猪链球菌毒力因子和鉴定方法的研究进展

猪链球菌(Streptococcus suis)是一种重要的人畜共患病病原体,能够引起猪疫病,人类感染该菌可导致脑膜炎、败血症甚至死亡。鉴于其对养猪业的巨大经济影响和对公共卫生事业的威胁,关于猪链球菌的研究日益深入,即对其毒力相关蛋白和鉴定方法的研究进展做一综述。     

用生物信息学方法预测猪链球菌2型05ZYH33株的脂蛋白

[目的]鉴别已公布基因组序列的SS2 05ZYH33株的脂蛋白(Lpp).[方法]首先从Genbank获取由 SS2 05ZYH33基因组推测的蛋白质氨基酸序列,然后利用Lpp预测软件PrositeScan和DOLOP预测05ZYH33株的Lpp,采用SignalP 3.0 HMM、PrediSi、Phobius、LipoP-HMM和TMHMMversion2.0分析预测的Lpp的信号肽,然后经"多数票决法"确定Lpp;最后利用InterProScan和BlastP服务器对鉴定的Lpp进行功能分析.[结果]鉴定出34种Lpp,占SS2致病株05ZYH33蛋白质组的1.555%.结构与功能分析结果表明,最大的一类Lpp是ABC运输蛋白的底物结合蛋白,共有16种,其中YP_001197586、YP_001197918、YP_001198449、YP_001199435、YP_001197482、YP_001199452和YP_001198914等7种基因可与其他成分组成完整的ABC运输蛋白操纵子,这些Lpp在SS2获取糖、氨基酸和金属离子等营养物质方面具有重要作用.YP_001197698与无乳链球菌的黏附素Lmb和化脓链球菌的黏附素Lbp及YP_001198710与肺炎链球菌的SlrA高度同源,推测它们与黏附功能有关,为SS2新的毒力因子;其他Lpp包括酶、参与蛋白质折叠过程的Lpp及功能未知的Lpp.[结论]这些资料提示Lpp在SS2的生理和致病性方面具有重要作用.     

Pro-inflammatory cytokine and chemokine release by human brain microvascular endothelial cells stimulated by Streptococcus suis serotype 2

Streptococcus suis serotype 2 is a world-wide agent of diseases among pigs including meningitis, septicemia and arthritis. This microorganism is also recognized as an important zoonotic agent. The pathogenesis of the meningitis caused by S. suis is poorly understood. We have previously shown that S. suis is able to adhere to human brain microvascular endothelial cells (BMEC), but not to human umbilical vein endothelial cells (HUVEC). The objective of this work was to study the ability of S. suis serotype 2 to induce the release of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1); IL-6 and the chemokines IL-8 and monocyte chemotactic protein-1 (MCP-1) by human BMEC and HUVEC, using a sandwich enzyme-linked immunosorbent assay. S. suis was able to stimulate the production of IL-6, IL-8 and MCP-1 by BMEC but not HUVEC, in a time- and concentration-dependent manner. Bacterial cell wall components were largely responsible for such stimulation. The human and pig origin of strains does not seem to affect the intensity of the response; indeed, a very heterogeneous pattern of cytokine and chemokine production was observed for the different strains tested in this study. In situ production of cytokines and chemokines by BMEC may be the result of specific adhesion of S. suis to this cell type, with several consequences such as increased recruitment of leukocytes and an increase in the blood-brain barrier permeability.     

多重聚合酶链反应检测猪链球菌7种主要毒力因子

目的 建立2个分开的多重聚合酶链反应(PCR)体系,以及对猪链球菌7种主要毒力因子的检测。方法 根据猪链球菌7种主要毒力因子mrp、epf、sly、gdh、gapdh、orf2和fbps的基因序列,设计和合成7对特异性引物,通过对它们在2个分开反应体系PCRⅠ和PCRⅡ中的组合和优化,建立猪链球菌7种主要毒力因子的2组多重PCR检测方法,并对实验室的29株背景明确的猪链球菌保存菌株进行检测。结果 29株猪链球菌菌株的检测结果和菌株的背景情况一致,阳性、阴性对照均成立。结论 此猪链球菌7种主要毒力因子的2组分开体系的多重PCR检测方法,特异性和敏感性均好,可用于快速诊断以及猪链球菌毒力因子的分子流行病调查。     

