Division of Bacterial, Parasitic and Allergenic Products1 and Division of Viral Products2, Center for Biologics Evaluation and Research, FDA, 8800 Rockville Pike, Bethesda, MD 20892, USA
National Microbiology Laboratory, Population and Public Health Branch, Health Canada, Canada3
Author for correspondence: Peixuan Zhu. Tel: +1 301 496 4177. Fax: +1 301 402 2776. e-mail: Zhu{at}cber.fda.gov
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ABSTRACT |
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Keywords: Neisseria meningitidis, Neisseria gonorrhoeae, glycosyltransferase, oligosaccharide structure, antigenic variation
Abbreviations: LOS, lipooligosaccharide
The GenBank accession numbers for the sequences reported in this paper are AF470655AF470685.
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INTRODUCTION |
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The lgt-2 locus includes lgtF and rfaK that are involved in the biosynthesis of and
chains, respectively (Fig. 1
) (Kahler et al., 1996a
, b
; van der Ley et al., 1997
). lgtF encodes a ß1,4 glucosyltransferase that is responsible for adding Glc to HepI in the inner core (
chain). rfaK encodes the
1,2-N-acetylglucosaminyltransferase that adds GlcNAc to HepII in the inner core (
chain). The lgt-3 locus has an lgtG gene encoding
1,3 glucosyltransferase for biosynthesis of the ß chain (Fig. 1
) (Banerjee et al., 1998
). Four genes at the lgt-1 and lgt-3 loci, lgtA, lgtC, lgtD and lgtG, have a homopolymeric tract that is thought to be involved in LOS phase variation by the slipped strand mispairing mechanism (Danaher et al., 1995
; Jennings et al., 1995
; Yang & Gotschlich, 1996
). Therefore, both the composition and expression of lgt will regulate the biosynthesis of LOS.
In N. gonorrhoeae, the lgt-1 locus has been sequenced in three strains (F62, FA1090 and 1291) and analysed in an additional four strains (15253, MS11, M94 and R10), by DNA hybridization (Erwin et al., 1996 ; Gotschlich, 1994
; Harvey et al., 2000
). The composition and organization of lgt-1 for N. gonorrhoeae is the same for all sequenced strains; only the hybridization data from strain 15253 suggests that there may be some heterogeneity (Erwin et al., 1996
). In N. meningitidis, lgt-1 has been sequenced in five strains [126E (L1), MC58 (L3), Z2491 (L9), A1 (L8) and M978 (L8)] and analysed for 10 representative strains of 10 LOS immunotypes (L1, L2, L3, L4, L5, L6, L8, L10, L11 and L12) by DNA hybridization (Jennings et al., 1995
, 1999
; Parkhill et al., 2000
; Tettelin et al., 2000
; Zhu et al., 2001
). These studies indicate that N. meningitidis has a small and variable repertoire at the lgt-1 locus. Both the composition and organization of lgt-1 differ among the N. meningitidis strains examined (Jennings et al., 1999
). The lgt-1 locus in two immunotypes, L7 and L9, has not been determined, and the lgt genetic organization and DNA sequences of 8 LOS immunotypes of N. meningitidis (L2, L4, L5, L6, L7, L9, L10, L11 and L12) have not been reported. In commensal Neisseria, only one lgt-1 sequence from strain 44 of Neisseria subflava has been reported (Arking et al., 2001
).
To understand the genetic basis of LOS heterogeneity and antigenic variation in Neisseria, nine lgt genes at three chromosomal loci were examined in 95 strains of N. meningitidis, N. gonorrhoeae and commensal Neisseria by DNA hybridization, PCR, restriction analysis and nucleotide sequencing.
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METHODS |
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PCR amplification of the lgt genes.
