Department of Microbiology and Parasitology, The University of Queensland, Brisbane, QLD 4072, Australia1
University of Oxford, Department of Paediatrics, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK2
National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands3
Author for correspondence: Michael P. Jennings. Tel: +61 7 3365 4879. Fax: +61 7 3365 4620. e-mail: jennings{at}biosci.uq.edu.au
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ABSTRACT |
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Keywords: phase variation, lgt genes, lipopolysaccharide, lipooligosaccharide, Neisseria meningitidis
The GenBank accession number for the sequence reported in this paper is U65788.
a Present address: SmithKline Beecham Biological s.a., Rue de IInstitut, 89 B-1330 Rixensart, Belgium.
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INTRODUCTION |
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The type of LPS structure expressed is a key factor in interactions with the host. For example, strains expressing the adhesin Opc and the L3 immunotype LPS structure (lacto-N-neotetraose, containing a terminal sialic acid) are non-invasive, relative to bacteria expressing the L8 immunotype, non-sialylated, LPS structure, which are invasive (Virji et al., 1995 ). The L3, 7 and 9 immunotypes are relatively serum resistant compared to the L8 immunotype (Moran et al., 1994
). Studies in the mouse model of infection reveal that the L8 immunotype predominates in the nasopharynx while the L3, 7 and 9 immunotypes are most commonly found in the blood of the infected mice (Mackinnon et al., 1993
). These studies are strongly suggestive of a key role for these terminal LPS structures, and the phase variation of their expression, in pathogenesis.
Over recent years there has been significant progress in the genetics of LPS biosynthesis (recently reviewed by Kahler & Stephens, 1998 ). A locus containing three genes, lgtABE, required for the biosynthesis of the terminal LPS structure lacto-N-neotetraose in Neisseria meningitidis strain MC58 has previously been described (Jennings et al., 1995
). This study also describes the mechanism that controls the phase-variable expression of this structure, which operates via high-frequency mutation in a homopolymeric tract of 14 G residues in the first gene of the locus, lgtA. Structural studies of LPS from lgt mutant strains and enzyme assays have confirmed that these three genes encode glycosyltransferases for the biosynthesis of lacto-N-neotetraose (Wakarchuk et al., 1996
). Prior to this work, Gotschlich (1994)
described a similar locus in Neisseria gonorrhoeae strain F62 which contained five LPS biosynthetic genes, lgtABCDE. The N. meningitidis genes described above are present in the same orientation and order as those in the N. gonorrhoeae locus, so that the major difference is the absence of lgtC and lgtD in the N. meningitidis locus. The lgtC and lgtD genes are involved in the biosynthesis of LPS structures that are not expressed by N. meningitidis strain MC58, which can express only the L3 or L8 immunotype (see Fig. 2
).
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METHODS |
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Reagents and enzymes.
Reagents were supplied by BDH with the exception of Tris (Boehringer Mannheim) and antibiotics (Sigma). Restriction and modification enzymes were obtained from Boehringer Mannheim.
Recombinant DNA techniques and nucleotide sequence analysis.
Most recombinant DNA techniques were as described in Sambrook et al. (1989) . Using methods described previously (Jennings et al., 1993
), a DNA fragment size enriched library was made to clone the lgt locus of strain 126E on a 6 kbp XbaIClaI fragment. The probe used was a PCR product specific for the lgtE gene (see below). Conditions for colony screening were the same as described below for Southern blotting. Nucleotide sequence analysis was performed using the PRISM Dye Terminator Sequencing Kit with AmpliTaq DNA polymerase FS (Perkin Elmer) in conjunction with a model 373a automated sequencer (Applied Biosystems). PCR was essentially as described by Saiki et al. (1988)
. Nucleotide sequence analysis was done using the GCG sequence analysis package (Devereux et al., 1984
). Oligonucleotide primers used for PCR and sequencing are listed in Table 1
. Sequence analysis of the number of residues in the homopolymeric tracts of the lgtA, lgtC, lgtD and lgtG genes was determined in a set of immunotype typing strains. The lgtD and lgtC homopolymeric tracts were sequenced from the cloned 126E lgt locus (this work). The lgtC gene of M978 was amplified with lgtCF and lgtCR, and sequenced with primers lgtCF and R99. The homopolymeric tract region of lgtA was amplified using lic31ext and lic16ext, and sequenced with the same primers. The lgtG homopolymeric tract region was amplified using primers LG1 and LG2, and sequenced with LG3 and LG4. All sequences reported were determined by nucleotide sequence analysis of pooled triplicate independent PCR products on both strands.
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Colony-immunoblots.
