1 Bacterial Microarray Group, St George's Hospital Medical School, London SW7 0RE, UK
2 Centre for Molecular Microbiology and Infection, Department of Infectious Diseases, Imperial College London, London SW7 2AZ, UK
3 The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge CB10 1SA, UK
Correspondence
Richard A. Stabler
Richard.Stabler{at}lshtm.ac.uk
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ABSTRACT |
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Supplementary microarray data are available with the online version of this paper.
Present address: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
Present address: Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK.
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INTRODUCTION |
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The epidemiology of N. meningitidis has been widely studied. The bacterium can be characterized on the antigenic properties of the polysaccharide capsule it expresses. Strains which express the A, B, C, or W135 serogroup capsules are most associated with human disease. Multilocus enzyme electrophoresis (MLEE) and more recently multilocus sequence typing (MLST) have been used to differentiate isolates on a phenotypic and genetic basis. The results have shown that there are several lineages of related strains that circulate in human populations and there is considerable diversity between lineages. An important feature of the bacterium, which has a considerable influence on its evolution, is its ability to take up and integrate exogenous DNA. The natural competence of N. meningitidis for DNA uptake has led to a mosaic gene structure, in which regions of individual open reading frames have been acquired from other bacteria. The impact of horizontal transfer is demonstrated by the mosaic structure of genes encoding housekeeping functions (Feil et al., 1996; Zhou & Spratt, 1992
; Zhou et al., 1997
) and structures that are targets of host immune responses (Feavers, 2000
). N. meningitidis has also acquired resistance to clinically important antibiotics such as penicillin through the transfer of alleles from Neisseria flavescens (Spratt et al., 1989
). Thus, differences in gene composition and content in pathogenic neisseriae may be important in determining the pathogenic potential in strains.
A growing number of pathogenic bacteria have been analysed by comparative genomics using DNA microarrays (Behr et al., 1999; Dorrell et al., 2001
; Marokhazi et al., 2003
; Salama et al., 2000
). In this way the information provided by genome sequencing projects can be expanded to provide an indication of variation in gene content between strains. At present four strains of Neisseria have been sequenced, with a further two more in progress. The sequences currently available cover the commonly used laboratory strains of N. meningitidis and N. gonorrhoeae, namely N. meningitidis MC58 serogroup B (Tettelin et al., 2000
), N. meningitidis Z2491 serogroup A (Parkhill et al., 2000
), N. meningitidis FAM18 serogroup C (The Wellcome Trust Sanger Institute, unpublished data) and N. gonorrhoeae FA1090 (ATCC 700825) (University of Oklahoma, unpublished data). Comparative analysis of the genome sequences reveals a propensity for large- to small-scale genome rearrangements.
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METHODS |
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Amplification of microarray reporter elements.
PCR primers were synthesized by MWG Biotech and supplied in 96-well format to enable high-throughput amplification of PCR products using a liquid handling and PCR amplification robot (RoboAmp 9600, MWG Biotech). PCRs were performed with 10 ng DNA template, 5 U HotStar Taq DNA polymerase (Qiagen), 0·5 µM primers, 1·5 mM MgCl2 and 200 µM dNTPs. Thermocycling was performed using denaturation of 95 °C for 15 min, 40 cycles of 95 °C for 1 min, 52 °C for 1 min and 72 °C for 1 min followed by a final extension of 72 °C for 5 min.
Subsequent rounds of PCR amplification with modified conditions were performed until a single product of predicted size was obtained for all genes that were not amplified successfully under standard conditions. Additional validation was undertaken by sequencing 5 % of the amplified genes. PCR products were concentrated by 2-propanol/glycogen precipitation and resuspended in 50 % DMSO print buffer. Microarrays were constructed by robotic spotting of the PCR products in duplicate on UltraGaps amino-silane coated glass slides (Corning) using a MicroGrid II (BioRobotics) (Hinds et al., 2002b). The microarrays were post-print processed according to the slide manufacturer's instructions using hydration and UV irradiation, and stored in a dark, dust-free environment.
Microarray hybridization.
