Elucidation of the Monoclonal Antibody 5G8-Reactive, Virulence-Associated Lipopolysaccharide Epitope of Haemophilus influenzae and Its Role in Bacterial Resistance to Complement-Mediated Killing

ABSTRACT The phase-variable locus lex2 is required for expression of a Haemophilus influenzae lipopolysaccharide (LPS) epitope of previously unknown structure. This epitope, which is reactive with monoclonal antibody (MAb) 5G8, has been associated with virulence of type b strains. When strain RM118 (from the same source as strain Rd), in which the lex2 locus and MAb 5G8 reactivity are absent, was transformed with lex2 DNA, transformants that were reactive with MAb 5G8 were obtained. Surprisingly, the 5G8 reactivity of these transformants was phase variable, although the lex2 locus lacked tetrameric repeats and was constitutively expressed. This phase variation was shown to be the result of phase-variable expression of phosphorylcholine (PCho) such that MAb 5G8 reacted only in the absence of PCho. Structural analysis showed that, compared to RM118, the lex2 transformant had acquired a tetrasaccharide, Gal-α1,4-Gal-β1,4-Glc-β1,4-Glc-β1,4, linked to the proximal heptose (HepI). A terminal GalNAc was detected in a minority of glycoforms. LPS derived from a mutant of RM7004, a virulent type b strain which naturally expresses lex2 and has LPS containing the same tetrasaccharide linked to HepI as the sole oligosaccharide extension from the inner core, confirmed that GalNAc is not a part of the MAb 5G8-reactive epitope. Thus, MAb 5G8 specifically binds to the structure Gal-α1,4-Gal-β1,4-Glc-β1,4-Glc-β attached via a 1,4 linkage to HepI of H. influenzae LPS, and we show that the ability to synthesize this novel tetrasaccharide was associated with enhanced bacterial resistance to complement-mediated killing.

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Structural elucidation of lipopolysaccharide core oligosaccharides from lic1 and lic1/lic2 mutants of Haemophilus influenzae type b strain Eagan

Canadian Journal of Microbiology ◽

10.1139/w08-009 ◽

2008 ◽

Vol 54 (4) ◽

pp. 281-290 ◽

Cited By ~ 2

Author(s):

Hussein Masoud ◽

E. Richard Moxon ◽

James C. Richards

Keyword(s):

Haemophilus Influenzae ◽

Inner Core ◽

Transcriptional Unit ◽

Haemophilus Influenzae Type ◽

Resonance Spectroscopy ◽

Ionization Mass ◽

Haemophilus Influenzae Type B ◽

Phase Variable ◽

Type B ◽

Variable Expression

The structures of lipopolysaccharides (LPSs) of lic1 and lic1/lic2 mutants from Haemophilus influenzae type b strain Eagan (RM153) were investigated using methylation analysis, electrospray ionization – mass spectrometry, and nuclear magnetic resonance spectroscopy on O-deacylated, O- and N-deacylated core oligosaccharide (OS); and deacylated, dephosphorylated, and terminally reduced samples. The backbone OS derived from the major LPS glycoforms were determined to consist of the inner-core triheptosyl unit, l-α-d-Hepp-(1-2)-l-α-d-Hepp-(1-3)-l-α-d-Hepp-(1-, common to all H. influenzae strains investigated to date that is linked to the lipid A region of the molecule via a Kdo residue to which β-d-Glcp and β-d-Galp residues are attached in 1,4 and 1,2 linkages to the proximal (HepI) and distal (HepIII) heptose residues, respectively. It was found that the lic1 mutant predominately elaborates the Hex4 LPS glycoforms previously identified in the parent strain where a β-d-Glcp-(1-4)-α-d-Glcp unit is linked in a 1,3 linkage to the central heptose (HepII) of the triheptosyl moiety. The lic1 locus consists of 4 genes (lic1A to lic1D) in a single transcriptional unit that directs phase variable expression of phosphocholine. The lic1A gene is phased off in the RM153 isolate of strain Eagan. LPS from the double mutant, lic1/lic2 had a similar structure to that of lic1 mutant except that there was no chain extension from the central heptose in the inner core (HepII). The lic2 locus consists of 4 genes (lic2A to lic2D). Our structural data were consistent with the proposed function of lic2C, providing the first definitive evidence for its role as the glycosyltransferase required for chain initiation from HepII. The presence of an O-acetyl group at O-3 of the distal heptose (HepIII) was elucidated by 1H NMR on the mild acid liberated core OS samples.