猪链球菌2型强毒株S.suis 05ZY和弱毒株S.suis 1940的比较转录谱分析

目的:比较猪链球菌2型强毒株S.suis 05ZY和弱毒株S.suis 1940毒力相关基因转录水平的差异,为进一步研究强毒株S.suis 05ZY毒力增强的原因提供实验基础。方法:分别提取S.suis 05ZY和S.suis 1940的RNA,反转录成cDNA并纯化,用Cy5或Cy3标记,与猪链球菌全基因组DNA芯片进行杂交,扫描芯片进行数据分析,比较二者在转录水平上的差异基因。结果:编码溶血素、精氨酸氨基肽酶的基因分别上调4.4和6.0倍,参与荚膜多糖合成的相关基因cps2H、cps2I、cps2J和一些可能的毒力相关基因ofs、dpr、SSU05₀196、SSU05₀272、SSU05₁408-1409均发生转录水平的上调。结论:溶血素、荚膜多糖、精氨酸氨基肽酶及一些可能的毒力因子在转录水平的上调很可能与S.suis 05ZY的毒力增强有关。     

猪链球菌2型感染仔猪致脑炎模型的建立

【背景】猪链球菌2型(Streptococcus suis type 2)是危害养猪业重要的病原,给我国养猪业造成了重大的损失,迄今为止仍缺乏猪链球菌2型感染仔猪致脑炎模型,建立合适的模型用于临床药物研发及机制研究为防控该病奠定基础。【目的】构建猪链球菌2型感染仔猪致脑炎模型,通过观察感染仔猪的临床变化、病理学变化、病原分离及PCR鉴定证实模型构建成功。【方法】选取6头健康的三元杂交猪仔猪,随机均分为A、B两组。A组仔猪接种猪链球菌,B组接种生理盐水作为空白对照,观察A组仔猪的临床症状、剖检变化、病理组织学变化。【结果】A组仔猪出现体温升高、精神沉郁、嗜睡以及严重的神经症状,甚至死亡;器官出现严重病变,如脊髓和大脑的神经元细胞的肿胀、坏死、溶解,小脑灰质区浦肯野细胞层和颗粒层见细胞灶状坏死并形成坏死后空腔。【结论】成功构建出猪链球菌2型感染仔猪的脑炎模型。     

猪链球菌2型荚膜唾液酸对细菌毒力和宿主炎症反应的影响

【目的】阐明猪链球菌2型荚膜唾液酸是否影响细菌毒力以及宿主对其炎症反应应答,为研究猪链球菌2型的致病机制奠定基础。【方法】比较实验菌株对BLAB/c小鼠模型的致病性;通过涂板计数的方法检测实验菌株在小鼠体内的分布;观察小鼠脑组织病理改变,分析实验菌株感染小鼠后中枢神经系统的病变差异;从小鼠体外全血细胞水平,运用ELISA法检测实验菌株感染后细胞炎性因子的分泌水平。【结果】荚膜唾液酸合成基因neuB缺失突变株ΔneuB相比野生株05ZYH33株,对小鼠毒力显著降低,回复突变株cΔneuB毒力回复至野生株水平;野生株和突变株在血液及脑组织中分布具有显著差异,均可致BLAB/c小鼠脑组织不同程度的损伤;与野生株组相比较,细菌/细胞相互作用不同时间点后,突变株组体外刺激小鼠全血细胞分泌MCP-1、IL-6的水平显著提高;【结论】荚膜唾液酸影响细菌的毒力及宿主细胞对其的炎症反应应答,它是猪链球菌2型穿透血脑屏障导致脑膜炎的重要毒力因子。     

Identification and characterization of four proteases produced by Streptococcus suis