Three lgt loci were examined by PCR using external primers at the flanking region and internal primers shown in Table 1. Primers for lgt-1, lgt-2 and lgt-3 were designed from the conserved regions identified by multiple alignment of eight [strain 126E, GenBank accession no. U65788; MC58, U25839; Z2491, AL162753.2; M978, AF355193; A1, AF355194; F62, U14554; 1291, AF121135; and FA1090, AE004969], four [strain CDC8201085, GenBank no. NMU58765; MC58, AE002520; Z2491, AL162757; and FA1090, AE004969] and four [strain 15253, GenBank no. AF076919; MC58, AE002553; FA1090, AE004969; and 44, AF241526] available lgt gene sequences, respectively.
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The PCR reaction mixtures contained 1 µl 10 mM dNTPs, 10 pmol each primer, 0·1 µg chromosomal DNA, 5 µl 10x PCR buffer, 1·5 U Taq DNA polymerase (Perkin-Elmer) and sterile redistilled H2O in a final volume of 50 µl. PCR amplification was performed on a GeneAmp 9700 cycler (Perkin-Elmer) using the following protocol: denaturation at 94 °C for 2 min, 30 cycles of amplification at 94 °C for 30 s, 56 °C for 30 s and 72 °C for 2 min, and a final extension at 72 °C for 4 min. The PCR products were analysed by electrophoresis on 1% agarose gels and stained with ethidium bromide.
Restriction analysis.
Amplified DNA fragments from the three lgt loci were subjected to digestion with restriction endonucleases AluI, HaeIII, RsaI and MspI (Roche Molecular Biochemicals). The digestion was performed using 8 µl PCR product, 1·0 µl 10xrestriction endonuclease buffer, 10U restriction endonuclease and sterile distilled water to a final volume of 10 µl. The mixture was incubated at 37 °C for 1 h and then separated on a 1·5% agarose gel and stained with ethidium bromide.
DNA dot blot hybridization.
Bacterial genomic DNA was boiled for 2 min and blotted to Hybond-N+ membranes (Amersham). The DNA probes for lgtA, lgtB, lgtC, lgtD, lgtE, lgtH, lgtF and lgtG were labelled with DIG-11-dUTP (PCR-DIG labelling mix, Roche Molecular Biochemicals) during PCR amplification using the primers in Table 1 and the genomic DNAs from strains F62 (lgtA, lgtB, lgtC, lgtD and lgtE probes), MC58 (lgtF and lgtG) and Z2491 (lgtH) as the templates. DNA hybridization and detection were performed as described by the manufacturer (DIG Nucleic Acid Detection Kit, Roche Molecular Biochemicals).
DNA sequencing.
The PCR products were purified by QIAquick spin columns (Qiagen). DNA sequences were determined from both strands of three independent PCR products for each strain using the primers in Table 1 and 28 additional internal primers (presented in a supplementary table at http://mic.sgmjournals.org). The sequences of homopolymeric regions in lgtA, lgtC and lgtG were determined again from cloned templates. The PCR products were cloned into pCRT7 vector (Invitrogen) and three clones for each strain were used for sequencing. DNA sequencing was performed using the ABI PRISM Dye Terminator Sequencing Kit with AmpliTaq DNA polymerase FS (Perkin Elmer) on a model 377 automated sequencer (Applied Biosystems). DNA sequences were analysed with the Genetics Computer Group package (GCG10.2-Unix, University of Wisconsin) and the Molecular Evolutionary Genetics Analysis software (MEGA2.1, Arizona State University) (Devereux et al., 1984
; Kumar et al., 1994
). The sequence data from this study have been submitted to the GenBank database with accession numbers AF470655AF470685.