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Lex2B, a Phase-Variable Glycosyltransferase, Adds either a Glucose or a Galactose to Haemophilus influenzae Lipopolysaccharide

Infection and Immunity ◽

10.1128/iai.01446-08 ◽

2009 ◽

Vol 77 (6) ◽

pp. 2376-2384 ◽

Cited By ~ 12

Author(s):

M. E. Deadman ◽

P. Hermant ◽

M. Engskog ◽

K. Makepeace ◽

E. R. Moxon ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Respiratory Tract Infections ◽

Inner Core ◽

Biological Properties ◽

Single Amino Acid ◽

Phase Variable ◽

Killing Effect ◽

Variable Expression ◽

Serotype B ◽

Tract Infections

ABSTRACT Nontypeable Haemophilus influenzae is a commensal that frequently causes otitis media and respiratory tract infections. The lex2 locus encodes a glycosyltransferase that is phase variably expressed and contributes to the significant intrastrain heterogeneity of lipopolysaccharide (LPS) composition in H. influenzae. In serotype b strains, Lex2B adds the second β-glucose in the oligosaccharide extension from the proximal heptose of the triheptose inner core backbone; this extension includes a digalactoside that plays a role in resistance of the bacteria to the killing effect of serum. As part of our studies of the structure and genetics of LPS in nontypeable H. influenzae, we show here that there are allelic polymorphisms in the lex2B sequence that correlate with addition of either a glucose or a galactose to the same position in the LPS molecule across strains. Through exchange of lex2 alleles between strains we show that alteration of a single amino acid at position 157 in Lex2B appears to be sufficient to direct the alternative glucosyl- or galactosyltransferase activities. Allelic exchange strains express LPS with altered structure and biological properties compared to the wild-type LPS. Thus, Lex2B contributes to both inter- and intrastrain LPS heterogeneity through its polymorphic sequences and phase-variable expression.

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The role of lex2 in lipopolysaccharide biosynthesis in Haemophilus influenzae strains RM7004 and RM153

Microbiology ◽

10.1099/mic.0.26387-0 ◽

2003 ◽

Vol 149 (11) ◽

pp. 3165-3175 ◽

Cited By ~ 23

Author(s):

Ruth Griffin ◽

Andrew D. Cox ◽

Katherine Makepeace ◽

James C. Richards ◽

E. Richard Moxon ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Inner Core ◽

Phase Variable ◽

Specific Monoclonal Antibody ◽

Variable Expression ◽

Sequence Identity ◽

Lipopolysaccharide Biosynthesis ◽

Serotype B ◽

Reading Frames

The locus lex2, comprising lex2A and lex2B, contributes to the phase-variable expression of lipopolysaccharide (LPS) of Haemophilus influenzae and was found to be present in 74 % of strains investigated. lex2A contains 5′-GCAA repeats which vary in number from 4 to 46 copies between strains. The locus was cloned from the serotype b strains RM7004 and RM153 and showed >99 % nucleotide sequence identity between these strains and the published lex2 sequence. Disruption of the lex2B gene in strain RM7004 resulted in truncation of some LPS glycoforms, shown by gel fractionation, with only one glycoform reacting with a digalactoside-specific monoclonal antibody, 4C4, compared with four LPS glycoforms in the more elongated LPS of the parent strain. Mass spectrometry and NMR analyses of LPS from the lex2B mutant revealed loss of the terminal digalactoside as well as the second β-glucose extending from the first heptose of the inner core. The authors conclude that Lex2B is the β-(1-4)-glucosyltransferase that adds the second β-glucose to the first β-glucose as part of the oligosaccharide extension from the first heptose of the LPS of strain RM7004. Investigation of the expression of the lex2 locus indicated that the genes are co-transcribed and that both reading frames are required for addition of this second β-glucose in a phase-variable manner.

