IDENTIFICATION AND CHARACTERIZATION OF THE DTDP-RHAMNOSE BIOSYNTHESISAND TRANSFER GENES OF THE LIPOPOLYSACCHARIDE-RELATED RFB LOCUS IN LEPTOSPIRA-INTERROGANS SEROVAR COPENHAGENI

Immunity to leptospirosis is principally humorally mediated and involv es opsonization of leptospires for phagocytosis by macrophages and neu trophils, The only protective antigen identified to date is the leptos piral lipopolysaccharide (LPS), which biochemically resembles typical gram-negative LPS but has greatly reduced endotoxic activity. Little i s known about the structure of leptospiral LPS. A 2.1-kb EcoRI fragmen t from the chromosome of serovar Copenhageni was cloned in pUC18 in Es cherichia coli, after which flanking regions were cloned from a genomi c library constructed in bacteriophage X GEM12. Sequence analysis iden tified four open reading frames which showed similarity to the rfbC, r fbD, rfbB, and rfbA genes, transcribed in that order, which encode the four enzymes involved in the biosynthesis of dTDP-rhamnose for the as sembly of LPS in Salmonella enterica, E. coli, and Shigella flexneri. An additional open reading frame downstream of the rfbCDBA locus showe d similarity with the rhamnosyltransferase genes of Shigella and Yersi nia enterocolitica but not Salmonella. Comparison of deduced amino aci d sequences showed up to 85% similarity of the leptospiral proteins wi th those of other gram-negative bacteria, Polyacrylamide gel electroph oresis of recombinant clones identified the putative RfbCDBA proteins, while reverse transcriptase-mediated PCR analysis indicated that the rfbCDBA gene cluster was expressed in Leptospira, Moreover, it could r estore normal LPS phenotype to a defined rfbB::Tn5 mutant of S. flexne ri which was deficient in all four genes, thereby confirming the funct ional identification of a part of the leptospiral rfb locus.