Hybridizations were performed by modifying a previously described protocol (Dorrell et al., 2001; Hinchliffe et al., 2003
). Briefly, 23 µg test genomic DNA was labelled with Cy3-dCTP and 2 µg Cy5-dCTP-labelled N. meningitidis MC58 genomic DNA was used as a common reference for all hybridizations. Microarray slides were prehybridized in 3·5x SSC, 0·1 % SDS, 10 mg BSA ml1 at 65 °C for 20 min before washing in distilled water for 1 min and a subsequent 1 min wash in 2-propanol. Test strain labelled DNA was mixed with reference strain labelled DNA, purified using a MiniElute kit (Qiagen), denatured at 95 °C and mixed to achieve a final 23 µl hybridization solution of 4x SSC, 0·3 % SDS. Using a 22x22 mm LifterSlips (Eyrie Scientific) a microarray was hybridized overnight sealed in a humidified hybridization chamber (Telechem International) and immersed in a water bath at 65 °C for 1620 h. Slides were washed once in 400 ml 1x SSC, 0·06 % SDS at 65 °C for 2 min and twice in 400 ml 0·06x SSC for 2 min. The microarrays were scanned using a 428 Array Scanner (Affymetrix) and intensity fluorescence data acquired using ImaGene 5.5 (BioDiscovery).
Data analysis.
Data were initially processed and normalized using GeneSpring (Silicon Genetics). For each spot the median pixel intensity for the local background of each spot was subtracted from the median pixel intensity of each spot. Background subtracted pixel intensities for the test strain channel were divided by those for the reference strain channel. The resulting log ratios were normalized by applying the LOWESS intensity-dependent normalization using 50 % of the data. If the value for the reference channel was less than 10 then a value of 10 was used instead. This normalization scheme counteracted any dye effects at low intensity and enabled inter-array comparisons by equalizing the median ratio on each array.
Designation of genes in each strain as present, divergent and absent (highly divergent) was determined using GACK software without the need for arbitrarily defined cut-offs (Fig. 1) (Kim et al., 2002
). GACK analysis used LOWESS normalized log ratio data from GeneSpring that was not flagged as absent by ImaGene and all genes were given equal weighting. GACK used the normal distribution to calculate an EPP (estimated probability of presence) value for each gene; in this way the overall genomic sequence deviation (data spread) was taken into account. A gene was designated present (assigned 1) if it had a calculated EPP of 100 %, as divergent (assigned 0) if it had an EPP between 0 % and 100 %, and as deleted (assigned 1) if it had an EPP of 0 %. An EPP of 0 % indicates a zero per cent chance of being falsely assigned as a divergent gene and an EPP of 100 % indicates a minimum assurance that a gene was present (Kim et al., 2002
). Divergence, although primarily indicating sequence divergence of a particular gene, can also indicate a change in copy number of members within a related gene family (e.g. IS elements and transposons). GACK software was also used to model presence of non-N. meningitidis MC58 (additional) genes in the test strain by performing GACK analysis on the inverse of the log ratio data. Strain-specific present genes were designated present (1) in both analyses (Fig. 1
).
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RESULTS |
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The phylogeny dendrogram based on GACK scores for every gene represented on the microarray (Fig. 2) showed that N. meningitidis strains clustered closely together, with commensal Neisseria species at greatest distance and N. gonorrhoeae occupying an intermediate position between the two groups. Within the N. meningitidis group, N. meningitidis serogroup A strains formed a distinct cluster, with the MLST clonal complex ST-4/subgroup IV strains (2059001 and Z2491) being marginally more closely related to each other than F6124 (MLST clonal complex ST-5/subgroup III). Strains of MLST clonal complex ST-41/44 complex/lineage 3 strains (BZ147, BZ198, NGH36 and NGH15) clustered with each other. Both strains 528 and 1000 are from the ST-18 clonal complex and occur close to the root of the dendrogram together with NGE28.