Streptococcus suis is an important worldwide swine pathogen. In this study, we investigated the production of proteases by S. suis serotype 2. Proteases were identified and characterized using chromogenic and fluorogenic assays and zymography. An Arg-aminopeptidase with a molecular mass of 55 kDa was found to be both cell-associated and extracellular. Cell-associated chymotrypsin-like and caseinase activities, belonging to the serine- and metalloprotease classes respectively, were also detected. Lastly, a dipeptidyl peptidase IV (DPP IV) with a molecular mass of 70 kDa was detected in both whole cells and culture supernatants of S. suis serotype 2. Arg-aminopeptidase, caseinase and DPP IV activities were detected in all strains of S. suis serotype 2 tested whereas the chymotrypsin-like activity was only detected in European virulent strains of serotype 2. The optimum pH for all four proteases was between 6 and 8, and the optimum temperature ranged from 25 to 42 degrees C. This is the first report on the production of proteases by S. suis. Further investigations will determine the possible contribution of these proteases in the pathogenicity of S. suis serotype 2.     

2型猪链球菌MocR家族转录调控因子SSU0562基因敲除突变体的构建及毒力分析

目的:构建2型猪链球菌强毒株05ZYH33中MocR家族转录调控因子SSU0562基因敲除的突变株,探索SSU0562基因缺失对细菌基本生物学特性和毒力的影响。方法:构建左右两侧为SSU0562基因上下游的同源序列,中间部分为壮观霉素抗性基因(Spcr)的基因敲除质粒,通过同源重组的方法筛选SSU0562基因敲除突变株Δ0562。对突变株与野生株的基本生物学特性进行系统的比较分析,并且将小鼠作为动物感染的模型来研究突变株的毒力。结果:组合PCR的分析及基因测序结果均表明Spcr完全取代了S.suis2中SSU0562基因位点,表明基因敲除突变体Δ0562构建成功,反转录PCR(RT-PCR)证实了突变株Δ0562中SSU0562基因在转录水平的缺失;在溶血活性、生长速率及对小鼠的致病力方面,突变株Δ0562与野生株05ZYH33相比均无显著差别,然而革兰染色实验显示突变株Δ0562的成链能力明显减弱。结论:猪链球菌强毒株05ZYH33的毒力并未因SSU0562基因的缺失而发生显著性改变,表明SSU0562基因并非猪链球菌的毒力决定因子,但很有可能参与猪链球菌成链能力的调控。     

2型猪链球菌新毒力因子PlcR的发现

<正>2型猪链球菌(SS2)是一种重要的人畜共患传染病病原体。SS2感染不仅可致猪急性败血症、脑膜炎、关节炎、心内膜炎及急性死亡,并且可通过伤口和呼吸道等传播途径,导致人的感染发病和死亡。1998年和2005年在我国江苏"苏中"地区和四川资阳等市县人群中曾先后两次暴发大规模SS2感染人的事件。人感染病例中出现了从未报道过的链球菌中毒性休克综合征(Streptococcal toxic shock syndrome,STSS),病死率高达80%以上,已成为重要的新发传染病病原体[1-4]。     

Studies on a murine model for evaluation of virulence of Streptococcus suis capsular type 2 isolates

Five different parameters, time of incubation of the culture, type of culture medium, inoculum, strain of inbred mice, and age of mice, were tested using the LD50 technique to standardize a murine model for the evaluation of the virulence of Streptococcus suis capsular type 2 isolates. A model using 28 day-old mice belonging to CF1 strain appeared to give the best results. The inoculum size was the parameter most influencing the 50% lethal dose obtained with mice. Inoculation with 1-ml volume of a bacterial suspension instead of 0.1 or 0.5 ml decreased the LD50. The standardized model was used to evaluate the virulence of some isolates of known pathogenicity for pigs. The minimum lethal dose was used in the model and it appeared that the virulence of Streptococcus suis capsular type 2 isolates can be measured from highly virulent to totally avirulent.     

