3 Department of Chemistry, University of New Hampshire, Durham, NH 03824, USA, and 4 Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA
Received on November 21, 2001; revised on February 28, 2002; accepted on February 28, 2002
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Abstract |
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Key words: carbohydrate epitopes/FACE analysis/lipooligosaccharide structure/LOS/mass spectrometry
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Introduction |
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Neisserial LOS has been examined by chemical (Gibson et al., 1993; Griffiss et al., 1987
; John et al., 1991
), biological (Griffiss et al., 1994
), and immunological techniques (Mandrell et al., 1988
; Schneider et al., 1985
), as well as through visualization by silver staining of samples analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) (Kim et al., 1988
; Schneider et al., 1984
). It is an amphipathic molecule that consists of a hydrophilic carbohydrate moiety and a hydrophobic lipid A moiety. These domains are linked together through the acidic sugar 3-deoxy-D-manno-2-octulosonic acid (KDO).
The oligosaccharide (OS) is multiantennaeary with chain extensions from each of the two heptose residues, forming three elongation centers defined as the , ß, and
chains (Gibson et al., 1993
; John et al., 1991
; Phillips et al., 1990
; Yamasaki et al., 1991
a,b). The
chain elongates from the first heptose (Hep I) and may contain several structures that are mimics of human carbohydrate epitopes (Apicella and Mandrell, 1989
; Moran et al., 1996
). Its synthesis is directed by a gene cluster (lgtA-E) (Gotschlich, 1994
), with structural variations being regulated by changes in the number of guanines contained in the polyguanine tract of three of the genes (lgtA, C, and D) (Burch et al., 1997
; Danaher et al., 1995
; Yang and Gotschlich, 1996
). ß chain (extending from the second heptose [Hep II]) expression is modulated by the expression of lgtG (Banerjee et al., 1998
) and may be composed of single glucose, lactose, or additional sugars added onto Glc (Gibson et al., 1989
; Yamasaki et al., 1994
). The
chain has been found in all strains examined and consists of a GlcNAc or GlcNAc (OAc) linked to Hep II. Occasionally, this chain is elongated by the addition of galactose (Griffiss et al., 2000
). Some positions of Hep I and Hep II are also available for phosphoethanolamine (PEA) or phosphate addition. Most of the genes that mediate gonococcal and meningococcal LOS biosynthesis have been cloned and characterized (Banerjee et al., 1998
; Drazek et al., 1995
; Gilbert et al., 1996
; Gotschlich, 1994
; Kahler et al., 1996
; Levin and Stein, 1996
; Sandlin et al., 1993
; Sandlin and Stein, 1994
; Stojiljkovic et al., 1997
; Zhou et al., 1994
).
N. gonorrhoeae strain PID2, isolated from a women suffering from pelvic inflammatory disease, produces at least six different LOS components when these molecules are separated on an SDSPAGE gel. The mobility of the four slowest-migrating components are similar in mobility to LOS components isolated from strain MS11mkC, however, LOS isolated from MS11mkC binds mAbs 2-1-L8 and B5, whereas LOS isolated from PID2 fails to bind these monoclonal antibodies (mAbs). This indicates that there are a structural differences between LOS isolated from PID2 and MS11mkC. This study was undertaken to determine the structure of the various LOS components isolated from PID2 and to identify key genes needed for its synthesis.
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Results |
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The DNA sequence of lgtB indicated that it should encode a functional protein. The polyguanine tract contained in lgtC was composed of nine guanines and should produce a frame shift mutation in LgtC. Additionally, at the 3' end of the lgtC coding sequence, there were some differences relative the DNA sequence seen for lgtC of F62 (Gotschlich, 1994). The DNA sequence obtained from the cloned fragment indicate that lgtA1 and lgtA2 have more than 99% identity with each other and would encode nonfunctional proteins (lgtA1 contained a polyguanine tract of 14 bp and lgtA2 contained a polyguanine tract of 9 bp). However, if lgtA1 contained a polyguanine tract of 13 guanines, it would produce an in-frame LgtA protein; lgtA2 would need a polyguanine tract of 10 guanines to produce a functional protein. The last gene in the cluster is a hybrid between lgtB and lgtE; the first 560 bp are identical to its own lgtB gene, and the last 300 bp would encode a peptide identical to F62lgtE at the amino acid sequence level.