To examine the potential for phase variation between immunotypes, colony-immunoblots were performed as described previously (Jennings et al., 1995 ). For each strain tested at least 10000 individual colonies were plated and examined for switching. mAbs Mn14F20-11, Mn4A8B2 and 171-L1 are described by Scholten et al. (1994)
. mAb Mn42F12.32 was isolated after immunization of mice with outer-membrane complexes of the L2 strain 3006 (B. Kuipers & P. van der Ley, unpublished).
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RESULTS |
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Variation in the poly-G regions of the lgtA gene and poly-C region of the lgtG gene in the immunotype typing strains
In previous work, we have described the mechanism of phase variation between the immunotypes, which is based on high-frequency frameshift mutations which occur in a homopolymeric tract of G residues in the lgtA gene (Jennings et al., 1995 ). This mechanism has been confirmed in independent studies in lgt genes of N. gonorrhoeae (Danaher et al., 1995
; Yang & Gotschlich, 1996
; Banerjee et al., 1998
). Variation between the L3 and L8 immunotypes, mediated by lgtA expression, results in the loss and gain of the lacto-N-neotetraose structure. Such variation has not been observed in the L2 and L4 immunotypes, i.e. a switch between either L2 or L4, and a structure lacking lacto-N-neotetraose (see Fig. 2
). To investigate whether this was due to differences in the homopolymeric tract of the lgtA gene, we determined the number of G residues in the lgtA genes of each of the immunotype strains. This was achieved by PCR amplification of the region followed by nucleotide sequence analysis of the homopolymeric tract region (Methods and Fig. 4
). As expected, MC58 and another L3 type strain, H44/76, contained 14 G residues consistent with lgtA expression. The L8 strain, M978, contains 13 G residues resulting with an (currently) inactive lgtA gene, consistent with the absence of the lacto-N-neotetraose moiety in the L8 immunotype structure (see Fig. 2
). Immunotypes L10, L11 and L12 contained 9, 12 and 10 G residues, respectively, all currently out of frame for expression. L11 and L12, which have lgtA, lgtB and lgtE (see above), presumably only required a single nucleotide change to express lacto-N-neotetraose. L10 was negative for lgtA in the Southern blot experiment, and presumably contains only the 5'-end of lgtA, outside the region of probe binding.
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In order to investigate the size of the homopolymeric tract of the lgtG gene of each strain, the lgtG gene was amplified by PCR and the tract region sequenced. The result, shown in Fig. 4, revealed that the L2 and L5 immunotypes contained 11 C residues, like the N. gonorrhoeae example (Banerjee et al., 1998
), which is in-frame for expression. Strain M992 (L6) had 14 C residues, which is also in-frame for expression of lgtG. These findings are consistent with the published structures for L2 and L5 immunotypes, which express the
13-linked glucose structure, but not the L6 strain, which is not reported to express this structure. The L4 and L3 immunotype strains were out of frame for expression, consistent with the absence of the
1-3-linked glucose extension in the structures of these immunotypes.
Colony-immunoblot analysis of the phase variation repertoire
To confirm the potential for structural variation that was predicted by the Southern blot data, the phase variation repertoire of several strains was tested for immunotype switching by colony-immunoblot experiments. The selection of strains that could be tested was restricted as only a subset of the mAbs used in immunotyping ELISA (Scholten et al., 1994 ) are suitable for colony-immunoblot experiments. The results of the colony-immunoblot experiments are displayed in Table 2
. Southern blots (see above) suggested that strain M978 (L8 immunotype) could potentially switch between three alternate immunotypes, L8, L1 and L3, as it contained the lgtA, lgtB, lgtC and lgtE genes (see Figs 2
and 3
). Using mAb Mn4A8B2, switching from - to + was observed, so from L8 to L3. Using mAb 17-1-L1 there was switching from - to +, so from L8 to L1. These results confirm the phase variation repertoire predicted for strain M978 from the Southern blot data. Strain 126E is predicted to be limited to variation between L1 and L8 as it contains only active lgtC and lgtE genes. With mAb 17-1-L1 there was a switch from + to -, so from L1 to L8. With mAb Mn4A8B2 all colonies were negative, so there was no switch from L1 to L3 consistent with absence of the lgtA gene in the Southern blot result, which was confirmed by the deletion of most of the lgtA and lgtB genes seen in the nucleotide sequence of strain 126E. Strain M992 (L6 immunotype) hybridized with the lgtB probe, suggesting the potential for extension of the L6 structure to the L4-like structure (see Figs 2
and 3
). With mAb Mn15A8-1 all colonies are negative, therefore no switch was detectable from L6 to L4. With mAb Mn4C1B all colonies remain positive. This result suggests that although strain M992 hybridizes with the lgtB probe, the gene may be inactive.