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RESULTS |
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The distributions of lgt genes at the three loci in different Neisseria species are shown in Table 2. At the lgt-1 locus, all 49 strains of N. gonorrhoeae had the same lgt genes as the reference strain FA1090: lgtA, lgtB, lgtC, lgtD and lgtE. The lgt genes for N. meningitidis were more variable. lgtD was present only in N. meningitidis strain 126E (L1). lgtE and lgtH in N. meningitidis were mutually exclusive. lgtA and lgtB were present in all but one and three strains of N. meningitidis, respectively. At the lgt-2 locus, all N. gonorrhoeae strains and all 25 N. meningitidis strains tested in this study had lgtF. However, only three commensal Neisseria strains had lgtF: Neisseria cinerea (strain 81176), Neisseria lactamica (81186) and Neisseria polysaccharea (87043). At the lgt-3 locus, 18 N. meningitidis strains, four N. lactamica strains, one N. subflava strain and all N. gonorrhoeae strains had lgtG. The lgt genes were not detected in two strains of N. subflava (81187 and 81201) and one strain each of Neisseria canis, Neisseria caviae, Neisseria flavescens, Neisseria mucosa, Neisseria ovis, Neisseria sicca and Neisseria weaveri.
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Genetic organization of the lgt loci
The lgt-1 locus of 12 N. meningitidis strains (35E, 6275, M986, 89I, M981, M992, 6155, M120, 7880, 7889, 7897 and BB305) was sequenced. For the other 66 strains that yielded positive signals in the hybridization and PCR, the lgt gene order was mapped by PCR using multiple combinations of flanking and internal primers. The data were compared to the reported lgt-1 from five strains of N. meningitidis (strains Z2491, MC58, 126E, A1 and M978) (Jennings et al., 1995 , 1999
; Parkhill et al., 2000
; Zhu et al., 2001
), seven strains of N. gonorrhoeae (FA1090, F62, 15253, 1291, MS11, M94 and R10) (Erwin et al., 1996
; Gotschlich 1994
; Harvey et al., 2000
) and one strain of N. subflava (44) (Arking et al., 2001
). Based on the genetic organization, the lgt-1 locus in these strains was classified into eight genetic types (Types IVIII; Fig. 2
). The organization of all N. gonorrhoeae strains was identical and designated Type I. This type was not observed in any other Neisseria species. Type II was found in both N. lactamica and N. subflava. Types IIIVIII were found in N. meningitidis as well as in commensal Neisseria (one N. subflava, VI; two N. polysaccharea, VII).
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Size variation and sequence polymorphism of the lgt genes
In addition to the lgt-1 sequences from 12 N. meningitidis strains, the sequences of lgtF were determined from 14 N. meningitidis strains, which included all 12 of the LOS prototype strains and two additional N. meningitidis strains (M986 and A1). The sequences of lgtG were determined from seven N. meningitidis strains (35E, 6275, M986, 89I, M981, M992 and A1) as well. lgtF and lgtG from N. gonorrhoeae and commensal Neisseria were analysed by digestion of PCR products with four restriction endonucleases (AluI, HaeIII, MspI and RsaI). Based on the distinct restriction patterns, five lgtF genes and six lgtG genes were also sequenced from N. gonorrhoeae strain 880140, and commensal Neisseria strains 81186, 93302, 81176 and 85071. The sequences obtained in this study were compared to the lgt sequences reported in the databases (Arking et al., 2001 ; Gotschlich, 1994
; Harvey et al., 2000
; Jennings et al., 1995
, 1999
; Kahler et al., 1996a
, b
; Parkhill et al., 2000
; Tettelin et al., 2000
; Zhu et al., 2001
). The size variation and sequence polymorphisms of the lgt genes are shown in Table 3
. The mean G+C content for each lgt gene varied from 45·09 mol% to 56·51 mol% (Table 3
). Six genes (lgtA, lgtB, lgtC, lgtD, lgtE and lgtG) showed size variations whereas three genes (lgtF, lgtH and lgtZ) had a conserved length (Table 3
). The size variations resulted from variations in the homopolymeric region, insertions and deletions, or presence of a premature stop codon. The genetic organization of the three lgt loci for 25 N. meningitidis strains and homopolymeric tract length for 17 N. meningitidis strains are summarized in Table 4
.