PID2 SDSPAGE profile and mAb binding
N. gonorrhoeae PID2 expresses at least six LOS components that can be separated on Tris-tricine gels. As shown in Figure 2, from top to bottom we have numbered them as 1 to 6. Bands 1 and band 3 react with mAb 1B2 (Figure 2b, lane 5). Others have shown that band 6 can bind mAb 4C4, which recognizes LOS with only one glucose on the chain (Noda et al., 2000
). None of the other bands were able to bind the mAbs 2-1-L8, 17-1-L1, 3G9, B5, and 25-1-LC1 (Figure 2b and data not shown).
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The insertional disruptions of these genes, contained on plasmids pF62lgtF and pF62lgtDE
, were introduced into the PID2 chromosome by transformation. Because these plasmids cannot replicate in the gonococcus, spectinomycin-resistant colonies can only arise via homologous recombination between the plasmid DNA and the chromosome. Because pF62lgtF
has the omega cassette inserted into the lgtF gene without transcription termination signals, this construct should have no polar effects; thus only the lgtF gene is nonfunctional because of the insertion and rfaK (necessary for
chain biosynthesis) retains its function. The plasmid pF62lgtDE
has the complete omega cassette (including the transcription termination signals) inserted between the lgtD and lgtE genes; thus the lgtE gene is not transcribed, due to the polar effect of the insertion. (We have shown that the insertion of this cassette at this point in a variety of gonococcal strains completely oblates the expression of lgtE; unpublished data).
After introducing the desired insertion into the gonococcal chromosome by DNA-mediated transformation, several transformants were isolated; each was screened for reactivity with various mAbs. All transformants generated by pF62lgtF possessed the same SDSPAGE profile and no longer reacted with any of the mAbs specific for the parental LOS. One mutant was named PID2F
(see Figure 2b, lane 2). When we tested transformants generated by pF62lgtDE
for reactivity with mAb 25-1-LC1, of the hundreds of spectinomycin-resistant transformants we obtained, we identified a single transformant that had acquired the ability to bind this mAb (see Figure 2b, lane 3). The acquisition of mAb 25-1-LC1 reactivity by this mutant, named PID2E
#13, probably arose via a spontaneous phase shift in lgtG. One mutant, representing the class that failed to bind mAb 25-1-LC1 was named as PID2E
#1 (see Figure 2b, lane 4). The SDSPAGE and western blot analysis of LOS isolated from these strains demonstrate that with a nonfunctional lgtF, as seen in strain PID2F
(Figure 2b, lane 2), only a single LOS component is expressed, and it possesses a mobility similar to F62
lgtA
lgtF LOS (Figure 2b, lane 1). This LOS did not bind mAb B5, indicating that this LOS does not contain a PEA on the 3-carbon of Hep II. Because it did not bind mAb 25-1-LC1, it indicated that this strain also lacked a glucose on the ß chain.
Of the two kinds of mutants generated when the omega cassette was inserted before the lgtE gene, only one of them, PID2E#13, was mAb 25-1-LC1 reactive; the others (represented by the strain PID2E
#1) all possessed the same SDSPAGE profile. Because the ß chain glucose is necessary for binding by mAb 25-1-LC1 (Tong et al., 2001
), we concluded that lgtG is out of frame in PID2, and the one transformant we isolated that acquired the ability to bind mAb 25-1-LC1 represented a spontaneous frame shift in the polycytosine tract of lgtG.
Exoglycosidase digestion of PID2 LOS
To further characterize the LOS made by PID2, two exoglycosidases were used to digest purified PID2 LOS. Jack bean meal ß-galactosidase can readily hydrolyze nonreducing terminal Gal ß1-6 GlcNAc and Gal ß1-4 GlcNAc. Jack bean meal ß-N-acetylhexosaminidase can cleave nonreducing terminal ß1-2, 1-3, 1-4, and 1-6 linked N-acetylglucosamine and N-acetylgalactosamine residues. However, this enzyme has different pH optima for the hydrolysis of p-nitrophenol (pNP) N-acetyl-ß-D-glucosaminide (pH 5.06.0) and pNP N-acetyl-ß-D-galactosaminide (pH 3.54.0). Thus, by controlling the digestion conditions, we could differentiate between terminal GlcNAc and GalNAc residues. Streptomyces plicatus ß-N-acetylhexosaminidase cleaves terminal GlcNAc and GalNAc with equal efficiency.