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DISCUSSION |
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Analysis of the set of immunotype type strains with probes based on the five lgt genes involved in -chain extension demonstrates that most strains contain only three genes: lgtA, lgtB and lgtE. This indicates that these strains can only make the lacto-N-neotetraose extension from the terminal glucose which extends from the first heptose (see Fig. 2
). The exceptions to this trend are the L1 type strain, 126E (discussed above), the L10 type strain, 7880, which contains only lgtE, and the L8 type strain, M978, which contains lgtA, lgtB, lgtC and lgtE. Colony-immunoblot experiments confirmed that the latter strain can make the L1, L3 and L8 immunotype structures. These data are consistent with our original observation of a restricted repertoire of terminal structures in N. meningitidis with only one of the 10 strains examined (M978) capable of more than one alternative
-chain extension.
The lgtG gene of N. gonorrhoeae which encodes a transferase for an 1-3-linked glucose extension from the second heptose (ß-chain extension) was reported during the preparation of this manuscript (Banerjee et al., 1998
). This structure is also present in the L2 and L5 immunotype structures, and our additional Southern blot experiments revealed that, as expected, the lgtG gene was present in the L2 and L5 type strains, and also in the L4, L6 and L3 type strains.
Southern blot analysis is limited to an indication of gene content, suggesting structures that may be expressed by an individual strain. To examine the current state of expression (in or out of reading frame) and the potential for switching between structures, we looked at the homopolymeric tract regions of lgtA and lgtG genes, in which the high-frequency mutation events occur that mediate phase variation. In this study, we determined that the L2, L4, L5 and L6 immunotype strains contain only five G residues in the homopolymeric tract of their lgtA genes. This suggests that in these strains, the expression of this gene is not subject to phase variation, and that the terminal lacto-N-neotetraose is constitutively expressed (see Fig. 2). The set of strains examined fell into two main groups with respect to phase variation: one group phase-vary
-chain extension via lgtA or lgtC but cannot make ß-chain (immunotype type strains for L1, L8, L11 and L12); the second group phase-vary the ß-chain extension via lgtG but presumably cannot vary
-chain, lacto-N-neotetraose, expression (immunotype type strains for L2, L4, L5 and L6). The exception to this grouping is the L3 strain, H44/76, which appears to have the capability of making extensions from both heptose molecules possessing both lgtA and lgtG, although the lgtG gene is out of frame for expression. We have demonstrated that H44/76 can switch from L3 to L2, and that this correlates with a change in predicted lgtG expression via an alteration in the poly-C tract. This finding is consistent with a recently published structural analysis of strain NMB (Rahman et al., 1998
), which reported that both the L2 and L3 immunotype structures are expressed by this strain. The rate of phase variation from L3 to L2 in H44/76 was observed to be 1 in 10005000. This is an order of magnitude lower than has been observed for other LPS phase variations, such as the L3 to L8 transition in strain MC58 which is 1 in 200 (M. P. Jennings, unpublished). This lower rate of variation may be due to the characteristics of the homopolymeric tract of lgtG or may indicate that other, lgtG-independent events are required prior to the addition of the
1-3-linked glucose structure.
As described above, the switching between L3 and L8 immunotypes in strain MC58, which is mediated by high-frequency mutation in the homopolymeric tract of 14 G residues in the lgtA gene, results in modulation of LPS sialylation: L3 immunotype LPS is sialylated; L8 immunotype LPS is not. This difference in LPS sialylation has been demonstrated to have profound effects on adherence and invasion in in vitro model systems (Virji et al., 1995 ). The level of surface sialylation has also been associated with serum resistance in both N. meningitidis and N. gonorrhoeae. As structural studies have reported that the strains which constitutively express lacto-N-neotetraose do contain sialic acid (Kogan et al., 1997
; Gamian et al., 1992
), the question of how these strains regulate the level of LPS sialylation is raised. Two likely possibilities are either environmental regulation of the lst gene, which encodes the sialyltransferase, or an alternative phase variation event which may modify the LPS acceptor molecule.
As described above, the advantage of examining LPS expression in N. meningitidis genetically rather than immunologically is the ability to classify strains by their phase variation repertoire, based on a combination of gene content and phase variation potential (homopolymeric tract presence and/or length). This is far more informative than determining the immunotype which happens to be expressed by a single colony picked at the time of isolation, and has the potential to reveal new relationships between aspects of meningococcal disease and this important virulence factor.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 February 1999;
revised 14 June 1999;
accepted 30 June 1999.