Conservation of the lipooligosaccharide synthesis locus lgt among strains of Neisseria gonorrhoeae: requirement for lgtE in synthesis of the 2C7 epitope and of the beta chain of strain 15253.

Erwin, Alice L.; Haynes, Paul A.; Rice, Peter A.; Gotschlich, Emil C.

doi:10.1084/jem.184.4.1233

Cited by 39 publications

(39 citation statements)

References 39 publications

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“…M13761) (42); hence, the pNGLGTF::erm construct was nonpolar, as it was devoid of the transcriptional terminator. Furthermore, the presence of glucosamine (GlcNAc is added by rfaK to make the ␥-chain) was determined by carbohydrate analysis of MS11 lgtF LOS by high-pH anion-exchange chromatography (2,16), confirming the nonpolar nature of the mutation (data not shown).…”

Section: Reagentsmentioning

confidence: 89%

Effect of α-Oligosaccharide Phenotype of Neisseria gonorrhoeae Strain MS11 on Invasion of Chang Conjunctival, HEC-1-B Endometrial, and ME-180 Cervical Cells

2000

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The genes encoding the glycosyltransferases responsible for the addition of the five sugars in the ␣-oligosaccharide (␣-OS) moiety of lipooligosaccharide (LOS) have been identified. Disruption of these glycosyltransferase genes singly or in combination results in corresponding truncations in LOS. In the present work we show that sequential deletion of the terminal four sugar residues of gonococcal ␣-OS had no discernible effect on the invasion of human conjunctival, endometrial, and cervical cell lines. However, deletion of the proximal glucose, which resulted in the complete deletion of ␣-OS, significantly impaired invasion of the gonococci into all three cell lines. The effect of deleting ␣-OS on invasion was independent of and additive to the known invasion-promoting factor OpaA. These data suggest that the proximal glucose residue of the ␣-OS chain of LOS is required for efficient invasion of gonococci into host mucosa.

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Section: Reagentsmentioning

confidence: 89%

Effect of α-Oligosaccharide Phenotype of Neisseria gonorrhoeae Strain MS11 on Invasion of Chang Conjunctival, HEC-1-B Endometrial, and ME-180 Cervical Cells

2000

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“…Anti-LPS antibodies were measured as follows: a suspension of LPS (purified from strain KW20 by hot phenol-water extraction as described [4]) was diluted to 10 g per ml in Dulbecco's phosphate-buffered saline (PBS; Hyclone, Logan, Utah) containing 10 mM MgCl 2 and was used to coat Immulon 2HB plates (Dynex Technologies, Inc., Chantilly, Va.) (100 l per well). Plates were incubated overnight at 4°C and then wells were filled with blocking solution consisting of 5% bovine serum albumin fraction V (Sigma) in PBS containing 0.2% Tween 20, and the plates were incubated for 1 h. The wells were washed once with PBS-0.2% Tween 20 using an automatic plate washer (model 96PW; TECAN U.S., Hillsborough, N.C.).…”

Section: Methodsmentioning

confidence: 99%

Role of Lipopolysaccharide Phase Variation in Susceptibility of Haemophilus influenzae to Bactericidal Immunoglobulin M Antibodies in Rabbit Sera

Erwin¹,

Brewah²,

Couchenour³

et al. 2000

Infect Immun

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The effect of phase variation of lipopolysaccharide (LPS) structure on the susceptibility of Haemophilus influenzae to complement-dependent killing by normal human sera and normal rat sera has been described previously. The phase-variable structure phosphorylcholine (ChoP) confers susceptibility to human serum, since ChoP on the bacterial cell surface binds to serum C-reactive protein and activates complement. In contrast, expression of gal␣1,4gal, a second phase-variable epitope that is also found on human glycoconjugates, confers resistance to human serum. We studied the role of phase variation of these structures in the susceptibilities of H. influenzae KW20 (Rd) and a clinical isolate of nontypeable H. influenzae to killing by rabbit sera, which often possess naturally acquired complement-dependent bactericidal activity for unencapsulated H. influenzae. Expression of ChoP increased the resistance of strain KW20 to killing by bactericidal rabbit sera. In contrast, the serum resistance of a clinical isolate, H233, was unaffected by ChoP expression but was reduced by gal␣1,4gal expression. The rabbit sera with bactericidal activity (but not the nonbactericidal sera) all contained immunoglobulin M (IgM) antibodies able to bind to the surface of H. influenzae bacteria, as detected by flow cytometry, and contained IgM antibodies to LPS purified from strain KW20. Preincubation of sera with LPS reduced their bactericidal activity. Bactericidal activity was recovered quantitatively in an IgM-enriched fraction of sera. It is concluded that naturally occuring bactericidal activity for unencapsulated H. influenzae is largely due to IgM antibodies directed against phase-variable structures of the LPS.