Purified LOS from different strains were digested by these exoglycosidases and analyzed on a SDSPAGE gel. The results shown in Figure 3A show that ß-N-acetylhexosaminidase (from jack bean meal, pH 5.0) could not excise the terminal GalNAc from F62 LOS (Figure 3A, lane 7), whereas S. plicatus ß-N-acetylhexosaminidase could (Figure 3A, lane 6). Jack bean meal ß-galactosidase was able to remove Gal from F62lgtD LOS (Figure 3A, lane 4), however, this ß-galactosidase could not cleave Gal from F62
lgtA (Figure 3A, lane 3). Figure 3B shows the digestion results from PID2 LOS. When treated with ß-galactosidase, PID2 LOS bands 1 and 3 disappeared and more of bands 2 and 4 appeared (Figure 3B, lane 4). When digested with jack bean meal ß-N-acetylhexosaminidase, bands 2 and 4 were degraded, and more of band 3 and 5 appeared on the gel (Figure 3B, lane 3). When treated with ß-galactosidase first and then ß-N-acetylhexosaminidase or vice versa, only bands 5 and 6 were seen on the gel (lane 5 and 6). Because PID2 LOS was degraded with jack bean meal ß-N-acetylhexosaminidase, but F62 LOS was not, we concluded that the PID2 band 2 and the larger F62 band have different terminal sugars, with the PID2 band terminating in GlcNAc and the F62 band terminating with GalNAc.
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The remote neutral losses that provide the chain sequence, m/z 1654.6/1449.9/1204.5, were unusual considering the presence of GlcNAc glycosidic linkages, which usually rupture to dominate a spectrum. To confirm the proposed sequence and structure of this ion (m/z 967.52+), an alternative product ion (m/z 838.5+2) was selected and activated, MS3 (Figure 5E). This ion represents the loss of a single HexNAc residue (tHexNAc), and the product ion spectrum clearly indicates this residue originates from the
chain. Subsequent glycosidic rupture proximal to the GlcNAc moiety, m/z 472.2/1204.5, dominates the product ion spectrum and confirms the
chain sequence.
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Discussion |
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DNA sequence analysis of a PCR-amplified chromosomal DNA fragment demonstrated that the PID2 lgt gene cluster consisted of lgtA1, lgtB, lgtC, lgtA2, and lgtB/E; there was no lgtD gene. Although the DNA sequence obtained indicated that both copies of lgtA would be nonfunctional due to the length of the polyguanine tracts contained within their coding sequences, we believe that this is an artifact of PCR amplification, because the LOS expressed by this strain appears to have been modified by LgtA. Others have shown that PCR amplification of these polyguanine tracts can produce spontaneous frame shifts (Jennings et al., 1995).
In N. gonorrhoeae strain F62, the genes lgtA and lgtD are more than 70% identical and have long stretches of sequence identity, especially at the 5' end of the genes. Likewise, the genes lgtB and lgtE share greater than 70% identity (Gotschlich, 1994). The data presented in Figure 6 depict a model that shows how the lgt gene cluster is organized in F62 and how the organization found in PID2 might arise. This model involves homologous recombination between conserved DNA sequences of lgtA and lgtD and lgtB and lgtE. This intramolecular recombination provides another mechanism that could allow for LOS antigenic variation to occur. However, the net result of this mechanism would be the loss of genetic information. In N. meningitidis, deletions within the lgt gene cluster are common (Jennings et al., 1995
, 1999; Tettelin et al., 2000
).
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The structure of LOS isolated from N. gonorrhoeae strain 15253 has been determined. In addition to the lacto-N-neotetraose on the chain, this strain also contains a ß chain consisting of lactose (Yamasaki et al., 1999
). Mutations in lgtE result in the production of an LOS that consists of a glucose on both the
and ß chains (Banerjee et al., 1998
), and this LOS binds the mAb 25-1-LC1 (Tong et al., 2001
). We generated a nonfunctional lgtE mutant of PID2. Because only truncated LOS molecules were expressed in the mutant strain and this LOS was not able to bind mAb 25-1-LC1, a mAb that requires a single ß-chain glucose and an
chain consisting of glucose or lactose to bind (Tong et al., 2001
), it indicated that this LOS did not possess a glucose on the ß chain. This LOS also failed to bind mAb B5, an antibody that requires the presence of PEA on C3 of Hep II for binding (Plested et al., 1999
). These data indicate that the structural differences between PID2 and MS11mkC are seen as differences in the addition of PEA. They further suggest that all of the LOS components found in PID2 are extended off of
chain. MS studies were readily able to identify the presence of a five-sugar OS extending from Hep I. These studies also clearly showed that these OS molecules lack PEA. Though these studies were unable to confirm the presence of a six-sugar oligosaccharide, we believe that this failure is due to the limitations of the methodology employed and the low abundance of the high-molecular-mass ion, relative to the large amount of the smaller ions.
