Mary E. Deadman

Sialic acid in the lipopolysaccharide of Haemophilus influenzae: strain distribution, influence on serum resistance and structural characterization.

A survey of Haemophilus influenzae strains indicated that around one‐third of capsular strains and over two‐thirds of non‐typeable strains included sialic acid in their lipopolysaccharides (LPS). Mutation of the CMP‐Neu5Ac synthetase gene (siaB) resulted in a sialylation‐deficient phenotype. Isogenic pairs, wild type and siaB mutant of two non‐typeable strains were used to demonstrate that sialic acid influences resistance to the killing effect of normal human serum but has little effect on attachment to, or invasion of, cultured human epithelial cells or neutrophils. We determine for the first time the site of attachment of sialic acid in the LPS of a non‐typeable strain and report that a small proportion of glycoforms include two sialic acid residues in a disaccharide unit.

Use of the complete genome sequence information of Haemophilus influenzae strain Rd to investigate lipopolysaccharide biosynthesis

The availability of the complete 1.83‐megabase‐pair sequence of the Haemophilus influenzae strain Rd genome has facilitated significant progress in investigating the biology of H. influenzae lipopolysaccharide (LPS), a major virulence determinant of this human pathogen. By searching the H. influenzae genomic database, with sequences of known LPS biosynthetic genes from other organisms, we identified and then cloned 25 candidate LPS genes. Construction of mutant strains and characterization of the LPS by reactivity with monoclonal antibodies, PAGE fractionation patterns and electrospray mass spectrometry comparative analysis have confirmed a potential role in LPS biosynthesis for the majority of these candidate genes. Virulence studies in the infant rat have allowed us to estimate the minimal LPS structure required for intravascular dissemination. This study is one of the first to demonstrate the rapidity, economy and completeness with which novel biological information can be accessed once the complete genome sequence of an organism is available.

Identification of a lipopolysaccharide alpha-2,3-sialyltransferase from Haemophilus influenzae.

We have identified a gene for the addition of N‐acetylneuraminic acid (Neu5Ac) in an α‐2,3‐linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenzae. The gene is one that was identified previously as a phase‐variable gene known as lic3A. Extracts of H. influenzae, as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N‐acetyl‐lactosaminyl moiety. In the RM118 strain of H. influenzae, Lic3A activity is modulated by the action of another phase‐variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118:lgtC mutant and the non‐typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl‐α‐(2–3)‐lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118:lgtC lic3A, indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A, encoding an α‐2,3‐sialyltransferase activity, is the first reported phase‐variable sialyltransferase gene.

The role of a repetitive DNA motif (5′‐CAAT‐3′) in the variable expression of the Haemophilus influenzae lipopolysaccharide epitope αGal(1–4)βGal

Haemophilus influenzae lipopolysaccharide (LPS) contains structures, defined by monoclonal antibodies, which undergo phase variation. This investigation reports the nucleotide sequence of lic2A, which is required for the expression of at least three phase‐variable LPS epitopes, one of which has the structure αGal(1–4)βGal. lic2A contains multiple tandem repeats of the tetramer 5′‐CAAT‐3′ Previous studies have correlated changes in the number of 5′‐CAAT‐3′ repeats with the phase‐variable expression of the αGal(1–4)βGal epitope. To obtain direct evidence for this, the 5′‐CAAT‐3′ repeat region from lic2A was amplified directly from immunostained colonies and sequenced. This demonstrated that the variable expression of LPS epitopes, including αGal(1–4)βGal, is in part directly dependent upon the number of copies of 5′‐CAAT‐3′ within lic2A.

Batten disease (Spielmeyer-Vogt disease, juvenile onset neuronal ceroid-lipofuscinosis) gene (CLN3) maps to human chromosome 16

The ceroid-lipofuscinoses are a group of inherited neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in neurons and other cell types. The underlying biochemical defect is unknown. Batten disease (Spielmeyer-Vogt disease, juvenile onset neuronal ceroid-lipofuscinosis) displays autosomal recessive inheritance. Genetic linkage studies were undertaken to determine the chromosomal location of the Batten disease mutation (CLN3). Following identification of linkage to the haptoglobin locus, linkage analysis has been carried out in 42 families by using DNA markers for loci on the long arm of human chromosome 16. The maximal lod score between Batten disease and the locus D16S148 calculated for combined sexes is 6.05 at a recombination fraction theta = 0.00. Multilocus analysis using five loci indicated the most likely order to be HP-D16S151-D16S150-CLN3-D16S148-D16S147. The maximal location score for CLN3 was 48 (equivalent to a lod score of 10.4) in that interval within this fixed marker map.