BZ83 and H44/76 are from the same ST-32 complex/ET-5 clonal complex as N. meningitidis MC58 and were remarkably conserved in gene content and sequence. N. meningitidis C311 clusters closely with BZ83 and H44/76 but is of unassigned clonal cluster. GACK analysis scored 36 N. meningitidis MC58 genes absent in BZ83 whereas H44/76 and C311 had 52 and 57 genes absent respectively. Microarray data of the absent genes showed that the majority of genes [12 (33 %), 23 (44 %), 37 (65 %) respectively] clustered in a region between NMB1123 and NMB1197 and had a ratio of approximately 0·5 (twofold decrease in signal intensity). This region contains a number of phage-related genes and was part of a larger section of the genome that showed high variability between strains. Therefore, this area probably represents a cryptic phage; the ratio data are consistent with this region being duplicated in MC58. BLAST analysis of the region confirms that the NMB11231159 region has been duplicated (at NMB11611197) but this region was only present as a single copy in the other sequenced isolates [N. meningitidis Z2491 (NMA13321369), FAM18 (NMC10631098) and N. gonorrhoeae FA1090 (NG08010775)]. The ratio data suggest that this duplication has been relatively recent, as the closely related species appear to have only a single copy, which may be a result of serial laboratory passage. NMB1123 (and hence NMB1161), encoding a hypothetical protein, shows regions of 98 % identity with at least seven other N. meningitidis MC58 genes; these may act as IS-like elements and allow genetic rearrangement.
Gene composition of pathogenic neisseriae
Analysis of the microarray data identified 1499 present and divergent genes in all N. meningitidis serogroup B strains (core genes), of which 1160 genes were present in all N. meningitidis and N. gonorrhoeae isolates examined. The remaining 339 genes were found in at least one of the non-serogroup B N. meningitidis or N. gonorrhoeae strains tested, but not all, and therefore no gene was specific to serogroup B.
Analysis of the commensal strains was restricted to N. cinerea, N. mucosa, N. flavescens, N. lactamica and N. polysaccharea (seven strains) due to the sequence divergence of N. cuniculi and N. elongata (including N. elongata var. glycolytica). The commensal present and divergent gene set consisted of 1225 genes (core gene set), 1024 of which were also present in the N. meningitidis serogroup B core gene set; 862 genes were common to all strains tested.
Within the commensal group, 105 genes were absent by GACK analysis in all strains, 55 of which were present in the N. meningitidis serogroup B core gene set (Table 3). All 55 genes were also confirmed to be absent from the N. lactamica ST-640 sequence available at the Sanger Centre by BLAST analysis, which at the time of analysis had a theoretical genome coverage of 99·99 %. The N. meningitidis serogroup B shared gene list has an over-representation of cell processes' and other functional categories compared to the N. meningitidis MC58 genome. The 55 N. meningitidis serogroup B shared genes contain 10 transposon-associated genes, 3 phage-related and 23 encoding conserved/hypothetical proteins of unknown function (FUN). Eight genes show homology to the non-functional pilS gene cluster. Additionally a gene encoding a putative TonB-dependent receptor (NMB0293
NMA2193, NMC1887) was present. TonB has a function in transport of iron from receptors on the outer membrane to the bacterial cytoplasm. A number of the hypothetical proteins possess integral membrane motifs and membrane transport motifs; in addition to these, the N. meningitidis serogroup B conserved genes include ones encoding a putative periplasmic type I secretion system protein, and a putative cytolysin secretion ABC transporter. Furthermore a capsule polysaccharide modification protein gene lipB was present. dca has evidence of phase variability and was previously shown to be absent from N. lactamica (Snyder et al., 2001
); it may have a role in competence.
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Analysis of the complete pilin locus (NMA0264-0272NMB00180026) (Fig. 3
) demonstrated that this locus is absent from all the commensal strains (except a possible copy of pilS7 which was classified as divergent by GACK). Of the N. meningitidis strains, N. meningitidis F6124 (serogroup A), N. meningitidis B16B6, NGE28 and 2996 (serogroup B) and N. meningitidis 90/18311 (serogroup C) show sequence divergence of the locus but to a lesser extent than the commensals (mean locus ratio of 0·5 compared to 0·2 respectively). GACK analysis of B16/B6 scored all pilS genes as divergent except pilS2, pilS5 and pilS6, which were absent; however these were slightly over the divergent/absent cut-off and the result could be due to a reduced copy number as well as sequence divergence. GACK analysis indicated that pilE was absent from NGE28 but divergent in N. lactamica L18. The ratio of pilE from NGE28 shows that the gene was close to the divergent cut-off and its apparent absence may be a result of sequence divergence, whereas pilE from L18 was just inside the divergent cut-off but has a lower microarray ratio than NGE28 due to the overall sequence divergence of the N. lactamica and may not be present.
