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The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection
D A Haake1, G Chao, R L Zuerner
1Division of Infectious Diseases, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073, USA. dhaake@ucla.edu
Abstract:
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 [(3)H]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. CaCl(2) (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.
Insights
Researchers cloned the gene for LipL32, a key outer membrane lipoprotein in Leptospira bacteria. This protein is expressed during infection and is conserved in pathogenic species, suggesting its importance in leptospirosis diagnosis and prevention.
Area of Science:
- Microbiology
- Molecular Biology
- Immunology
Background:
- Leptospira are pathogenic bacteria causing leptospirosis.
- LipL32 is the most abundant protein in Leptospira.
- Understanding LipL32's role is crucial for disease management.
Purpose of the Study:
- To clone and characterize the gene encoding the LipL32 protein.
- To investigate the expression and localization of LipL32.
- To assess LipL32's potential as a diagnostic and preventive target.
Main Methods:
- Gene cloning using a Lambda-Zap II library and oligonucleotide probes.
- Amino acid sequencing and analysis of the LipL32 gene.
- Lipid modification analysis using radiolabeling and Triton X-114 extraction.
- Immunohistochemistry to detect LipL32 in infected tissues.
Main Results:
- The gene for the 32-kDa lipoprotein LipL32 was successfully cloned from Leptospira kirschneri.
- LipL32 is a lipid-modified outer membrane protein, confirmed by palmitoylation and detergent partitioning.
- LipL32 is expressed during bacterial cultivation and in infected mammalian tissues, notably hamster kidneys.
- LipL32 is a significant immunogen in human leptospirosis and is conserved across pathogenic Leptospira species.
Conclusions:
- LipL32 is a highly conserved, lipid-modified outer membrane lipoprotein in pathogenic Leptospira.
- Its expression during infection and immunogenicity suggest LipL32's critical role in leptospirosis pathogenesis.
- LipL32 holds potential as a target for diagnosing and preventing leptospirosis.