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

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3165-3175 ◽  
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.

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

2009 ◽  
Vol 77 (6) ◽  
pp. 2376-2384 ◽  
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.

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

2005 ◽  
Vol 73 (4) ◽  
pp. 2213-2221 ◽  
Author(s):  
Ruth Griffin ◽  
Andrew D. Cox ◽  
Katherine Makepeace ◽  
James C. Richards ◽  
E. Richard Moxon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Haemophilus Influenzae ◽  
Bacterial Resistance ◽  
Inner Core ◽  
Phase Variation ◽  
Phase Variable ◽  
Type B ◽  
Variable Expression ◽  
Virulent Type ◽  
Unknown Structure

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.

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

2008 ◽  
Vol 54 (4) ◽  
pp. 281-290 ◽  
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.

scholarly journals Role of CCAA Nucleotide Repeats in Regulation of Hemoglobin and Hemoglobin-Haptoglobin Binding Protein Genes ofHaemophilus influenzae

1999 ◽  
Vol 181 (18) ◽  
pp. 5865-5870 ◽  
Author(s):  
Zhen Ren ◽  
Hongfan Jin ◽  
Paul W. Whitby ◽  
Daniel J. Morton ◽  
Terrence L. Stull
Keyword(s):  
Haemophilus Influenzae ◽  
Gene Fusion ◽  
Binding Protein ◽  
Phase Variable ◽  
Repeat Region ◽  
Variable Expression ◽  
Ofhaemophilus Influenzae ◽  
Nucleotide Repeats

ABSTRACT Haemophilus influenzae utilizes hemoglobin and hemoglobin-haptoglobin as heme sources. The H. influenzaehemoglobin- and hemoglobin-haptoglobin binding protein genes,hgpA, hgpB, and hgpC, contain lengths of tetrameric CCAA repeats. Using an hgpA-lacZtranslational gene fusion, we demonstrate phase-variable expression oflacZ associated with alteration in the length of the CCAA repeat region.

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

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3361-3369 ◽  
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.

Haemophilus influenzae Type b in a Nursery School: The Value of Biotyping

PEDIATRICS ◽  
1982 ◽  
Vol 69 (2) ◽  
pp. 215-218
Author(s):  
Charles G. Prober ◽  
Moshe M. Ipp ◽  
Robert M. Bannatyne
Keyword(s):  
Haemophilus Influenzae ◽  
Nursery School ◽  
Colonization Rate ◽  
Haemophilus Influenzae Type ◽  
Type B ◽  
Nasal Swabs ◽  
Serotype B ◽  
Culture Site

Meningitis due to Haemophilus influenzae serotype b biotype II occurred in a 2-year-old child who attended a nursery school along with 26 other 2-year-old children. Nasal swabs from these 26 contacts revealed a H influenzae type b colonization rate of 50% (13/26); simultaneously performed throat swabs detected a colonization rate of 4% (1/26). Biotyping of the H influenzae type b isolates revealed that only 46% (6/13) were the same biotype as the index case; the remaining seven isolates were biotype III. All children received treatment with 20 mg/kg/day of rifampin administered by the nursery school attendant as a single dose for four days before the results of the cultures were known. Eradication of H influenzae type b carriage was successful in three of the six biotype II carriers and five of the six biotype III carriers available for follow-up culture. It was concluded that: (1) the culture site utilized in determining H influenzae type b colonization rates may markedly influence the results obtained, (2) biotyping may be a valuable epidemiologic tool in investigating the contacts of patients with H influenzae type b disease, and (3) failures of rifampin to eradicate the carriage of H influenzae type b from the nasopharynx may occur. The prudent approach to the management of young contacts of patients with serious H influenzae type b disease is to recognize their high risk status and to maintain close surveillance of them. The role of chemoprophylaxis with rifampin remains mains to be established.

scholarly journals OpsX from Haemophilus influenzae Represents a Novel Type of Heptosyltransferase I in Lipopolysaccharide Biosynthesis

2005 ◽  
Vol 187 (17) ◽  
pp. 6242-6247 ◽  
Author(s):  
Sabine Gronow ◽  
Werner Brabetz ◽  
Buko Lindner ◽  
Helmut Brade
Keyword(s):  
Substrate Specificity ◽  
Haemophilus Influenzae ◽  
Inner Core ◽  
Core Region ◽  
The Other ◽  
Lipopolysaccharide Biosynthesis

ABSTRACT The inner core region of the lipopolysaccharide (LPS) of Haemophilus influenzae is characterized by the presence of a phosphorylated 3-deoxy-α-d-manno-octulosonic acid (Kdo). In this study, we show that the heptosyltransferase I adding the first l-glycero-d-manno-heptose residue to this acceptor is encoded by the gene opsX, which differs in substrate specificity from the other heptosyltransferase I, known as WaaC.

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