Cullen Paul A, Haake David A, Bulach Dieter M, Zuerner Richard L, Adler Ben
Bacterial Pathogenesis Research Group, Department of Microbiology, Monash University, Melbourne, Victoria 3800, Australia.
Infect Immun. 2003 May;71(5):2414-21. doi: 10.1128/IAI.71.5.2414-2421.2003.
Leptospira is the etiologic agent of leptospirosis, a bacterial zoonosis distributed worldwide. Leptospiral lipopolysaccharide is a protective immunogen, but the extensive serological diversity of leptospires has inspired a search for conserved outer membrane proteins (OMPs) that may stimulate heterologous immunity. Previously, a global analysis of leptospiral OMPs (P. A. Cullen, S. J. Cordwell, D. M. Bulach, D. A. Haake, and B. Adler, Infect. Immun. 70:2311-2318, 2002) identified pL21, a novel 21-kDa protein that is the second most abundant constituent of the Leptospira interrogans serovar Lai outer membrane proteome. In this study, we identified the gene encoding pL21 and found it to encode a putative lipoprotein; accordingly, the protein was renamed LipL21. Southern hybridization analysis revealed the presence of lipL21 in all of the pathogenic species but in none of the saprophytic species examined. Alignment of the LipL21 sequence from six strains of Leptospira revealed 96 to 100% identity. When specific polyclonal antisera to recombinant LipL21 were used, LipL21 was isolated together with other known leptospiral OMPs by both Triton X-114 extraction and sucrose density gradient membrane fractionation. All nine strains of pathogenic leptospires investigated by Western blotting, whether culture attenuated or virulent, were found to express LipL21. In contrast, the expression of LipL21 or an antigenically related protein could not be detected in nonpathogenic L. biflexa. Infected hamster sera and two of eight human leptospirosis sera tested were found to react with recombinant LipL21. Native LipL21 was found to incorporate tritiated palmitic acid, consistent with the prediction of a lipoprotein signal peptidase cleavage site. Biotinylation of the leptospiral surface resulted in selective labeling of LipL21 and the previously known OMPs LipL32 and LipL41. These findings show that LipL21 is a surface-exposed, abundant outer membrane lipoprotein that is expressed during infection and conserved among pathogenic Leptospira species.
钩端螺旋体是钩端螺旋体病的病原体，这是一种在全球范围内传播的细菌性人畜共患病。钩端螺旋体脂多糖是一种保护性免疫原，但钩端螺旋体广泛的血清学多样性促使人们寻找可能刺激异源免疫的保守外膜蛋白（OMP）。此前，对钩端螺旋体OMP的一项全面分析（P.A.卡伦、S.J.科德韦尔、D.M.布拉奇、D.A.哈克和B.阿德勒，《感染与免疫》70:2311 - 2318，2002年）鉴定出pL21，一种新的21 kDa蛋白，它是问号钩端螺旋体赖型外膜蛋白质组中第二丰富的成分。在本研究中，我们鉴定了编码pL21的基因，发现它编码一种假定的脂蛋白；因此，该蛋白被重新命名为LipL21。Southern杂交分析显示lipL21存在于所有致病菌种中，但在所检测的腐生菌种中均不存在。来自六种钩端螺旋体菌株的LipL21序列比对显示同一性为96%至100%。当使用针对重组LipL21的特异性多克隆抗血清时，通过Triton X - 114提取和蔗糖密度梯度膜分级分离，LipL21与其他已知的钩端螺旋体OMP一起被分离出来。通过蛋白质印迹法研究的所有九种致病钩端螺旋体菌株，无论培养减毒的还是有毒力的，均发现表达LipL21。相比之下，在非致病性双曲钩端螺旋体中未检测到LipL21或抗原相关蛋白的表达。在受感染仓鼠血清以及所检测的八份人类钩端螺旋体病血清中的两份血清中发现与重组LipL21发生反应。发现天然LipL21掺入了氚标记的棕榈酸，这与脂蛋白信号肽酶切割位点的预测一致。钩端螺旋体表面的生物素化导致LipL21以及先前已知的OMP LipL32和LipL41被选择性标记。这些发现表明LipL21是一种表面暴露的、丰富的外膜脂蛋白，在感染期间表达且在致病钩端螺旋体菌种中保守。
