LIPOPOLYSACCHARIDE O-ANTIGEN EXPRESSION AND THE EFFECT OF ITS ABSENCEON VIRULENCE IN RFB MUTANTS OF VIBRIO-CHOLERAE O1

Using defined rfb mutants, defective in the biosynthesis of the O-anti gen of the lipopolysaccharide (LPS), and monoclonal antibodies (MAbs) to the A, B and C LPS antigens, we have examined the distribution of t he antigens and the effects of their loss. By immunogold electron micr oscopy, it has been possible to determine the relative amounts of the A, B and C antigens on Inaba and Ogawa cells, confirming previous stud ies based upon bacterial agglutination and hemagglutination inhibition s. These antigens are absent from rfb ::Tn mutants selected as resista nt to phages which have been shown to use the O-antigen as their recep tor. These mutants were severely attenuated as measured by both LD50 a nd their ability to compete with the wildtype parents when analyzed in the infant mouse cholera model. These mutants were unchanged in the e xport of cholera toxin or other secreted proteins but revealed an alte red outer membrane protein profile. The competition defect suggested a n effect on TCP (toxin-coregulated pilus). An analysis of the rfb::Tn mutants revealed that they were unable to assemble TCP on their surfac e, but the major subunit, TcpA, could be found as an intracellular poo l. These mutants could be complemented back to wild-type using the clo ned rfb region, implying that functional TCP assembly is dependent upo n an intact LPS. (C) 1998 Federation of European Microbiological Socie ties. Published by Elsevier Science B.V.