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Phase-variable PilC is required for pilus assembly and pilus-mediated adherence, and most strains carry two pilC loci (pilC1 and pilC2) which are homologous but not identical; however, only PilC1 mediates adherence of N. meningitidis (Rytkonen et al., 2004). Analyses of pilC1 and pilC2 using microarray data and/or GACK scores (Table 4
) are confounded by cross-hybridization between genes and between species due to the slight sequence divergence. pilC1 was represented on the microarray by three reporters, NMA0609, NMB1847 (
NMC0371) and NG0046, and pilC2 by NMB0049 (
NMA0293), NMC0033 and NG1812. pilC1 and pilC2 were absent from all commensal strains tested except that a marginally positive GACK score was detected for pilC1NMA with both N. lactamica strains and N. mucosa, and pilC1NMB/C with N. lactamica L18 (Table 4
). pilC is probably absent given the lack of the remainder of the pilin biogenesis locus; additionally, BLAST analysis against the N. lactamica ST-640 sequence failed to identify a pilC2 homologue. Of the pilC1 reporters, pilC1NMB/C and pilC1NG gave weak homologies with N. lactamica ST-640; however, the pilC1NMA reporter showed a region of identity up to 522/633 bp (82 %), indicating a potential orthologue and explaining the divergent GACK prediction.
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DNA adenine methyltransferase (Dam) is involved in the bacterial mismatch repair system; the loss or absence of the gene impairs this repair system, which may increase the occurrence of frameshifts in homopolymeric tracts or simple base repeats (Bucci et al., 1999). This is an important bacterial mechanism for alteration of gene expression, allowing the onoff switching of loci that can confer heritable niche-specific adaptation. N. meningitidis FAM18 possess a functional dam gene (NMC0327); however in N. meningitidis MC58 and N. meningitidis Z2491 only a non-functional dam gene remnant remains the rest has been replaced by the gene drg (dam-replacing gene), which encodes a restriction enzyme. This genetic exchange has been linked with increased virulence and conserved within particular ST types (Jolley et al., 2004
). The pan-Neisseria microarray had a reporter for dam (NMC0327) and drg (NMB1896) from which it was possible to establish whether a strain was dam+ or drg+ (Table 5
). Martin et al. (2004)
listed a number of dam+ and dam strains which correlated with the microarray data (Table 5
), and Jolley et al. (2004)
identified ST lineages that were drg+, which was also consistent with the microarray data (Table 5
).
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Sun et al. (2000) identified through signature-tagged mutagenesis 16 N. meningitidis MC58 hypothetical genes essential for bacteraemic disease; an additional 10 hypothetical genes have since been identified as being required for colonization of the nasopharynx (unpublished results). Twenty-one of the genes were present in the N. meningitidis serogroup B core gene set but none of these were absent from all commensal strains (Fig. 5
). NMB0103 (putative bacteriocin resistance protein) was not found in the commensal strains but was present in all N. meningitidis strains except NGE28. Thirteen genes were present in all strains tested and may be part of general stress-response pathways. NMB1596 was present only in ST-32 complex/ET-5 complex cluster strains, and NMB0065 was absent from all serogroup A strains but also serogroup B strains 528 and 297-0.
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DISCUSSION |
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Barratt & Sneath (1994) used 155 phenotypic tests to characterize four major groups within the Neisseriaceae. The majority (33/38) of strains tested for our work fall within the area A, which includes N. meningitidis, N. gonorrhoeae, N. polysaccharea, N. lactamica and N. cinerea. Area B species included N. mucosa while area C was represented by N. elongata (including var. glycolytica) and N. flavescens. Area D, which is described as the false Neisseria, was represented by N. cuniculi [synonym Moraxella (Branhamella) cuniculi]. Our microarray analysis of N. cuniculi and N. elongata (including var. glycolytica) is entirely consistent with this classification of strains. The DNA from strains of areas C and D produced poor hybridization signals to a large number of microarray reporters. Interestingly, for one isolate, there is a discrepancy between the phenotypic analysis and our microarray results. N. flavescens has been classified as an area C strain but microarray analysis showed no apparent significant sequence divergence from area A strains.
