【作者】 王欢；

【导师】 严杰；

【作者基本信息】 浙江大学 ， 病原生物学， 2012， 博士

【摘要】 钩端螺旋体病(Leptospirosis,简称钩体病)是一种全球分布的、洪涝时重点监控的自然疫源性传染病,是我国重点防疫和监控的主要传染病之一。致病钩体赖株基因组中存在众多编码溶血素的基因表明溶血素在钩端螺旋体病中发挥重要作用,但溶血素在钩体感染巨噬细胞后基因表达水平和分泌性尚未阐明。本研究中采用T-A克隆等分子生物学方法构建钩体溶血素基因sphl (LA1027)、 sph2(LA1029)、sph3(LA4004)、sph4(LA3050)、 hlpA(LA1650)、 hly(LA3937)、 hlyX(LA0378)和tlyA(LA0327)的原核表达系统及Ni-NTA亲和层析法提纯各重组溶血素蛋白,分光光度法检测重组溶血素蛋白对绵羊红细胞的溶血活性,实时荧光定量PCR检测钩体赖株感染THP-1和J774A.1细胞后溶血素基因mRNA水平变化及Western-blot检测感染这两种细胞后溶血素蛋白的分泌性。结果成功地构建了8个高效表达目的重组蛋白的表达系统及重组蛋白具备不同程度的溶血活性,感染巨噬细胞后8个溶血素基因水平上调,其中以sph2和hlpA上调更为显著,在两种细胞中rSphl、 rSph2、 rSph3、 rH1pA和rT1yA均能够分泌到胞外。这为后续深入研究8个溶血素的致病机理奠定了良好的基础。人感染致病性钩端螺旋体后可引起严重的全身性炎症反应,但是钩体病人血清中完整的炎症因子谱未见报道。钩体赖株基因组中存在众多编码溶血素的基因表明溶血素在钩端螺旋体致病中发挥作用,但关于钩端螺旋体溶血素能否诱导炎性细胞因子分泌及相关的信号通路研究未见报道。本研究中,采用炎性相关细胞因子抗体芯片技术检测钩体病人和感染钩体后的小鼠血清中的完整炎性因子谱。与钩体病人血清相比,感染的小鼠血清中细胞因子出现相对较低的水平,而IL-1β,IL-6和TNF-α三种促炎性细胞因子是钩体病人和感染后小鼠血清均升高的细胞因子。钩端螺旋体重组鞘磷脂类溶血素蛋白(rSph1-rSph4)和非鞘磷脂类溶血素蛋白(rH1pA、rH1yC、rH1yX和rT1yA)均可诱导入和小鼠巨噬细胞分泌IL-1β,IL-6和TNF-α,但在小鼠巨噬细胞中水平相对较低。TLR2和TLR4受体封闭后,溶血素诱导人和小鼠巨噬细胞释放IL-1β,IL-6和TNF-α的能力减弱,这一结论在TLR敲除小鼠的单核巨噬细胞中也得到验证。胞内信号通路NF-κB和JNK抑制剂处理后,人和小鼠巨噬细胞在溶血素刺激下释放上述三种细胞因子的能力减弱。本研究发现8个钩端螺旋体重组溶血素蛋白能通过TLR2和TLR4受体介导的JNK和NF-κB途径诱导人和小鼠巨噬细胞分泌炎性细胞因子。为了探讨钩端螺旋体(简称钩体)侵入人或鼠单核-巨噬细胞方式及其吞噬泡形成差异性。本研究中采用透射电镜观察钩体赖株侵入小鼠单核-巨噬样细胞J774A.1和佛波酯(PMA)激活的人单核细胞THP-1后吞噬泡形成情况。采用免疫荧光联合激光共聚焦显微镜及荧光分光光度仪等方法,观察细胞内吞抑制剂单丹磺酰尸胺(MDC)、氧化酚砷(PAO)阻断及网格蛋白抗体封闭前后,J774A.1细胞和THP-1细胞内钩体赖株数量的变化。结果发现774A.1细胞内钩体存在于吞噬泡内,THP-1细胞内钩体无吞噬泡膜包绕。MDC和PAO能以剂量依赖方式抑制J774A.1和THP-1细胞内吞钩体,其中10μmol/L以上MDC和1μmol/L以上PAO阻断的J774A.1和THP-1细胞内钩体数量明显少于未阻断细胞(P<0.05)。网格蛋白抗体封闭后,J774A.1和THP-1细胞内钩体数量也明显减少(P<0.05)。从上述结果中得出如下结论：钩体以网格蛋白依赖性内吞途径侵入人或鼠单核-巨噬细胞。人或鼠单核-巨噬细胞内钩体吞噬泡形成有明显差异,这可能是人或鼠感染钩体后发病情况不同的原因之一。钩体病是由致病性钩体引起的全世界流行的人兽共患病,但致病性钩体感染人和家禽(或野生动物)的症状不同。钩体在感染人和动物时迅速通过皮肤和粘膜进入血流,吞噬过程是最重要的天然免疫防护机制之一,因此单核细胞和巨噬细胞在清除侵入病原菌的吞噬功能对钩体感染的天然免疫保护中起到关键作用。钩体与人和小鼠巨噬细胞相互作用的结局不一样,大多数进入小鼠原代与传代细胞的钩体局限在吞噬泡内,而感染人原代与传代巨噬细胞内大多数钩体分布在细胞质内,且不被吞噬泡膜所包裹；含钩体的小鼠吞噬细胞膜能够与溶酶体融合而被杀灭,但人巨噬细胞内钩体反而有繁殖的迹象。新生的吞噬体缺乏将病原体或摄入物质杀死降解的能力,从吞噬体产生到成为具有杀死并降解被吞噬物的吞噬溶酶体的过程称为吞噬体的成熟。微生物的逃逸机制是通过寄生在巨噬细胞内的吞噬体中的细菌,干扰内体的发生和成熟,导致形成的吞噬体不能获得溶酶体性质。致病性钩体感染人巨噬细胞后是如何逃逸宿主天然防疫系统而在其内存活和繁殖以及钩体溶血素是否参与此干扰吞噬体成熟的过程,均未见报道。本研究中,我们通过透射电镜对THP-1和J774A.1细胞中吞噬泡的形成及演变的研究,结果发现THP-1和J774A.1细胞在内吞开始均形成吞噬泡膜,THP-1中吞噬泡膜出现消失的过程,但J774A.1细胞中的吞噬泡膜没有出现消失的过程。通过对THP-1和J774A.1细胞吞噬泡成熟过程中的表面标志分子进行免疫荧光及激光共聚焦观察,结果显示THP-1细胞中,溶血素主要与吞噬泡膜早期标志物(Rab5)共定位,与晚期标志物(Rab7和LAMP-1)共定位较少；J774A.1细胞中,溶血素可与不同时期吞噬泡膜表面标志物发生共定位。上述结果初步得出钩体溶血素能够与吞噬泡膜发生作用,通过溶解吞噬泡膜参与干扰吞噬泡成熟的过程,从而介导钩体逃逸人巨噬细胞的吞噬杀灭作用。这可能是人或鼠感染问号钩体后发病情况不同的原因之一更多还原

【Abstract】 Leptospira interrogans is a global distribution zoontic diseases when flood outbreak, which is one of the major infectious diseases in China’s key prevention and control. There are many genes that encode putative hemolysin Leptospira interrogans strain Lai genome, hemolysin may contribute to the pathogenesis of leptospirosis, but leptospira gene expression and secretion of hemolysin during infected with macrophages have not been reported.In this study, first, we constructed Leptospira hemolysin genes including sphl (LA1027), sph2(LA1029), sph3(LA4004), sph4(LA3050), hlpA (LA1650), hlyC (LA3937), hlyX(LA0378) and tlyA (LA0327) prokaryotic expression system by cloning, then the recombinant proteins were extracted and purified by Ni-NTA affinity chromatography, and hemolytic activity of recombinant proteins were measured by hemolytic test in sheep blood by spectrophotometry. Finally, real-time fluorescence quantitative PCR was applied to detect the change of hemolysin genes in mRNA levels and