M16-Type Metallopeptidases Are Involved in Virulence for Invasiveness and Diffusion of Leptospira interrogans and Transmission of Leptospirosis
M16-Type Metallopeptidases Are Involved in Virulence for Invasiveness and Diffusion of Leptospira interrogans and Transmission of Leptospirosis
复制标题
M16 型金属肽酶参与询问钩端螺旋体的侵袭和扩散以及钩端螺旋体病传播的毒力。
DOI：
10.1093/infdis/jiaa176
复制
发表时间：
2020-09-15
影响因子：
6.4
通讯作者：
Yan, Jie
中科院分区：
文献类型：
--
作者：
Ge, Yu-Mei;Sun, Ai-Hua;Yan, Jie
场景
钩端螺旋体病是由问号钩端螺旋体引起的一种全球性人畜共患传染病。病原体迅速侵入宿主，从血液扩散到内脏，并从尿液中排泄，引起钩端螺旋体病的传播。然而，钩端螺旋体的侵袭机制仍然知之甚少。
方法
L. M16型金属肽酶（Lep-MP 1/2/3）的蛋白水解活性。用分光光度法测定问号。通过qRT-PCR、Western Blot和共聚焦显微镜检测Lep-MP 1/2/3在感染细胞过程中的表达和分泌。通过构建Lep-MP 1/2/3的缺失突变体和互补突变体，用transwell法研究Lep-MP 1/2/3在侵袭力和毒力中的作用。
结果
L.而不是saperitic L. biflexa菌株可检测到Lep-MP-1/2/3编码基因。rLep-MP 1/2/3水解ECM蛋白，但rLep-MP 1/3比rLep-MP2更能水解ECM蛋白，Km值为123.179/340.136 μmol·L-1，Kcat值为0.154/0.159 s-1。Lep-MP 1/2/3的表达、分泌和易位在感染细胞过程中增加。Δ MP 1/3突变体（而非ΔMP2突变体）通过细胞单层的迁移减弱，血液、肺、肝、肾和尿液中的钩端螺旋体载量降低，LD 50降低10/13倍，组织病理学损伤减轻。
结论
Lep-MP 1/3与L.问号在钩体入侵宿主和体内扩散中的作用以及钩体病的传播。
BACKGROUD
Leptospirosis is a global zoonotic infectious disease caused by Leptospira interrogans. The pathogen rapidly invades into hosts and diffuses from bloodstream into internal organs and excretes from urine to cause transmission of leptospirosis. However, the mechanism of leptospiral invasiveness remains poorly understood.
METHODS
Proteolytic activity of M16-type metallopeptidases (Lep-MP1/2/3) of L. interrogans was determined by spectrophotometry. Expression and secretion of Lep-MP1/2/3 during infection of cells were detected by qRT-PCR, Western Blot assay and confocal microscopy. Deletion and complementation mutants of the genes encoding Lep-MP1/2/3 were generated to determine the roles of Lep-MP1/2/3 in invasiveness using transwell assay and virulence in hamsters.
RESULTS
L. interrogans but not saprophytic L. biflexa strains were detectable for Lep-MP-1/2/3-encoding genes. rLep-MP1/2/3 hydrolyzed ECM proteins, but rLep-MP1/3 displayed stronger proteolysis than rLep-MP2, with 123.179/340.136 µmol•L-1 Km and 0.154/0.159 s-1 Kcat values. Expression, secretion and translocation of Lep-MP1/2/3 during infection of cells were increased. ΔMP1/3 but not ΔMP2 mutant presented attenuated transmigration through cell monolayers, decreased leptospiral loading in the blood, lungs, liver, kidneys and urine, and 10/13-fold-decreased LD50 and milder histopathologic injury in hamsters.
CONCLUSIONS
Lep-MP1/3 are involved in virulence of L. interrogans in invasion into hosts and diffusion in vivo, and transmission of leptospirosis.