Smith et al. (1999) analysed Neisseria species relatedness using MLST of three housekeeping genes (recA, argF and rho) and 16S rRNA, and placed them in five related groups. Group 1 (meningitidis group) consists of the species closely related to and including N. meningitidis: N. gonorrhoeae, N. lactamica, N. polysaccharea and N. perflava M7. Group 2 (flavescens group) was represented by N. flavescens K3, group 3 (cinerea group) by N. cinerea F12, and group 5 (elongata group) by N. elongata I1 and N. elongata var. glycolytica J3 and J15. The strains used for our microarray analysis included four of these five groups. Strains from groups 2 and 3 did not differ sufficiently from group 1 to affect the microarray hybridizations. This finding was supported by 16S rRNA sequence typing, which places groups 2 and 3 in the same sub-branch as N. meningitidis group 1 strains (Smith et al., 1999
). Dendrograms based on phenotypic traits of argF and recA place N. cinerea as a distant relative of N. meningitidis serogroup, whereas rho and 16S RNA identify the species as a close relative (Smith et al., 1999
); our data are consistent with N. cinerea being closely related. Our results confirm that the group 5 species are highly divergent from pathogenic neisseriae.
Our microarray phylogeny was concordant with those obtained by MLST analysis, as strains of the same clonal complex still clustered together. The MLST dendrogram indicates clustering together of the hypervirulent MLST type strains, which is also reproduced with the microarray phylogeny. However, in the MLST phylogeny SWZ107 appears as a close relative to the ST-32/ET-5 clonal complex (e.g. MC58) whereas the nearest relatives of NG3/88 and NG6/88 belong to ET-37 complex (e.g. serogroup C); on the microarray phylogeny SWZ107 also clustered with the ST-32/ET-5 clonal complex but had greatest similarity with NG3/88 and NG6/88.
Interestingly we also detected a major split within the strains tested. One group contains the majority (12/18) of serogroup B strains plus E32 (serogroup Z) (B arm), with the second group containing, contains all the non-serogroup B strains including the commensals (A arm), as well as three serogroup B strains.
On the microarray phylogeny the ET-37 complex strain (9018311) occurred on a different major branch (A arm) of the dendrogram to the ST-32/ET-5 clonal complex (B arm). A further MLST cluster consists of BZ198, NGH15, NGH36, BZ147, NGH38, BZ232 and 297-0, which is replicated within the microarray phylogeny, down to sub-branch levels, except 297-0, which showed divergence from this group.
Congruence of MLST clonal complexes with the array data was surprising given the high degree of lateral gene transfer and genetic rearrangements that occur within Neisseria species (Feavers, 2000). This shows that MLST conservation is a good estimation of the conservation within the whole genome but not necessarily of gene arrangement.
Pathogen-specific genes
Bioinformatic analysis of the complete genome sequence identified 55 potential virulence genes in N. meningitidis. These genes were universally present in the N. meningitidis serogroup B strains analysed, and absent from commensal strains and the available N. lactamica genome sequence. The majority of known genes have some role on the bacterial cell surface, including pilin genes, secretion system proteins, capsule modification, and an adhesion and invasion protein. The presence of 13 transposon and phage genes may indicate a route of acquisition. The pilin locus cluster consists of a number of pseudogenes that have the potential to recombine with pilE to form functional pilin subunits and may have a role in altering the surface antigens to assist in avoiding the host cell immune system. These genes appear to have some sequence divergence between strains, which is likely to have evolved through host immune pressure; this is borne out by the fact that N. gonorrhoeae has a distinct pilin locus from N. meningitidis. Although pili are an important virulence factor for pathogenic neisseriae the presence in commensals is less established and our data suggest that they lack the pilE pilS loci; however, other pili-related genes appear to be conserved. In all commensal strains pilD (NMB0332), pilF (NMB0329), pilT (NMB0052) and pilT2 (NMB0768) were scored as present and pilQ (NMB1812) was scored either present or divergent by GACK. Although pilE (NMB0018) was found to be absent from all commensals, an orthologue found in N. meningitidis FAM18 (NMC0210) was found to be present in both N. lactamica strains and was confirmed to be present in N. lactamica ST-640 by BLAST analysis. Expressed pilin can be categorized into two structurally distinct classes: class I (e.g. N. gonorrhoeae MS11, N. meningitidis MC58) and class II (e.g. N. meningitidis FAM18). The monoclonal antibody SM1, used to distinguish these two classess, binds to a highly conserved region within the 5' end of class I PilE but this epitope was lacking from the more structurally divergent class II PilE (Aho et al., 2000; Kahler et al., 2001
). The SM1 minimum epitope has been determined as the linear peptide EYYLN (Virji et al., 1989
). Analysis of the translated genome sequences indicates that this was present in the class I pilin strains N. meningitidis NM58 and N. gonorrhoeae NMNG but absent in the class II strain N. meningitidis FAM18. In N. meningitidis MC58 the SM1 epitope occurred in pilE and four of the silent pilS genes; this was also replicated in N. meningitidis Z2491. The pilE reporters on the pan-Neisseria cDNA microarray cannot distinguish between these slight sequence changes, especially as the gene has a number of highly variable regions, but identification could be achieved using specifically designed oligonucleotides on an oligo-microarray.
