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.
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 greater than 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. coli K-12 LPS core.
Spinola
S M
SM
Department of Medicine, School of Medicine, State University of New York, Buffalo.
Kwaik
Y A
YA
Lesse
A J
AJ
Campagnari
A A
AA
Apicella
M A
MA
D000942:Antigens, Bacterial / Q000235:genetics*; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D000939:Epitopes; D005798:Genes, Bacterial; D006193:Haemophilus influenzae / Q000235:genetics*; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D015183:Restriction Mapping; D013400:Sugar Acids / Q000276:immunology / Q000378:metabolism*
D000942:Antigens, Bacterial; D004269:DNA, Bacterial; D000939:Epitopes; D008070:Lipopolysaccharides; D013400:Sugar Acids; C023023:lipid-linked oligosaccharides; C002532:2-keto-3-deoxyoctonate