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The number of synonymous substitutions per synonymous site (Ks) and nonsynonymous substitutions per nonsynonymous site (Ka) were estimated for all pairwise comparisons by the Li method using the DIVERGE in the GCG 10.2 program package (Devereux et al., 1984 ; Li et al., 1985
; Li, 1993
). Ks varied from 1·94% to 11·52% and Ka from 0·73% to 4·71%. Based on the amino acid substitution frequencies, lgtF, lgtH and lgtZ were the most conserved genes. The Ka/Ks ratio, which is a measure of the selective constraints on a gene, varied from 0·14 to 0·83. In the lgtA, lgtB, lgtC, lgtD and lgtE genes at the lgt-1 locus, the levels of synonymous and nonsynonymous site variation were elevated, and similarly, the Ka/Ks ratios (0·38 to 0·83) were extremely high (Table 3
).
Phylogenetic analysis of the lgt genes
A phylogenetic tree was constructed using the neighbour-joining method by program MEGA 2.1 from the nucleotide sequences for each distinct allele of the lgtB, lgtE and lgtH genes (Kumar et al., 1994 ; Saitou & Nei, 1987
)(Fig. 4A
). The phylogenetic tree reveals that lgtH was related to but separated from lgtB and lgtE. The genetic distances among lgtH alleles are shorter than that among lgtB and lgtE in the phylogenetic tree, suggesting that lgtH might be a new paralogue resulting from a recent gene duplication event or imported from other bacterial sources. It was also noted that lgtB and lgtE from three strains of N. gonorrhoeae (F62, 1291 and FA1090) clustered into a group, similar to their relationship in a tree of lgtA genes (not shown).
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The lgtG gene from strain MC58 was an intermediate branch between the N. meningitidis group and the commensal Neisseria. The 5' end of lgtG from MC58 was similar to other N. meningitidis strains but the 3' 51 bp was identical to the lgtG genes from the commensal Neisseria, suggesting intragenic recombination between commensal Neisseria and N. meningitidis. The reported lgtG from N. subflava strain 44 (Arking et al., 2001 ) was closely related to N. meningitidis strain 89I (Fig. 4C
). These two strains formed an intermediate branch between the N. gonorrhoeae and N. meningitidis groups. The 5' and 3' ends of lgtG from these two strains were similar to N. meningitidis but five polymorphic sites in the internal region were identical to lgtG from N. gonorrhoeae. These data implied an independent evolutionary history for the genes at three lgt loci involved in LOS biosynthesis.
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DISCUSSION |
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In 51 N. gonorrhoeae strains examined in this study, only one genetic type of lgt-1, Type I, was found. This type contained five genes: lgtA, lgtB, lgtC, lgtD and lgtE. These results agree with all reports in the literature except for strain 15253 (Erwin et al., 1996 ). In this study we have examined a variety of clinical isolates including urethritis, disseminated gonococcal infection and reference strains. Therefore, it appears that the lgt-1 organization of 15253 is not common and lgt-1 of N. gonorrhoeae strains is not diverse. At the lgt-2 and lgt-3 loci, all 51 strains of N. gonorrhoeae contain lgtF and lgtG, respectively. N. gonorrhoeae strains showed only Type I organization at the lgt-3 locus. These results revealed that most N. gonorrhoeae strains have a common lgt composition and arrangement at three chromosomal loci for biosynthesis of LOS. The LOS heterogeneity and antigenic variation in N. gonorrhoeae may be controlled at the transcriptional or the translational level.
