Laboratorio Especial de Desenvolvimento de Vacinas-Centro de Biotecnologia, Instituto Butantan, Avenida Vital Brazil, 1500, 05503-900, Sao Paulo, SP, Brazil; Programa de Pós-Graduação Interunidades em Biotecnologia, ICB, USP, Avenida Prof. LineuPrestes, 1730, 05508-900, Sao Paulo, SP, Brazil.
Laboratorio Especial de Desenvolvimento de Vacinas-Centro de Biotecnologia, Instituto Butantan, Avenida Vital Brazil, 1500, 05503-900, Sao Paulo, SP, Brazil.
J Microbiol Immunol Infect. 2020 Feb;53(1):163-175. doi: 10.1016/j.jmii.2018.12.012. Epub 2019 Jan 22.
Leptospirosis is an infectious disease that affects humans and animals worldwide. The etiological agents of this disease are the pathogenic species of the genus Leptospira. The mechanisms involved in the leptospiral pathogenesis are not full understood. The elucidation of novel mediators of host-pathogen interaction is important in the detection of virulence factors involved in the pathogenesis of leptospirosis.
This work focused on identification and characterization of a hypothetical protein of Leptospira encoded by the gene LIC10920.
The protein of unknown function was predicted to be surface exposed. Therefore, the LIC10920 gene was cloned and the protein expressed in Escherichia coli BL21 (DE3) Star pLysS strain. The recombinant protein was purified by metal affinity chromatography and evaluated with leptospirosis human serum samples. The interaction with host components was also performed.
The recombinant protein was recognized by antibodies present in leptopsirosis human serum, suggesting its expression during infection. Immunofluorescence and intact bacteria assays indicated that the bacterial protein is surface-exposed. The recombinant protein interacted with human laminin, in a dose-dependent and saturable manner and was named Lsa24.9, for Leptospiral surface adhesin, followed by its molecular mass. Lsa24.9 also binds plasminogen (PLG) in a dose-dependent and saturable fashion, fulfilling receptor ligand interaction. Moreover, Lsa24.9 has the ability to acquire PLG from normal human serum, exhibiting similar profile as observed with the human purified component. PLG bound Lsa24.9 was able of generating plasmin, which could increase the proteolytic power of the bacteria.
This novel leptospiral protein may function as an adhesin at the colonization steps and may help the invasion process by plasmin generation at the bacterial cell surface.
钩端螺旋体病是一种影响全球人类和动物的传染病。该病的病原体是钩端螺旋体属的致病性种。钩端螺旋体病发病机制中涉及的机制尚未完全了解。阐明宿主-病原体相互作用的新介质对于检测参与钩端螺旋体病发病机制的毒力因子非常重要。
本工作重点鉴定和表征由基因 LIC10920 编码的钩端螺旋体的一个假定蛋白。
预测该未知功能蛋白是表面暴露的。因此，克隆了 LIC10920 基因，并在大肠杆菌 BL21（DE3）Star pLysS 菌株中表达该蛋白。通过金属亲和层析纯化重组蛋白，并与钩端螺旋体患者血清样本进行评估。还进行了与宿主成分的相互作用。
重组蛋白被钩端螺旋体患者血清中的抗体识别，表明其在感染期间表达。免疫荧光和完整细菌检测表明，细菌蛋白是表面暴露的。重组蛋白与人层粘连蛋白以剂量依赖和饱和的方式相互作用，以 Leptospiral surface adhesin（Lsa24.9）命名，后面跟着它的分子量。Lsa24.9 还以剂量依赖和饱和的方式与纤溶酶原（PLG）结合，满足受体配体相互作用。此外，Lsa24.9 能够从正常人血清中获取 PLG，表现出与人类纯化成分相似的谱。结合 PLG 的 Lsa24.9 能够生成纤溶酶，从而增加细菌的蛋白水解能力。
这种新型钩端螺旋体蛋白可能在定植阶段作为黏附素发挥作用，并通过在细菌细胞表面生成纤溶酶来帮助入侵过程。
