IDENTIFICATION OF A NEW LOCUS INVOLVED IN EXPRESSION OF HAEMOPHILUS-INFLUENZAE TYPE-B LIPOOLIGOSACCHARIDE

Previous studies have shown that changes in the expression of the Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS) epitope reactive with monoclonal antibody (MAb) 5G8 can be correlated with alterations in the virulence of some Hib strains. To identify the locus involved in expression of this particular LOS epitope, a genomic library was constructed in the plasmid shuttle vector pGJB103 from Hib strain DLA2, which constitutively expressed LOS reactive with MAb 5G8. This library was used to transform a second Hib strain (DL180) that normally does not express this LOS epitope, and a recombinant clone was identified that bound MAb 5G8. Subcloning of different regions of the Hib DL42 DNA insert in this recombinant plasmid determined that a 1,9-kb EcoRI fragment, designated lex-2, was responsible for transforming Hib strain DL180 to reactivity,vith MAb 5G8. Nucleotide sequence analysis revealed the presence of two contiguous open reading frames (ORFs) in lex-2, the first of which contained 18 tandem repeats of the nucleotide tetramer GCAA near its 5' end. Sequence analysis of PCR-derived products from MAb 5G8-reactive and -nonreactive Hib DL180 colonies established that 18 GCAA repeats were associated with expression of the LOS epitope that bound MAb 5G8. Mutational analysis determined that a functional ORF 2 was essential for expression of the MAb 5G8-reactive LOS epitope. The nucleotide tetramer GCAA repeat present in ORF 1 was also detected in at least two different chromosomal regions in ail Hib strains tested. The availability of the cloned len-2 locus should facilitate future analysis of the complex regulatory mechanisms involved in expression of LOS epitopes by this pathogen.