Western-blot was used to detect the secreted of hemolysin proteins during Leptospira interrogans infected THP-1and J774A.1cells.The results showed that eight recombinant proteins with varying degrees of hemolytic activity has been constructed with high-level expression, eight hemolysin genes mRNA levels upregulated after infected with macrophages, especaily for sph2and hlpA show more significant increase, rSphl, rSph2, rSph3, rH1pA and rTlyA can be secreted into the extracellular in THP-1and J774A.1cells.All these results lead us to have a conclusion that a prokaryotic expression system with high efficiency of L. interrogans hemolysin genes were constructed successfully and the hemolysin genes change in mRNA and protein levels during infected with macrophages, which lay a foundation for further study of pathogenic of leptospira hemolysin. Infection of pathogenic Leptospira species causes serious systemic inflammation in patients but the profile of proinflammatory cytokines has never been characterized. Although the presence of numerous hemolysin-encoding genes in the genomes of pathogenic L.interrogans strains suggests that the hemolysins may contribute to pathogenesis in leptospirosis, the ability of leptospiral hemolysins to induce proinflammatory cytokines and stimulate TLR-dependent signaling pathways has never been reported. In the present study, we measured the cytokine profile in sera from leptospirosis patients and leptospire-infected mice using cytokine protein microarrays. The infected mice provided less elevated serum cytokines with relatively lower levels compared to the patients. However, IL-1β, IL-6and TNF-a were the main proinflammatory cytokines in sera of both the patients and infected mice. The tested leptospiral recombinant sphingomyelinic hemolysins (rSphl to rSph4) and non-sphingomyelinic hemolysins (rHlpA, rHlyC, rHlyX and rTlyA) could induce the production of IL-1(3, IL-6and TNF-a in human macrophages, while the production of cytokines in mouse macrophages was relatively lower. Inhibition of TLR2and TLR4as well as the NF-kB and JNK pathways in the two species of macrophages significantly reduced the production of hemolysin-induced IL-1β,IL-6and TNF-a. Macrophages isolated from TLR2-, TLR4-or double TLR2-and4-deficient mice confirmed the TLR2-and TLR4-dependent regulation of proinflammatory cytokine production induced by leptospiral hemolysins. Taken together, we demonstrated that all eight leptospiral hemolysins have the ability to induce inflammatory cytokines via TLR2and TLR4through the JNK and NF-kB pathways. This study is aim to determine the modality of Leptospira interrogans invading human and murine mononuclear-macrophages and diversity of leptospiral phagocytotic vesicle formation. Transmission electron microscopy was applied to observe the invasion of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai into murine mononuclear-macrophage-like cell line J774A.1and PMA-activated human monocyte line THP-1and the formation of leptospiral phagocytotic vesicles. By using immunofluorescence plus either laser confocal microscopy or fluorescence spectrophotometry, the changes of intracellular leptospiral numbers in J774A.1and PMA-activated THP-1cells before and after block with endocytosis inhibitors monodansylcadaverin (MDC), phenylarsine oxide (PAO) and clathrin antibody were investigated.The leptospires in J774A.1cells were located in phagocytotic vesicles while the leptospires in THP-1cells had no package with phagocytotic