Epithelial invasion and cell lysis by virulent strains of Streptococcus suis is enhanced by the presence of suilysin

Streptococcus suis is an important pathogen of pigs causing arthritis, pneumonia and meningitis and is an occupational disease of farmers and those in the meat industry. As with other streptococci, both virulent and avirulent strains of S. suis are frequently carried asymptomatically in the tonsillar crypts and nasal cavities. Little is known about the process by which virulent strains cross the mucosal epithelia to generate systemic disease and whether this process requires expression of specific bacterial virulence factors. Although putative virulence factors have been postulated, no specific role in the disease process has yet been demonstrated for these factors. This study is the first demonstration that virulent strains of S. suis both invade and lyse HEp-2 cells, a continuous laryngeal epithelial cell line, and that at least one bacterial virulence factor, suilysin, is involved in this process.     

猪链球菌2型烯醇化酶的分子克隆与免疫学特性

对新近测定的猪链球菌2型(S. suis 2) 05ZYH33全基因序列进行生物信息学分析, 并与相关家族蛋白进行同源性比较, 设计合成引物, PCR法扩增出约1.3 kb的烯醇化酶编码基因 (enolase, eno), 将其克隆入pMD-18T载体中, 进一步亚克隆入表达载体pET32a。将重组表达质粒pET32a::eno转化E. coli BL21 (DE3), 经IPTG诱导表达后, SDS-PAGE初步检测到分子量约为75kD的蛋白带。通过His-Tag亲和层析纯化, 获得融合蛋白His-ENO。Western-blot表明该表达产物具有免疫原性。基于ELISA进行的细胞定位实验证实了Enolase可以部分存在S. suis 2 05ZYH33细菌的表面。这提示了Enolase作为一种新发现的抗原对于引发猪链球菌相关疾病可能发挥着重要的作用。     

猪链球菌2型烯醇化酶的分子克隆与免疫学特性

对新近测定的猪链球菌2型(S.suis 2)05ZYH33全基因序列进行生物信息学分析,并与相关家族蛋白进行同源性比较,设计合成引物,PCR法扩增出约1.3 kb的烯醇化酶编码基因(enolase,eno),将其克隆入pMD-18T载体中,进一步亚克隆入表达载体pET32a.将重组表达质粒pET32a::eno转化E.coli BL21(DE3),经IPTG诱导表达后,SDS-PAGE初步检测到分子量约为75kD的蛋白带.通过His-Tag亲和层析纯化,获得融合蛋白His-ENO.Western-blot表明该表达产物具有免疫原性.基于ELISA进行的细胞定位实验证实了Enolase可以部分存在S.suis 2 05ZYH33细菌的表面.这提示了Enolase作为一种新发现的抗原对于引发猪链球菌相关疾病可能发挥着重要的作用.     

2型猪链球菌假想转运蛋白89k/pTP与毒力的相关分析

目的：构建2型猪链球菌假想转运蛋白89k/pTP的基因插入失活突变体并进行毒力相关分析。方法：首先将同源臂片段克隆到pSET4s质粒上,构建插入失活载体pSET4s-pTP,并通过对单交换的筛选获得89k/pTP的基因插入失活突变体,再用小鼠腹腔感染模型对突变株和野生株的毒力进行比较。结果：获得了89k/pTP的基因插入失活突变体,并发现其毒力与野生型相比明显下降。结论：2型猪链球菌假想转运蛋白89k/pTP与其毒力有关,其作用和机制值得进一步分析。     

Allelic variation in srtAs of Streptococcus suis strains

Streptococcus suis NCTC10234 possesses five srtA homologs: srtA encodes sortase, which anchors surface proteins with an LPXTG motif to the cell wall, while the functions of the other four homologs (the srtBCD cluster and srtE) remain unknown. The genetic organization of the srtA region was found to be conserved in the 59 S. suis strains examined in this study. Although the srtAs in three of these strains showed strong sequence divergence, their functions were verified to be overlapping by genetic complementation, indicating the functional conservation of srtAs during the evolution of these strains. These results indicate the importance of an srtA-mediated cell wall sorting system for displaying proteins on the surface of S. suis.