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Specific detection of Haemophilus influenzae type b lipooligosaccharide by a polymyxin B monoclonal antibody assay

Journal of Immunological Methods ◽

10.1016/0022-1759(89)90267-6 ◽

1989 ◽

Vol 122 (2) ◽

pp. 219-226 ◽

Cited By ~ 3

Author(s):

Jussi Mertsola ◽

Octavio Ramilo ◽

Xavier Sáez-Llorens ◽

Mark S. Hanson ◽

George H. McCracken ◽

...

Keyword(s):

Monoclonal Antibody ◽

Haemophilus Influenzae ◽

Polymyxin B ◽

Haemophilus Influenzae Type ◽

Haemophilus Influenzae Type B ◽

Type B ◽

Specific Detection ◽

Antibody Assay

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The role of Dam methylation in phase variation of Haemophilus influenzae genes involved in defence against phage infection

Microbiology ◽

10.1099/mic.0.28184-0 ◽

2005 ◽

Vol 151 (10) ◽

pp. 3361-3369 ◽

Cited By ~ 62

Author(s):

Piotr Zaleski ◽

Marek Wojciechowski ◽

Andrzej Piekarowicz

Keyword(s):

Haemophilus Influenzae ◽

Phase Variation ◽

Phage Infection ◽

Phase Variable ◽

Repeat Tract ◽

Insertional Inactivation ◽

Phage Dna ◽

Dam Methylation ◽

Tetranucleotide Repeats

Haemophilus influenzae uses phase variation (PV) to modulate the activity of its defence systems against phage infection. The PV of the restriction–modification (R-M) system HindI, the main defence system against phage infection and incoming chromosomal and phage DNA in H. influenzae Rd, is driven by changes of the pentanucleotide repeat tract within the coding sequence of the hsdM gene and is influenced by lack of Dam methylation. Phase-variable resistance/sensitivity to phage infection correlates with changes in lipooligosaccharide (LOS) structure and occurs by slippage of tetranucleotide repeats within the gene lic2A, coding for a step in the biosynthesis of LOS. The lack of Dam activity destabilizes the tetranuclotide (5′-CAAT) repeat tract and increases the frequency of switching from sensitivity to resistance to phage infection more than in the opposite direction. The PV of the lgtC gene does not influence resistance or sensitivity to phage infection. Insertional inactivation of lic2A, but not lgtC or lgtF, leads to resistance to phage infection and to the same structure of the LOS as observed among phase-variable phage-resistant variants. This indicates that in the H. influenzae Rd LOS only the first two sugars (Glc-Gal) extending from the third heptose are part of bacterial phage receptors.

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Phase-Variable Expression of an Operon Encoding Extracellular Alkaline Protease, a Serine Protease Homolog, and Lipase in Pseudomonas brassicacearum

Journal of Bacteriology ◽

10.1128/jb.183.6.2117-2120.2001 ◽

2001 ◽

Vol 183 (6) ◽

pp. 2117-2120 ◽

Cited By ~ 49

Author(s):

Philippe Chabeaud ◽

Arjan de Groot ◽

Wilbert Bitter ◽

Jan Tommassen ◽

Thierry Heulin ◽

...

Keyword(s):

Serine Protease ◽

Lipase Activity ◽

Alkaline Protease ◽

Phase Variation ◽

Extracellular Protease ◽

Type I ◽

Phase Variable ◽

Gene Fusions ◽

Variable Expression ◽

Pseudomonas Brassicacearum

ABSTRACT The rhizobacterium Pseudomonas brassicacearum forms phenotypic variants which do not show extracellular protease and lipase activity. The operon encoding these enzymes, a serine protease homolog, and a type I secretion machinery was characterized. Transcriptional lacZ gene fusions revealed that the expression of the operon is under the control of phase variation.

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Identification of Haemophilus influenzae type b by a monoclonal antibody coagglutination assay.

Journal of Clinical Microbiology ◽

10.1128/jcm.25.12.2434-2436.1987 ◽

1987 ◽

Vol 25 (12) ◽

pp. 2434-2436 ◽

Cited By ~ 7

Author(s):

J Hamel ◽

B R Brodeur ◽

A Belmaaza ◽

S Montplaisir ◽

J M Musser ◽

...