The acquisition of a polysaccharide capsule has been suggested as a vital step in the evolution of non-pathogenic neisseriae into pathogenic neisseriae. N. meningitidis MC58 and N. meningitidis FAM18 have identical capsule genes with the exception of siaD. The N. meningitidis Z2491 capsule region contained a number of different serogroup A specific capsule biosynthesis genes. This enables the microarray to distinguish between the three N. meningitidis serogroup capsules. This is currently achieved by examining the capsule biosynthesis locus, where the presence of different alleles of the transferase genes (siaD) can differentiate between serogroup B and C strains (Borrow et al., 1997) and the presence of sacB for serogroup A. None of the capsule loci were present in the commensal species but they were also absent from N. gonorrhoeae NMNG and N. meningitidis E26 (serogroup X). This was consistent with N. gonorrhoeae not possessing a capsule and the unique serogroup X capsule genes not being present on the microarray. The microarray was able to distinguish serogroups A, B and C using what is known about the capsule locus, but with the addition of reporters from the variable region of ctrA and serotype X specific capsule genes it may also be possible to distinguish the other serogroups. Additionally analysing a number of strains from the rarer serogroups may identify other genes that may discriminate between the serogroups.
The pan-Neisseria microarray has been used to analyse the genomic content of a number of documented pathogenic Neisseria strains plus comparison with a number of commensal strains. The microarray was able to identify highly divergent strains through lack of hybridization across the majority of reporters. Reduction of the stringent hybridization condition could allow for greater identification of divergent genes but the data would be less reliable due to increased cross-hybridization. Previous phylogenetic studies using MLST and MLEE were comparable with the microarray data, highlighting both the usefulness of these typing methods and the accuracy of the microarray data. The co-clustering was surprising given the natural competence of Neisseria and evidence of gene acquisition through G+C content variation. This may indicate that virulent strains rarely encounter enhancing exogenous DNA and use the propensity for genetic rearrangement for phase variation and evasion of host immunity. The microarray data also concurred with other virulence determinants, such as dam/drg genotype, characterizing which opc orthologue was present, selectively identifying which pilin subunits and pilC orthologue were present and identifying capsule type in one test. As further virulence determinants are identified the dataset can be used retrospectively, so long as the gene existed in the four design species, to assess all strains tested, and the microarray can be expanded to include additional genes. The microarray can also be used to assess the gene content of a clinical specimen; however, with the need for rapid diagnosis due to the rapid onset of the disease it would be unlikely a microarray would have any benefit over the developing diagnostic TaqMan real-time PCR. Microarrays have been used to identify unique characteristics of an outbreak strain and follow the epidemiology (Rajakumar et al., 2004).
The microarray data were used to identify a subset of genes that appear to be present in all Neisseria species; this may be an overestimate, as only the closely related species produced usable microarray data under the stringent conditions used. Potentially more importantly, the identification of pathogen-specific genes has potential for targeted drug and vaccine design without reducing the beneficial commensal neisseriae. The majority of these genes with known function are related to the cell surface and pathogenicity, which would be expected; from this we could speculate that the FUN genes within this group must also have important roles in pathogenicity and could provide further insights into Neisseria pathogenesis.
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ACKNOWLEDGEMENTS |
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Received 7 April 2005;
revised 6 June 2005;
accepted 8 June 2005.
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