vesicle membrane. Both MDC and PAO presented the effect inhibiting endocytosis of L. interrogans into J774A.1and THP-1cells in dose-dependent manner. The numbers of leptospires in J774A.1and THP-1cells that pre-blocked with10μmol/L or above MDC and1μmol/L or above PAO were significantly less than that in the two cells untreated with MDC and PAO (P＜0.05). After J774A.1and THP-1cells were blocked with clathrin antibody, the numbers of intracellular leptospires were also remarkbly decreased (P<0.05).Leptospira interrogans can invade into both human and murine mononuclear-macrophages through the way of clathrin-dependent endocytosis. There is an opposite diversity of leptospiral phagocytotic vesicle formations in human and murine mononuclear-macrophages, which may result in the difference of pathogenesis in humans and mice after infected with L. interrogans. Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira, but the symptoms of human and poultry infected with leptospira are different. Leptospira can enter the bloodstream through the skin and mucous membranes quickly, Phagocytic process is the most important innate immune protection mechanisms, so the monocytes and macrophages play a key role in removing invasive pathogen. However, Leptospira invaded human and mouse macrophages occured different phenomenons, most of leptospira limited in the phagocytic vacuole of mouse primary and passage macrophages and distributed in the cytoplasm of human primary and passage macrophages. Furthermore, phagocytic membrane containing leptospira fused with lysosomes and leptospira were killed in mouse, while fertile signs of leptospira within human macrophages.Cells of the innate immune system ingest and destroy invading microorganisms by initially engulfing them into a specialized vacuole, known as the phagosome. The nascent phagosome is not competent to kill and eliminate the ingested microorganisms. Phagosome maturation is the process by which internalized particles (such asbacteria and apoptotic cells) are trafficked into a series of increasingly acidified membrane-bound structures, leading to particle degradation. The basic evasion mechanism is to mediate the process of phagosome, resulting in the formation of the mature phagosome-lysosome could not be obtained to produce within the phagosome, such as Mycobacterium tuberculosis. However, the mechanisms of pathogenic leptospira in human macrophages escaped from the host immune system and reproducted in the organization, and whether the leptospira hemolysins involved in the phagosome maturation process are not reported.In this study, we found that THP-1and J774A.1cells formed phatocytic vesicle membrane at the beginning of infection, then the phatocytic vesicle membrane diappeared in THP-1cells while not in J774A.1cells using TEM. Then using laser confocal immunofluorescence, we found that the major hemolysin co-localizated with the early markers of phagocytic vesicle membrane (Rab5),less co-localizated with the late markers (Rab7and LAMP-1) in THP-1cells, hemolysins could co-localizated with different periods of surface markers of vesicle membrane in J774A.1cells.We can conclude preliminarily that leptospira hemolysins interfered with the process of vacuolar maturation and mediated the evasion of leptospira from human macrophages for pathogen survival which id different with mouse macrophages, which may result in the difference of pathogenesis in humans and mice after infected with L. interrogans更多还原