We still do not understand the mechanism that allows for the surface expression of LOS biosynthetic intermediates, as seen in strains like PID2 and MS11mkC. Burch et al. (1997) demonstrated that limiting the amount of the LgtA allowed strain FA19 to surface express two related LOS components, one that was able to bind mAb 2-1-L8 and one that was able to bind mAb 1B2. They suggested that the transcription/trasnslation of each of the proteins encoded by the lgt gene cluster is highly regulated and that small shifts in the amount a enzyme made can dramatically effect the surface expressed phenotype. If the hybrid LgtB/E had a lower Kcat than the wild-type LgtE, the amount of LgtE activity would be limiting in PID2. This would allow for the surface expression of small LOS biosynthetic intermediates. This same mechanism could explain how the gonococcus expresses the lactosamine repeat. In PID2, lgtA is duplicated. In theory, this could double the amount of this enzyme made, relative to the other enzymes. Because this enzyme mediates the addition of GlcNAc to Gal, if it were in excess, it could act to add sugars onto the terminal Gal of the lacto-N-neotetraose, which could then be further elongated by the lgtB gene product. This would produce the lactosamine repeat seen in PID2 and MS11mkC. However, we do not favor this hypothesis because if it is true, we would expect to see limited amounts of lactosamine repeats in all strains. We believe that additional as yet uncharacterized genes mediate the addition of polylactosamine.
The absence of phosphoethanolamine in neisserial LOS has not been reported. PID2 could surface express high-molecular-mass LOS components in the absence of this modification, and this indicates that this decoration is added later in biosynthesis and plays no role in the surface localization of LOS. The LOS components that lack this modification possess the same electrophoretic mobility as those that contain it. Because mAb 1B2 is able to bind LOS, irrespective of the presence of the PEA modification, whereas mAb 2-1-L8 is only able to bind when this modification is present, it serves as a further reminder of the dangers of ascribing structural features to an LOS molecule based solely on the binding of a mAb or an SDSPAGE profile.
Though a systematic study of LOS structures found on strains causing PID has not yet been performed, strains associated with PID are generally serum-sensitive and possess an LOS that can be modified by gonococcal sialytransferase (Rice et al., 1994). Sialyltransferase competes with LgtD for the terminal galactose found on the lacto-N-neotetraose. It is possible that the increased virulence associated with PID2 may be due to this loss in ability to cap the lacto-N-neotetraose structure through the action of LgtD. The data presented herein serve as a starting point for this line of investigation because they demonstrate that FACE analysis, combined with enzymatic digestion of LOS, can be used to rapidly define the nature of the sugars that are found at the termini of surface expressed LOS.
The data reported here represent the first application of FACE technology to the identification of sugar components in lipopolysaccharides. This technology will be invaluable in future studies because, in combination with enzymatic digestions, it will allow one to rapidly identify the nature of terminal sugars in gonococci. This will allow for the systematic study of LOSs isolated from strains associated with PID. The loss of lgtD in PID2 results in the loss of the ability to cap an LOS molecule with N-acetylgalactosamine. N. meningitidis has increased virulence, relative to the gonococcus. All of these strains characterized to date lack the ability to add this modification (Jennings et al., 1999), suggesting that this modification reduces virulence.
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Materials and methods |
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LOS purification
LOSs were purified from broth-grown cells using acetone-powdered organisms by the hot phenolwater method (Westphal and Jann, 1972). Methylation of LOS for use in MS experiments was achieved by dissolving samples in a NaOH/dimethyl sulfoxide suspension, prepared by vortex mixing dimethyl sulfoxide and powdered NaOH (Ciucanu and Kerek, 1984
). After 1 h incubation at room temperature, 50 µg of methyliodide was added and the suspension incubated for an additional hour at room temperature with occasional vortexing (Reinhold et al., 1996
). The methylated product was back-extracted by adding 1 ml chloroform, and the suspensions were back washed four times with 23 ml 30% acetic acid. The chloroform layer was taken to dryness and stored at 20°C. Methylated samples were redissolved to a concentration of 10 µM in a 1 mM solution of sodium acetate in 70:30 methanol/water just prior to analysis.
FACE monosaccharide composition analysis
Purified LOS (~5 g) was hydrolyzed in 1% acetic acid for 2 h at 80°C. The hydrolysate was centrifuged (12,000 x g, 20 min) and the supernatant containing the OS collected. For sugar composition analysis, the OS was treated following the procedure provided by Glyko, with the only difference being that the OS was hydrolyzed with 4 N HCl for 2 h instead of with 2 N trifluoracetic acid for 5 h. For exoglycosidase digestion, the samples were labeled after digestion.
