8013, were aligned using ClustalW and presented using Boxshade. (B) and (C) Class II pilins. The predicted mature pilin sequences deduced from the 29 clonal complex ST-8 N. meningitidis isolates (B) and from the 29 clonal complex ST11 N. meningitidis isolates and sequenced strain FAM18 (C) were aligned using the described methods. A schematic representation of the predicted secondary structure 29 based on the structure of gonococcal pilin [51] is shown. Wavy lines represent αhelices and arrows represent ß-sheets. The D-region defined by two cysteine residues is indicated. The D-region which is hypervariable in class I pilins is highly conserved in the class II pilins. Figure 3. Phylogram of mature peptide sequence of class I and class II pilins from all meningococcal isolates used in this study as well as previously sequenced N. meningitidis strains (Table S1-S3). Isolate identification number or name and clonal complex (ST) are shown for each sequence. Figure 4. Alignment of ComP minor pilins. To improve the readability of this figure, shown are only the different aa sequence types that were identified in the 56 meningococcal isolates and in the five sequenced meningococcal genomes. Representative isolate number or name of sequenced strain is shown together with ST. White, grey or black shading indicates identical, similar or different residue from consensus, respectively. The predicted secondary structure is indicated at the bottom of the alignment with waves and arrows corresponding to α-helices and β-sheets, respectively. Figure 5. The PilX minor pilin. (A) Alignment of PilX minor pilins. Only the different aa sequence types that were identified in 56 isolates and in the 5 sequenced meningococcal genomes are shown. Representative isolate number or name of sequenced strain is shown together with ST. White, grey or black shading indicates identical, similar or different residue, respectively. The secondary structure is indicated at the bottom of the alignment with waves and arrows corresponding to α- 30 helices and β-sheets, respectively. (B) Neighbour-joining phylogenetic tree of PilX. Three major variants of PilX (indicated) were identified. Figure 6. The PilV minor pilin. (A) Alignment of PilV minor pilins. Only the different aa sequence types that were identified in 56 isolates and in the 5 sequenced meningococcal genomes are shown. Representative isolate number or name of sequenced strain is shown together with ST. White, grey or black shading indicates identical, similar or different residue, respectively. The predicted secondary structure is indicated at the bottom of the alignment with waves and arrows corresponding to αhelices and β-sheets, respectively. (B) Neighbour-joining phylogenetic tree of PilV. Three major variants of PilV (indicated) were identified. 31