TN5-INDUCED LIPOPOLYSACCHARIDE MUTATIONS IN BORDETELLA-PERTUSSIS THATAFFECT OUTER-MEMBRANE FUNCTION

An LPSB-specific mAb was used to screen for ten Tn5 insertion mutants of Bordetella pertussis which have LPS which is phenotypically distinc t from either wild-type LPSAB or LPSB. Silver-stained SDS-PAGE gels sh owed nine different LPS phenotypes, six of which contain two clinicall y undocumented LPS bands, designated IntA and IntB based on their prox imity to the LPSA and LPSB bands, respectively. Binding assays with LP SA- and LPSB-specific mAbs established changes in epitope exposure for the various mutant LPS, both in cell-free form and as presented on th e surface of whole cells. The possible involvement of a number of gene s, both structural and regulatory, was indicated in production of the altered phenotypes. PFGE and Southern blotting showed that the Tn5 ins erts of seven mutants mapped to a region of the B. pertussis chromosom e shown previously to encode the bpl gene products of LPS biosynthesis . Mutants MLT3, MLT5 and MLT8, however, mapped to distinctly different parts of the chromosome. In addition, mutants MLT2 and MLT3 contribut ed to an accelerated frequency in the appearance of avirulent phase or ganisms despite their Tn5 inserts being over 1000 bp from the bvg/ASR locus. the alterations in LPS structure in the mutants changed their r eactivity to strain-specific mAbs and their sensitivity to hydrophobic and hydrophilic antibiotics.