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“…The biochemical functions of LgtABCD have been demonstrated and correspond to the functions inferred from analysis of mutations in these genes (3,30,31). While there is significant genetic evidence supporting the function of LgtE as the glycosyltransferase responsible for the addition of galactose ␤-1,4 to glucose (6,11,14,26), biochemical data supporting this assignment have been lacking. Furthermore, since the addition of galactose ␤-1,4 to glucose can occur on both the ␣ and ␤ chains, it is unclear if LgtE is responsible for both of these biosynthetic processes.…”

Section: Discussionmentioning

confidence: 99%

“…However, purified LgtE was unable to mediate the transfer of galactose to synthetic LOS biosynthesis intermediates (30). Erwin et al (6) showed that when lgtE was nonfunctional, galactose was not added to the ␤ chain. However, no direct biochemical evidence was presented to implicate LgtE directly in this addition.…”

mentioning

confidence: 99%

“…The biochemical properties of LgtB and LgtC have been examined similarly (21,30,31). Both enzyme possessed the predicted galactosyltransferase activities.Genetic evidence supporting the function of lgtE as encoding a glycosyl transferase responsible for the addition of galactose ␤-1,4 to glucose has been reported in a variety of publications (6,11,14,26). However, purified LgtE was unable to mediate the transfer of galactose to synthetic LOS biosynthesis intermediates (30).…”

mentioning

confidence: 99%

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Biochemical Properties of Neisseria gonorrhoeae LgtE

Piekarowicz

Stein

2002

J Bacteriol

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A fragment of chromosomal DNA encoding the lgtE gene of Neisseria gonorrhoeae strain F62 was amplified by PCR and cloned into the expression vector pET15b. Functional LgtE was purified and its biochemical properties were determined. The purified enzyme was maximally active in buffer containing manganese; minimal activity was obtained in buffer containing other divalent cations. LgtE was only able to mediate the addition of UDP-galactose into neisserial lipooligosaccharides (LOSs). We used a variety of genetically defined and chemically verified LOS structures to determine acceptor specificity. LgtE was able to mediate the addition of galactose into a variety of LOS structures, indicating the this enzyme possesses broad acceptor specificity. Furthermore, it was able to add multiple galactose residues onto LOS. We also determined that this enzyme was capable of adding galactose onto both the ␣ and ␤ chains of neisserial LOS.Lipooligosaccharide (LOS) is an important virulence determinant of the pathogenic neisseriae (12,16). It consists of an oligosaccharide component that is attached to lipid A via a Kdo (3-deoxy-D-manno-octulosonic acid) linkage. The genes involved in the synthesis of the oligosaccharide portion of this molecule from a variety of species have been identified and characterized (1,2,11,13,17,28). A common feature of LOS expression in all of these species is the expression of multiple phase-variable LOS structures. Most of this variability is attributed to changes in the carbohydrate composition of the molecule. The genetic basis for this variation has been well characterized. Key genes in the biosynthetic pathway contain homopolymeric runs of guanine (2, 11). Changes in the number of guanines result in reading frame shifts, with the end result being the truncation or elongation of a particular LOS molecule, depending on the nature of the starting reading frame (4,5,37).The data presented in Fig. 1 summarize the genetic potential and reported carbohydrate structures that have been identified in Neisseria gonorrhoeae (7,9,10,15,18,(34)(35)(36). The genes responsible for the addition of most of these sugars have been defined genetically; loss of gene function results in the truncation of an LOS structure. Biochemical characterization of several of these gene products has been performed by measuring the incorporation of sugars from various UDP-sugars into a variety of synthetic carbohydrates. LgtA possessed broad substrate specificity towards ␣ and ␤ galactosides. Depending on the acceptor, this enzyme could mediate the transfer of GlcNAc from UDP-GlcNAc and GalNAc from UDP-GalNAc (3). However, this broad specificity was not seen in vivo (27). The biochemical properties of LgtB and LgtC have been examined similarly (21,30,31). Both enzyme possessed the predicted galactosyltransferase activities.Genetic evidence supporting the function of lgtE as encoding a glycosyl transferase responsible for the addition of galactose ␤-1,4 to glucose has been reported in a variety of publications (6,11,14,26). However...

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Conservation of the lipooligosaccharide synthesis locus lgt among strains of Neisseria gonorrhoeae: requirement for lgtE in synthesis of the 2C7 epitope and of the beta chain of strain 15253.

Cited by 39 publications

References 39 publications

Effect of α-Oligosaccharide Phenotype of Neisseria gonorrhoeae Strain MS11 on Invasion of Chang Conjunctival, HEC-1-B Endometrial, and ME-180 Cervical Cells

Effect of α-Oligosaccharide Phenotype of Neisseria gonorrhoeae Strain MS11 on Invasion of Chang Conjunctival, HEC-1-B Endometrial, and ME-180 Cervical Cells

Role of Lipopolysaccharide Phase Variation in Susceptibility of Haemophilus influenzae to Bactericidal Immunoglobulin M Antibodies in Rabbit Sera

Biochemical Properties of Neisseria gonorrhoeae LgtE

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