Tn5-induced lipopolysaccharide mutations in Bordetella pertussis that affect outer membrane function

1 Department of Medical Microbiology and Immunology, University of Alberta, 1-69 Medical Sciences Bldg, Edmonton, Alberta, Canada T6G 2H7 2 Unite de recherche en vaccinologie, Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Québec, Sainte-Foy, Québec, Canada G1V 4G2 *Author for correspondence: Mark S. Peppler. Tel: +1 403 492 2304. Fax: + 1 403 492 7521. e-mail: mark.peppler@ualberta.ca ABSTRACT Summary: An LPSB-specific mAb was used to screen for ten Tn 5 insertion mutants of Bordetella pertussis which have LPS which is phenotypically distinct from either wild-type LPSAB or LPSB. Silver-stained SDS-PAGE gels showed nine different LPS phenotypes, six of which contain two clinically undocumented LPS bands, designated IntA and IntB based on their proximity to the LPSA and LPSB bands, respectively. Binding assays with LPSA- and LPSB-specific mAbs established changes in epitope exposure for the various mutant LPS, both in cell-free form and as presented on the surface of whole cells. The possible involvement of a number of genes, both structural and regulatory, was indicated in production of the altered phenotypes. PFGE and Southern blotting showed that the Tn 5 inserts of seven mutants mapped to a region of the B. pertussis chromosome 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 contributed to an accelerated frequency in the appearance of avirulent phase organisms despite their Tn 5 inserts being over 1000 bp from the bvgl ASR locus. The alterations in LPS structure in the mutants changed their reactivity to strain-specific mAbs and their sensitivity to hydrophobic and hydrophilic antibiotics. Keywords: Bordetella pertussis, lipopolysaccharide mutants, outer membrane integrity