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Infection and Immunity, April 2000, p. 2276-2285, Vol. 68, No. 4
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Leptospiral Major Outer Membrane Protein LipL32
Is a Lipoprotein Expressed during Mammalian Infection
David A.
Haake,1,2,*
Garlo
Chao,1
Richard L.
Zuerner,3
Jeanne K.
Barnett,4
Dean
Barnett,4
Mary
Mazel,1
James
Matsunaga,1,2
Paul N.
Levett,5 and
Carole A.
Bolin3
Division of Infectious Diseases, Veterans
Affairs Greater Los Angeles Healthcare System, Los Angeles,
California 900731; Department of
Medicine, UCLA School of Medicine, Los Angeles, California
900952; National Animal Disease Center,
Agricultural Research Service, U.S. Department of Agriculture,
Ames, Iowa 500103; Biology
Department, University of Southern Indiana, Evansville, Indiana
477124; and University of the West
Indies School of Clinical Medicine and Research,
Barbados5
Received 2 September 1999/Returned for modification 3 November
1999/Accepted 4 January 2000
We report the cloning of the gene encoding the 32-kDa lipoprotein,
designated LipL32, the most prominent protein in the leptospiral protein profile. We obtained the N-terminal amino acid sequence of a
staphylococcal V8 proteolytic-digest fragment to design an oligonucleotide probe. A Lambda-Zap II library containing
EcoRI fragments of Leptospira kirschneri DNA
was screened, and a 5.0-kb DNA fragment which contained the entire
structural lipL32 gene was identified. Several lines of
evidence indicate that LipL32 is lipid modified in a manner similar to
that of other procaryotic lipoproteins. The deduced amino acid sequence
of LipL32 would encode a 272-amino-acid polypeptide with a
19-amino-acid signal peptide, followed by a lipoprotein signal
peptidase cleavage site. LipL32 is intrinsically labeled during
incubation of L. kirschneri in media containing
[3H]palmitate. The linkage of palmitate and the
amino-terminal cysteine of LipL32 is acid labile. LipL32 is completely
solubilized by Triton X-114 extraction of L. kirschneri;
phase separation results in partitioning of LipL32 exclusively into the
hydrophobic, detergent phase, indicating that it is a component of the
leptospiral outer membrane. CaCl2 (20 mM) must be present
during phase separation for recovery of LipL32. LipL32 is expressed not
only during cultivation but also during mammalian infection.
Immunohistochemistry demonstrated intense LipL32 reactivity with
L. kirschneri infecting proximal tubules of hamster
kidneys. LipL32 is also a prominent immunogen during human
leptospirosis. The sequence and expression of LipL32 is highly
conserved among pathogenic Leptospira species. These findings indicate that LipL32 may be important in the pathogenesis, diagnosis, and prevention of leptospirosis.
*
Corresponding author. Mailing address: Division of
Infectious Diseases, 111F, VA Greater LA Healthcare System, Los
Angeles, CA 90073. Phone: (310) 478-3711, x40267. Fax: (310) 268-4928. E-mail: dhaake{at}ucla.edu.
Infection and Immunity, April 2000, p. 2276-2285, Vol. 68, No. 4
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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