Six genetic types of lgt-1 from Type III to Type VIII were recognized in 25 N. meningitidis strains, representing 12 LOS phenotypes. The common lgt-1 types in N. meningitidis were Types VI and VII, which contained three genes, lgtABE or lgtABH. At the lgt-3 locus, N. meningitidis strains had a variable organization from Type I to IV. This suggests that the lgt composition and arrangement in N. meningitidis are important factors in LOS heterogeneity and antigenic variation. In two N. meningitidis strains, 7880 and 7889, the genes at the lgt-1 locus detected in this study differ from the previous report (Jennings et al., 1999 ). The PCR, DNA hybridization and nucleotide sequencing have been repeated to verify the data. The reproducible results showed that these two strains contain lgtABH and lgtAH at the lgt-1 locus, respectively.
The N. meningitidis LOS is classified into 12 immunotypes (Mandrell & Zollinger, 1977 ; Zollinger et al., 1977
, 1980
). However, N. meningitidis LOS is heterogeneous and its expression is subject to antigenic variation (Burch et al., 1997
; Danaher et al., 1995
; Jennings et al., 1995
; Yang & Gotschlich, 1996
), which complicate the LOS phenotype as a marker for epidemiological purposes. On the basis of genetic organization of the three lgt loci, 25 N. meningitidis strains examined in this study can be divided into 10 LOS genotypes (Table 4
). The eight Canadian strains all had LOS genotype 3. Furthermore, the sequence comparison showed that N. meningitidis strains with the same LOS genotype had variable combinations of the lgt gene alleles. Therefore, they could be further divided into LOS genetic subtypes.
No absolute correlation was observed in the relationships between the LOS genotypes and LOS phenotypes (Table 4). LOS phenotyping detects the antigenic variation of LOS products whereas the LOS genotyping detects the genetic basis for the LOS biosynthesis. Many N. meningitidis strains express a LOS mixture shown as multiple bands on SDS-PAGE gels, while some strains express only a single LOS band. LOS phenotyping classifies N. meningitidis strains into a major LOS immunotype plus one or more minor LOS immunotypes. LOS genotyping could distinguish N. meningitidis strains with the potential for variation in LOS expression. For example, strain A1 has the same L8 major immunotype as M978 but lacks expression of L3,7 (Zhu et al., 2001
).
In some cases, differences in expression of lgt genes may result in different phenotypes among strains with the same genotype. For example the homopolymeric tract of lgtG is in-frame in M981 (C11) but out-of-frame in MC58 (C12). Extension of the LOS ß chain controlled by lgtG might be one of the reasons for the phenotype difference. Strain 7889 (L11) has the same organization at the lgt-1 locus as A1 (L8) but expresses a different LOS immunotype. LOS from strain 7889 does not contain the terminal Gal (unpublished data), indicating that lgtH is not expressed or non-functional. Comparison of the LgtH protein sequences showed three amino acids difference between the two strains. At the lgt-3 locus, strain A1 has lgtG for extension of the ß chain but strain 7889 does not. This suggests that the antigenic difference between L8 and L11 LOS may be influenced by both the and ß chains.
Therefore, while LOS genotyping cannot replace LOS phenotyping, it provides complementary information that will contribute to our understanding of the mechanisms of LOS antigenic variation. Also, since LOS genotype subdivided several common LOS phenotypes and is not phase variable, it may serve as a useful epidemiologic marker.
Interspecies gene transfer and frequent DNA recombination in pathogenic and commensal Neisseria species has been observed at several chromosomal loci, e.g. the penA and tbpB genes (Linz et al., 2000 ; Spratt et al., 1992
). In this study, the genes lgtA and H were detected in six species, lgtB and F in five species, lgtC and G in four species and lgtD in three species of Neisseria (Table 2
). The distribution of the lgt genes in multiple species provides evidence that pathogenic Neisseria and some commensal Neisseria share a common lgt gene pool for LOS biosynthesis. Some genes seem specific for certain species, e.g. lgtE was detected almost exclusively in the pathogenic species N. gonorrhoeae and N. meningitidis, and the mosaic lgtZ was detected only in N. meningitidis. No lgt genes were detected in seven species: N. canis, N. caviae, N. flavescens, N. mucosa, N. ovis, N. sicca and N. weaveri. Only the hybridization signals of lgtA, H and G were observed in N. elongata and no specific products were obtained in the PCR amplification of this strain. These commensal Neisseria strains do express LOS and therefore must have lgt genes with low homology in the primer-binding regions or differ in other ways from those examined in this paper.
