Da. Haake et al., CHARACTERIZATION OF LEPTOSPIRAL OUTER-MEMBRANE LIPOPROTEIN LIPL36 - DOWN-REGULATION ASSOCIATED WITH LATE-LOG-PHASE GROWTH AND MAMMALIAN INFECTION, Infection and immunity, 66(4), 1998, pp. 1579-1587
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.