Novel lipopolysaccharide biosynthetic genes containing tetranucleotide repeats in Haemophilus influenzae, identification of a gene for adding O‐acetyl groups

Many of the genes for lipopolysaccharide (LPS) biosynthesis in Haemophilus influenzae are phase variable. The mechanism of this variable expression involves slippage of tetranucleotide repeats located within the reading frame of these genes. Based on this, we hypothesized that tetranucleotide repeat sequences might be used to identify as yet unrecognized LPS biosynthetic genes. Synthetic oligonucleotides (20 bases), representing all previously reported LPS‐related tetranucleotide repeat sequences in H. influenzae, were used to probe a collection of 25 genetically and epidemiologically diverse strains of non‐typeable H. influenzae. A novel gene identified through this strategy was a homologue of oafA, a putative O‐antigen LPS acetylase of Salmonella typhimurium, that was present in all 25 non‐typeable H. influenzae, 19 of which contained multiple copies of the tetranucleotide 5′‐GCAA. Using lacZ fusions, we showed that these tetranucleotide repeats could mediate phase variation of this gene. Structural analysis of LPS showed that a major site of acetylation was the distal heptose (HepIII) of the LPS inner‐core. An oafA deletion mutant showed absence of O‐acetylation of HepIII. When compared with wild type, oafA mutants displayed increased susceptibility to complement‐mediated killing by human serum, evidence that O‐acetylation of LPS facilitates resistance to host immune clearance mechanisms. These results provide genetic and structural evidence that H. influenzae oafA is required for phase variable O‐acetylation of LPS and functional evidence to support the role of O‐acetylation of LPS in pathogenesis.

Three genes, lgtF, lic2C and lpsA, have a primary role in determining the pattern of oligosaccharide extension from the inner core of Haemophilus influenzae LPS

Lipopolysaccharide (LPS) is a virulence determinant of Haemophilus influenzae and exhibits substantial heterogeneity in structure within and between strains. Key factors contributing to this heterogeneity are the genes required to add the first glycose to each of the three heptose residues of the LPS inner core. In each case this addition can facilitate further oligosaccharide extension. lgtF is invariably present in strains and the product has a function in adding the glucose to the first heptose. lic2C is present in half the strains and was found to add a glucose to the second heptose. Insertion of lic2C into a strain that does not naturally contain it resulted in hexose incorporation from the second heptose of the LPS. The product of the lpsA gene can add a glucose or galactose to the third heptose. By allelic replacement of lpsA between strains it is shown that the sequence of the gene can be the sole determinant of this specificity. Thus, lgtF, lic2C and lpsA make significant but very distinct contributions to the conservation and variable patterns of oligosaccharide extensions seen in H. influenzae LPS.

The gal locus from Haemophilus influenzae: cloning, sequencing and the use of gal mutants to study lipopolysaccharide

The gal locus from Haemophilus influenzae was cloned and sequenced. Four genes were identified by amino acid homology: galT, galK, galM an6 galR. The coding direction of galT, galK and galM is divergent from that of galR. There are non‐coding intergenic regions between gaIR and galT, galT and galK, and galK and galM. Deletion‐insertion mutations constructed in galK and galE, which is in Iic3, were moved into the H. influenzae chromosome generating each of the single mutants as well as the double gal mutant. Even when grown on complex media, the double mutant failed to react with an anti‐lipopolysac‐charide monoclonal antibody known to react with a digalactoside epitope. Both the galE single and the galEgalK double mutants were serum‐sensitive and relatively avirulent in infant rats, indicating a critical role for galactose metabolism, and providing evidence to support a central role for lipopolysaccharide, in H. influenzae virulence.

Haemophilus influenzae biofilms: hypothesis or fact?

Many publications state that nontypeable Haemophilus influenzae (NTHi) produces biofilms. Here, we review many of the publications that have led to acceptance by some that NTHi expresses a biofilm-specific phenotype as a distinct part of its life cycle. Biofilm formation was originally invoked to explain the failure to culture NTHi from middle-ear effusions, recalcitrance to antibiotics and its pathogenic behaviour. We argue that the current evidence for NTHi biofilm formation in vitro and in vivo is inconclusive. We consider that NTHi biofilm is hypothesis not fact, and although it might yet prove to be correct, there has been little or no consideration of alternative interpretations for the in vitro and in vivo observations. Uncritical acceptance of a distinctive NTHi biofilm phenotype has the potential to mislead and could confuse and compromise research efforts aimed at improving management and prevention of NTHi diseases of the human respiratory tract.

Genes required for the synthesis of heptose-containing oligosaccharide outer core extensions in Haemophilus influenzae lipopolysaccharide

Heptose-containing oligosaccharides (OSs) are found in the outer core of the lipopolysaccharide (LPS) of a subset of non-typable Haemophilus influenzae (NTHi) strains. Candidate genes for the addition of either l-glycero-d-manno-heptose (ld-Hep) or d-glycero-d-manno-heptose (dd-Hep) and subsequent hexose sugars to these OSs have been identified from the recently completed genome sequences available for NTHi strains. losA1/losB1 and losA2/losB2 are two sets of related genes in which losA has homology to genes encoding glycosyltransferases and losB to genes encoding heptosyltransferases. Each set of genes is variably present across NTHi strains and is located in a region of the genome with an alternative gene organization between strains that contributes to LPS heterogeneity. Dependent upon the strain background, the LPS phenotype, structure and serum resistance of strains mutated in these genes were altered when compared with the relevant parent strain. Our studies confirm that losB1 and losB2 usually encode dd-heptosyl- and ld-heptosyl transferases, respectively, and that losA1 and losA2 encode glycosyltransferases that play a role in OS extensions of NTHi LPS.