Keyword(s):

Monoclonal Antibody ◽

Haemophilus Influenzae ◽

Haemophilus Influenzae Type ◽

Haemophilus Influenzae Type B ◽

Type B

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Bypassing Phase Variation of Lipooligosaccharide (LOS): Using Heptose 1 Glycan Mutants To Establish Widespread Efficacy of Gonococcal Anti-LOS Monoclonal Antibody 2C7

Infection and Immunity ◽

10.1128/iai.00862-19 ◽

2019 ◽

Vol 88 (2) ◽

Author(s):

Srinjoy Chakraborti ◽

Sunita Gulati ◽

Bo Zheng ◽

Frank J. Beurskens ◽

Janine Schuurman ◽

...

Keyword(s):

Monoclonal Antibody ◽

Polymorphonuclear Leukocytes ◽

Phase Variation ◽

Human Infection ◽

Phase Variable ◽

Content Type ◽

Human Igg1 ◽

And Function

ABSTRACT The sialylatable lacto-N-neotetraose (LNnT; Gal-GlcNAc-Gal-Glc) moiety from heptose I (HepI) of the lipooligosaccharide (LOS) of Neisseria gonorrhoeae undergoes positive selection during human infection. Lactose (Gal-Glc) from HepII, although phase variable, is commonly expressed in humans; loss of HepII lactose compromises gonococcal fitness in mice. Anti-LOS monoclonal antibody (MAb) 2C7, a promising antigonococcal immunotherapeutic that elicits complement-dependent bactericidal activity and attenuates gonococcal colonization in mice, recognizes an epitope comprised of lactoses expressed simultaneously from HepI and HepII. Glycan extensions beyond lactose on HepI modulate binding and function of MAb 2C7 in vitro. Here, four gonococcal LOS mutants, each with lactose from HepII but fixed (unable to phase-vary) LOS HepI glycans extended beyond the lactose substitution of HepI (lactose alone, Gal-lactose, LNnT, or GalNAc-LNnT), were used to define how HepI glycan extensions affect (i) mouse vaginal colonization and (ii) efficacy in vitro and in vivo of a human IgG1 chimeric derivative of MAb 2C7 (2C7-Ximab) with a complement-enhancing E-to-G Fc mutation at position 430 (2C7-Ximab-E430G). About 10-fold lower 2C7-Ximab-E430G concentrations achieved similar complement-dependent killing of three gonococcal mutants with glycan extensions beyond lactose-substituted HepI (lactose alone, LNnT, or GalNAc-LNnT) as 2C7-Ximab (unmodified Fc). The fourth mutant (Gal-lactose) resisted direct complement-dependent killing but was killed approximately 70% by 2C7-Ximab-E430G in the presence of polymorphonuclear leukocytes and complement. Only mutants with (sialylatable) LNnT from HepI colonized mice for >3 days, reiterating the importance of LNnT sialylation for infection. 2C7-Ximab-E430G significantly attenuated colonization caused by the virulent mutants.

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Coupled Phase-Variable Expression and Epitope Masking of Selective Surface Lipoproteins Increase Surface Phenotypic Diversity in Mycoplasma hominis

Infection and Immunity ◽

10.1128/iai.69.8.5177-5181.2001 ◽

2001 ◽

Vol 69 (8) ◽

pp. 5177-5181 ◽

Cited By ~ 18

Author(s):

Qijing Zhang ◽

Kim S. Wise

Keyword(s):

Membrane Protein ◽

High Frequency ◽

Phenotypic Diversity ◽

Phase Variation ◽

Mycoplasma Hominis ◽

Phase Variable ◽

Variable Expression ◽

Adaptive Capabilities ◽

Frequency Phase

ABSTRACT A new mechanism expanding mycoplasmal surface diversity is described. Exposure of surface epitopes on a constitutively expressed membrane protein (P56) of Mycoplasma hominis was subject to high-frequency phase variation due to phase-variable expression of the P120 antigen and its selective masking of P56 epitopes. Phase-variable masking may confer previously unrealized adaptive capabilities on mycoplasmas.

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