Two ORFs, lgtH and lgtZ (Zhu et al., 2001 ), are homologues to the lgt genes. The lgtH gene reported in strain Z2491 and strain A1 is closely related to two known genes, lgtB and lgtE. This ORF was originally described as lgtB2 in the genome of N. meningitidis strain Z2491 (Parkhill et al., 2000
) based on its close relationship to lgtB. However, its 3' end differs from that of both lgtB and lgtE. In this study, the lgtH gene was found in seven additional N. meningitidis strains and in three species of commensal Neisseria. Eight distinct lgtH alleles were identified from nine lgtH sequences from N. meningitidis. Phylogenetic analysis revealed that lgtH is a gene cluster separated from lgtB and lgtE (Fig. 4A
). The location of lgtH and its mutual exclusivity with lgtE suggests that it may be an allele of lgtE, so further discussion regarding the nomenclature of this gene may be warranted. lgtZ is a mosaic structure consisting of parts of lgtA and lgtB. It was observed in three N. meningitidis strains, 126E (Jennings et al., 1999
), M978 (Zhu et al., 2001
) and BB305. The biological function of lgtH and lgtZ is currently being evaluated.
Both the genetic organization and DNA sequence of the lgt-2 locus were more conserved than in other lgt loci. The G+C content of lgtF (45 mol%) was remarkably lower than the mean G+C content of two N. meningitidis genomes (52 mol%) (Parkhill et al., 2000 ; Tettelin et al., 2000
). Furthermore, all strains of N. gonorrhoeae and N. meningitidis contained lgtF at the lgt-2 locus and the Ka/Ks ratio of lgtF (0·14) was the lowest of the lgt genes (Table 4
). This suggests that there are functional constraints on the LgtF protein in these two pathogenic species. It has been reported that the proximal glucose residue of the
chain catalysed by LgtF protein is required for efficient invasion of gonococci into the host mucosa (Minor et al., 2000
). The conservation of lgtF in N. meningitidis and N. gonorrhoeae may reflect the LOS structural requirement of the proximal glucose for interaction with the human host.
Horizontal gene transfer through interspecies and intraspecies DNA recombination appears to be one of the factors responsible for diverse genetic arrangements of the lgt locus. It is interesting to observe in the phylogenetic tree of lgtG that N. meningitidis strain MC58 was separated from other N. meningitidis strains. The 3' end of lgtG from MC58 was identical to that of lgtG from three strains of commensal Neisseria (Cn group, Fig. 4C). This suggests that lgtG in MC58 is a hybrid gene that resulted from horizontal gene transfer via DNA recombination. In addition, the genetic organizations of regions surrounding lgtG are also similar between N. meningitidis strain MC58 and four N. lactamica strains (Type II of lgt-3, Fig. 2
), consistent with interspecies gene transfer at the MC58 lgt-3 locus.
On the basis of comparisons of genetic organizations, sequence variations, G+C content and patterns of synonymous and nonsynonymous substitution, we conclude that Neisseria shares a common lgt gene pool for biosynthesis of LOS. The lgt-1 and lgt-3 loci are hypervariable genomic regions in N. meningitidis, whereas lgt-2 is conserved in both pathogenic Neisseria species. The typical polymorphisms of lgt appear to have arisen through horizontal gene transfer and homologous recombination in addition to mutation events. This study of the genetic diversity of the lgt loci provides fundamental information for understanding the heterogeneity and antigenic variation of Neisseria LOS.
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ACKNOWLEDGEMENTS |
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Received 6 November 2001;
revised 11 February 2002;
accepted 21 February 2002.