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.

[1]  D. Field,et al.  High rates of recombination in otitis media isolates of non-typeable Haemophilus influenzae. , 2003, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[2]  D. Hood,et al.  Structural diversity in lipopolysaccharide expression in nontypeable Haemophilus influenzae. Identification of L-glycerol-D-manno-heptose in the outer-core region in three clinical isolates. , 2003, European journal of biochemistry.

[3]  C. Frasch,et al.  Effect of O Acetylation of Neisseria meningitidis Serogroup A Capsular Polysaccharide on Development of Functional Immune Responses , 2002, Infection and Immunity.

[4]  Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 1003. , 2002, European journal of biochemistry.

[5]  E. Moxon,et al.  Glycine is a common substituent of the inner core in Haemophilus influenzae lipopolysaccharide. , 2001, Glycobiology.

[6]  M. Golomb,et al.  Evolution of an Autotransporter: Domain Shuffling and Lateral Transfer from Pathogenic Haemophilus toNeisseria , 2001, Journal of bacteriology.

[7]  D. Hood,et al.  A new structural type for Haemophilus influenzae lipopolysaccharide. Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 486. , 2001, European journal of biochemistry.

[8]  E. Miller,et al.  Evaluation of De-O-Acetylated Meningococcal C Polysaccharide-Tetanus Toxoid Conjugate Vaccine in Infancy: Reactogenicity, Immunogenicity, Immunologic Priming, and Bactericidal Activity against O-Acetylated and De-O-Acetylated Serogroup C Strains , 2001, Infection and Immunity.

[9]  J. Brisson,et al.  Identification of a lipopolysaccharide α‐2,3‐sialyltransferase from Haemophilus influenzae , 2001, Molecular microbiology.

[10]  J. Brisson,et al.  Characterization of the phosphocholine-substituted oligosaccharide in lipopolysaccharides of type b Haemophilus influenzae. , 2000, European journal of biochemistry.

[11]  D. Field,et al.  The length of a tetranucleotide repeat tract in Haemophilus influenzae determines the phase variation rate of a gene with homology to type III DNA methyltransferases , 2000, Molecular microbiology.

[12]  E. Schweda,et al.  Structural analysis of the lipopolysaccharide oligosaccharide epitopes expressed by Haemophilus influenzae strain RM.118-26. , 2001, European journal of biochemistry.

[13]  P. Thibault,et al.  Sialic acid in the lipopolysaccharide of Haemophilus influenzae: strain distribution, influence on serum resistance and structural characterization , 1999, Molecular microbiology.

[14]  E. Moxon,et al.  Structural analysis of the lipopolysaccharide oligosaccharide epitopes expressed by a capsule-deficient strain of Haemophilus influenzae Rd. , 1999, European journal of biochemistry.

[15]  J. Weiser,et al.  Decoration of lipopolysaccharide with phosphorylcholine: a phase-variable characteristic of Haemophilus influenzae , 1997, Infection and immunity.

[16]  E. Moxon,et al.  Structure of the variable and conserved lipopolysaccharide oligosaccharide epitopes expressed by Haemophilus influenzae serotype b strain Eagan. , 1997, Biochemistry.

[17]  R. Fleischmann,et al.  Use of the complete genome sequence information of Haemophilus influenzae strain Rd to investigate lipopolysaccharide biosynthesis , 1996, Molecular microbiology.

[18]  J. Mekalanos,et al.  Molecular characterization of the oafA locus responsible for acetylation of Salmonella typhimurium O-antigen: oafA is a member of a family of integral membrane trans-acylases , 1996, Journal of bacteriology.

[19]  E. Hansen,et al.  Identification of a new locus involved in expression of Haemophilus influenzae type b lipooligosaccharide , 1994, Infection and immunity.

[20]  H. Tsunekawa,et al.  Cloning of the macrolide antibiotic biosynthesis gene acyA, which encodes 3-O-acyltransferase, from Streptomyces thermotolerans and its use for direct fermentative production of a hybrid macrolide antibiotic , 1994, Applied and environmental microbiology.

[21]  G. Walker,et al.  The acetyl substituent of succinoglycan is not necessary for alfalfa nodule invasion by Rhizobium meliloti Rm1021 , 1993, Journal of bacteriology.

[22]  J. Griffiss,et al.  Structural studies of the lipooligosaccharides from Haemophilus influenzae type b strain A2. , 1993, Biochemistry.

[23]  C. Hutchinson,et al.  A macrolide 3-O-acyltransferase gene from the midecamycin-producing species Streptomyces mycarofaciens , 1992, Journal of bacteriology.

[24]  K. Niehaus,et al.  The Rhizobium meliloti exoZl exoB fragment of megaplasmid 2: ExoB functions as a UDP‐glucose 4‐epimerase and ExoZ shows homology to NodX of Rhizobium leguminosarum biovar viciae strain TOM , 1991, Molecular microbiology.

[25]  D. Maskell,et al.  Characterization of repetitive sequences controlling phase variation of Haemophilus influenzae lipopolysaccharide , 1990, Journal of bacteriology.

[26]  M. Apicella,et al.  Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. , 1990, Journal of immunological methods.

[27]  T. Goodson,et al.  Production of a hybrid macrolide antibiotic in Streptomyces ambofaciens and Streptomyces lividans by introduction of a cloned carbomycin biosynthetic gene from Streptomyces thermotolerans. , 1989, Gene.

[28]  E. Moxon,et al.  The molecular mechanism of phase variation of H. influenzae lipopolysaccharide , 1989, Cell.

[29]  E. Hansen,et al.  Identification of a chromosomal locus for expression of lipopolysaccharide epitopes in Haemophilus influenzae , 1989, Infection and immunity.

[30]  G. Gutman,et al.  Slipped-strand mispairing: a major mechanism for DNA sequence evolution. , 1987, Molecular biology and evolution.

[31]  J. Devereux,et al.  A comprehensive set of sequence analysis programs for the VAX , 1984, Nucleic Acids Res..

[32]  L. van Alphen,et al.  Homogeneity of cell envelope protein subtypes, lipopolysaccharide serotypes, and biotypes among Haemophilus influenzae type b from patients with meningitis in The Netherlands. , 1983, The Journal of infectious diseases.

[33]  C. A. Thomas,et al.  Molecular cloning. , 1977, Advances in pathobiology.

[34]  P. Anderson,et al.  Human serum activities against Hemophilus influenzae, type b. , 1972, The Journal of clinical investigation.

[35]  M. Vogt,et al.  Defined Nongrowth Media for Stage II Development of Competence in Haemophilus influenzae , 1970, Journal of bacteriology.

[36]  D. Maskell,et al.  Characterization of Repetitive Sequences Controlling Phase , 2022 .