Cloning and expression in Escherichia coli of a Haemophilus influenzae type b lipooligosaccharide synthesis gene(s) that encodes a 2-keto-3-deoxyoctulosonic acid epitope

Abstract

The composition of lipooligosaccharide (LOS) can modify the virulence of Haemophilus influenzae type b (Hib). A genomic library of Hib strain A2 was constructed in the lambda bacteriophage EMBL3. Twenty-six phage clones expressed a Hib LOS oligosaccharide epitope in Escherichia coli that was detected by the monoclonal antibody (MAb) 6E4. None of the clones bound a polyclonal sera specific for Hib A2 LOS or an anti-H. influenzae lipid A MAb. One clone, designated EMBLOS-1, assembled an oligosaccharide with an apparent molecular weight of 1,400 (the 1.4K oligosaccharide) on a 4.1K lipopolysaccharide (LPS) species in E. coli LE392 and produced a novel 5.5K LPS that bound 6E4. Binding of 6E4 to the 5.5K EMBLOS-1 LPS band was abolished by treatment with sodium metaperiodate but was not affected by digestion with proteinase K, confirming the carbohydrate nature of the epitope. The EMBLOS-1 Haemophilus insert hybridized to similar restriction fragments in type b and nontypeable strains regardless of whether they expressed the 6E4 epitope. The 6E4 epitope did not undergo phase variation in Hib strain A2 at a frequency of >10-3. The oligosaccharide of the Salmonella minnesota Re mutant and 2-keto-3-deoxyoctulosonic acid (KDO) inhibited binding of 6E4 to Hib A2 LOS. We conclude that a gene(s) encoding an enzyme(s) that assembles a stable Hib LOS epitope containing KDO is conserved in H. influenzae and that the cloned Hib LOS synthesis gene products assemble a Hib LOS epitope on an E. coil K-12 LPS core.H. influenzae LOS and S. minnesota LPS were prepared by the phenol-water method of Westphal and Jann (37) or by the microphenol method of Inzana (13). LPS was prepared from recombinant phage lysates by centrifugation at 100,000 x g 1558 on July 16, 2020 by guest http://iai.asm.org/ Downloaded from

Lipooligosaccharides (LOS) of some Haemophilus species mimic human glycosphingolipids, and some LOS are sialylated

“…Bacterial strains and culture conditions. Hib A2, Neisseria meningitidis serogroup B strain 6275, and Neisseria gonorrhoeae F62 have been described previously (13,14,18). The other 19 Hib strains were cerebrospinal fluid or nasopharyngeal isolates obtained from our culture collection.…”

“…Recombinant phage and plasmids. Hib A2 was the source of chromosomal DNA used to construct the recombinant phage EMBLOS-1 (18). When transfected in Escherichia coli LE392, EMBLOS-1 encodes the synthesis of an oligosaccharide with an apparent molecular mass of 1,400 (1.4 kDa) on a 4.1-kDa lipopolysaccharide (LPS) structure, producing a chimeric 5.5-kDa LPS band.…”

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

“…1). These included the lic genes (Weiser et al, 1989;High et al, 1993) and the lex2 gene (Jarosik and Hansen, 1994) involved with LPS phase variation, the lsg locus (Spinola et al, 1990), galE (Maskell et al, 1992), and rfaE (Lee et al, 1995), rfaD (A. Preston, personal communication) and isn (Preston et al, 1996;Zwahlen et al, 1985) genes which are involved with heptose biosynthesis. The majority of biosynthetic functions remain to be Identification of LPS-related genes from the H. influenzae strain Rd genome sequence The sequences of a wide range of LPS biosynthetic genes from Gram-negative bacteria were used as 'database probes' to screen for homologous genes, by DNA or amino acid similarity, in the H. influenzae strain Rd genome database prior to its annotation.…”

Identification of a new locus involved in expression of Haemophilus influenzae type b lipooligosaccharide

“…Over the past few years, several genes involved in the biosynthesis of Hib LOS have been characterized (6,13,16,26,28,30,32), and some of these have been shown to be involved in expression of surface-exposed LOS epitopes. Four of these surface-exposed epitopes, defined by their reactivity with monoclonal antibodies (MAbs) 12D9, 6A2, 4C4, and 5G8 (9, 20), were first noted because of the correlation between phase variation of their expression and changes in the virulence potential of Hib (11,12).…”