SDSPAGE analysis
Proteinase Ktreated whole cell lysates were prepared from 18 to 20 h cultures by the procedure of Hitchcock and Brown (1983). Approximately 0.1 g LOS was subjected to SDSPAGE on a 16.5% Tris-tricine gel (Bio-Rad) in Tris-tricine running buffer following the protocol suggested by the manufacturer. The gel was fixed overnight in 40% ethanol, 5% acetic acid, and the LOS visualized by silver staining (Tsai and Frasch, 1982
).
Western blot
After separation on an SDSPAGE gel, LOSs were electrotransferred onto Immobilon-P membrane (Millipore, MA) in a Tris-glycine-methanol buffer (0.025 M Tris, 0.192 M glycine, 20% methanol) at a constant voltage of 100 V for 1 h. After air-drying for 10 min, membranes were blocked with a filler solution (2% casein, 0.2% NaN3, and 0.002% phenol red, in 100 mM Na2HPO4 pH 7.5) for 30 min. The membranes were incubated in primary antibody (mAb 25-1-LC1, B5, 3G9, 17-1-L1, or 1B2) with gentle shaking for at least 2 h. The membranes were washed with 100 mM Na2HPO4, pH 8.0, three times (10 min each) and incubated in filler solution containing secondary antibody (goat anti-mouse IgG conjugated with horseradish peroxidase; goat anti-mouse IgM conjugated with horseradish peroxidase when mAb 1B2 was used) for at least 2 h. After three washes, antibody binding on the membranes was visualized by incubating the membranes in developing solution (50 mM TrisHCl, pH 8.0, 1% 4-chloro-1-napthol, 0.86% H2O2).
Transformation
Recombinant DNA transformation into E. coli DH5MCR was done according to the standard protocols (Sambrook et al., 1989
). Recombinant DNA transformation into N. gonorrhoeae were done by resuspending piliated cells to a density of approximately 1 x 108 cells/ml in GCP broth containing growth supplements, 0.042% NaHCO3, 10 mM MgCl2 and 1 µg of the DNA of interest. Cells were incubated for about 5 h with shaking at 37°C. Cells were plated onto GCK plates containing spectinomycin.
PCR
PCR was used to generate the DNA fragments employed in gene cloning experiments and for mutant N. gonorrhoeae strain verification. Primers were purchased from Bioserve Biotechnologies (Laurel, MD). DNA amplifications were performed by using the PCR supermix kit (Life Technologies, Grand Island, NY) following the procedure provided by the company. Purified chromosome DNA or plasmid DNA was used as a template for gene cloning. For strain construction verifications, DNA was isolated directly from colonies by the following procedure. A small colony was added to 5 µl 0.5 M NaOH, the cell mixture allowed to incubate at room temperature for 10 min, and the solution neutralized with 5 µl 1 M TrisHCl, pH 7.5. After adding 90 µl H2O, 3 µl of this solution was used for PCR amplifications.
DNA sequencing
All DNA sequencing was done using nested overlapping primers. The DNA sequence of the lgt gene region for PID2 has been submitted to GenBank under accession number AF313394.
Insertion of the omega cassette into various genes
To insert the omega cassette into F62lgtF, primer lgtF-PstIF and lgtF-PstIR were designed to amplify pRFAK2-1, inserting PstI site into the F62lgtF gene. Primer omega-F and omega-R, containing terminal of PstI sites, were used to amplify the omega cassette from pHP45 (Prentki and Krisch, 1984
). After digestion with PstI, the two fragments were ligated and transformed into E. coli DH5
giving pF62lgtF
. To insert the omega cassette into the lgt gene cluster, primer lgtDE-F and lgtDE-R were designed to amplify pF62lgt, inserting an EcoRI site just before lgtE start codon. The omega cassette was inserted into this site, giving pF62lgtDE
.
Electrospray ionization MS
Mass measurements were performed on an ion trap mass spectrometer (LCQ, Finnigan-MAT, San Jose, CA) equipped with electrospray ionization. Samples were dissolved in methanol:water solutions (6:4, v/v) containing 0.25 mM NaOH and analyzed by syringe pump flow injection (1.5 µl/min directly into the electrospray chamber. Ions were injected axially into the ion trap by a gate lens and a trapping field was established with a 1001100 kHz radio frequency applied to the ring electrode.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed; E-mail: ds64@mail.umd.edu
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References |
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