The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection
The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection
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DOI：
10.1128/iai.68.4.2276-2285.2000
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发表时间：
2000-04-01
影响因子：
3.1
通讯作者：
Bolin, CA
中科院分区：
文献类型：
--
作者：
Haake, DA;Chao, G;Bolin, CA
我们报告的基因编码的32 kDa的脂蛋白，命名为LipL 32，最突出的蛋白质在钩端螺旋体蛋白质谱的克隆。我们获得了葡萄球菌V8蛋白酶切片段的N-末端氨基酸序列，设计了寡核苷酸探针。筛选了含有克氏钩端螺旋体DNA的EcoRI片段的Lambda-Zap II文库，并鉴定了含有完整结构lipL 32基因的5.0-kb DNA片段。一些证据表明，LipL 32是脂质修饰的方式类似于其他原核脂蛋白。LipL 32的推导氨基酸序列将编码一个272个氨基酸的多肽与一个19个氨基酸的信号肽，其次是脂蛋白信号肽酶切割位点。LipL 32在L. kirschneri在含有[H-3]棕榈酸酯的培养基中。LipL 32的棕榈酸酯与氨基端半胱氨酸的连接是酸不稳定的，LipL 32被Triton X-114萃取L. kirschneri;相分离导致LipL 32专门分配到疏水的去污剂相中，表明它是钩端螺旋体外膜的组分。为了回收LipL 32，在相分离期间必须存在CaCl 2（20 mM）。LipL 32不仅在培养过程中表达，而且在哺乳动物感染过程中表达。免疫组化显示LipL 32与L.感染仓鼠肾近端小管的kirshneri。LipL 32也是人类钩端螺旋体病的主要免疫原。LipL 32的序列和表达在致病性钩端螺旋体物种中高度保守。这些结果表明LipL 32可能在钩端螺旋体病的发病机制、诊断和预防中具有重要意义。
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 poly-peptide 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 [H-3]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. kirshneri 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 my be important in the pathogenesis, diagnosis, and prevention of Leptospirosis.