CHARACTERIZATION OF LEPTOSPIRAL OUTER-MEMBRANE LIPOPROTEIN LIPL36 - DOWN-REGULATION ASSOCIATED WITH LATE-LOG-PHASE GROWTH AND MAMMALIAN INFECTION

We report the cloning of the gene encoding a 36-kDa leptospiral outer membrane lipoprotein, designated LipL36. We obtained the N-terminal am ino acid sequence of a staphylococcal V8 proteolytic-digest fragment i n order to design an oligonucleotide probe. A Lambda-Zap II library co ntaining EcoRI fragments of Leptospira kirschneri DNA was screened, an d a 2.3-kb DNA fragment which contained the entire structural lipL36 g ene was identified. Several lines of evidence indicate that LipL36 is lipid modified in a manner similar to that of LipL41, a leptospiral ou ter membrane lipoprotein we described in a previous study (E. S. Shang , T. A. Summers, and D. A. Haake, Infect. Immun, 64:2322-2330, 1996). The deduced amino acid sequence of LipL36 would constitute a 364-amino -acid polypeptide,vith a 20-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. LipL36 is solubili zed by Triton X-114 extraction of L. kirschneri; phase separation resu lts in partitioning of LipL36 exclusively into the hydrophobic, deterg ent phase. LipL36 is intrinsically labeled during incubation of L. kir schneri in media containing [H-3]palmitate. Processing of LipL36 is in hibited by globomycin, a selective inhibitor of lipoprotein signal pep tidase. After processing, LipL36 is exported to the outer membrane alo ng with LipL41 and lipopolysaccharide. Unlike Lip41, there appears to be differential expression of LipL36. In early-log-phase cultures, Lip L36 is one of the most abundant L. kirschneri proteins. However, LipL3 6 levels drop considerably beginning in mid-log phase. LipL36 expressi on in vivo was evaluated by examining the humoral immune response to l eptospiral antigens in the hamster model of leptospirosis. Hamsters su rviving challenge with culture-adapted virulent L. kirschneri generate a strong antibody response to LipL36. In contrast, sera from hamsters surviving challenge with host-adapted L. kirschneri do not recognize LipL36. These findings suggest that LipL36 expression is downregulated during mammalian infection, providing a marker for studying the mecha nisms by which pathogenic Leptospira species adapt to the host environ ment.