猪链球菌2型对扁桃腺上皮细胞的黏附和侵袭作用

猪链球菌2型(SS2)是重要的人畜共患病病原体,溶菌酶释放蛋白（MRP）是SS2的主要毒力因子之一。用天然表达MRP的江苏分离株HA9801和不表达MRP的上海分离株SH006444,研究SS2对仔兔扁桃腺上皮细胞的黏附和侵袭作用。黏附计数结果表明,HA9801（MRP+）和SH006444（MRP-）均能对扁桃腺上皮细胞高水平黏附。扫描和透射电镜均观察到HA9801的高水平黏附现象,黏附部位是细胞膜和细胞微绒毛,并观察到细胞膜上有链球菌正处于内化过程中。裂解记数结果表明,HA9801有低度侵袭力,SH006444未检测到侵袭力。结果提示,扁桃腺是SS2的定殖器官和感染门户;MRP＋菌株黏附后直接侵入细胞内是其穿过扁桃腺上皮细胞屏障的机制之一。     

猪链球菌2型疫苗研究进展

猪链球菌2型可引起人、猪急性败血型、脑膜炎型和关节炎型等传染病,致死率极高,是近年来致病性最强、危害性最大的猪链球菌类型之一。目前因该菌的致病机理不清、抗原类型复杂,限制了疫苗研究的顺利进行。当前在研的猪链球菌2型疫苗类型有死疫苗、弱毒苗、蛋白亚单位疫苗等。     

猪链球菌2型感染实验动物模型的研究进展

猪链球菌为革兰阳性兼性厌氧性球菌,感染后可引起猪的关节炎、脑膜炎、肺炎、败血症甚至死亡,严重影响着世界各国的生猪养殖业,导致极大的经济损失。近年来,人感染猪链球菌的病例时有报道,并于1998年和2005年在我国引起两次大规模流行,危害人类健康。建立和选择理想的动物模型,对于研究猪链球菌的致病机理、与宿主间的相互作用及研发防治猪链球菌感染的新药、疫苗及诊断试剂都有着十分重要的意义。本文就已建立的各种猪链球菌感染动物模型展开综述,希望能帮助人们根据不同的试验目的与研究方向选择合适的动物模型。     

寨卡病毒及其与宿主蛋白相互作用研究进展

寨卡病毒(Zika virus,ZIKV)作为一种新出现的蚊媒病原体,与成人格林-巴利综合征(Guillain–Barré syndrome,GBS)、新生儿小头畸形以及神经系统缺陷等疾病有关。2016年世界卫生组织将寨卡疫情列为国际关注的公共卫生突发事件,近年来关于ZIKV病原学、流行病学、感染与致病、抗病毒免疫应答、疫苗与药物的研究越来越多,为有效防控该病提供了理论依据和具体实践。本文就ZIKV蛋白组成及相应功能、感染周期、ZIKV受体及ZIKV-宿主相互作用等的最新研究进展进行综述,旨在正确认识和深入理解ZIKV致病与机体抗病相关机制,为设计有效的预防或治疗疫苗或药物提供参考资料。     

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。     

杆状病毒与昆虫宿主相互作用的研究进展

杆状病毒与昆虫宿主相互作用是一种基本的分子和生态问题, 不仅在农业上, 而且在真核表达系统、 基因治疗、 蛋白表面展示 系统以及基因工程疫苗等方面都有重要的实际应用。杆状病毒还是一种很有潜力的病毒杀虫剂, 而且对环境来说是安全的。研究这些相互 作用也产生了许多重要和有价值的发现。杆状病毒生命循环中存在两种不同形式的病毒, 即包埋型病毒粒子(occlusion derived virus, ODV) 和出芽型病毒粒子(budded virus, BV)。ODV包裹于多角体中, 主要负责宿主的原发感染; 而BV由感染的宿主细胞释放后引发继发 感染。病毒侵染起始于敏感的昆虫宿主食用了污染包涵体病毒的植物。在宿主中肠的碱性环境中, 多角体溶解释放ODV, ODV与宿主肠道 柱状上皮细胞细胞膜融合, 通过内吞体进入细胞。之后核衣壳从内吞体中逃脱并被转运到细胞核。病毒转录和复制在细胞核进行, 新生 的BV粒子从基底膜出芽引起全身感染。杆状病毒与宿主细胞相互作用包括从病毒结合和进入时的相互作用, 到宿主基因表达调节, 以及 修饰与调节细胞和机体所发生的生理和防御的相互作用的复杂和微妙的机制。本文主要以杆状病毒侵染昆虫宿主的过程为线索, 总结和评 述了杆状病毒与昆虫宿主相互作用方面研究的最新进展, 特别是杆状病毒基因在病毒入侵过程中所起的作用。     

猪链球菌2型基因工程疫苗研究进展

猪链球菌2型引起的链球菌病是一种重要的人兽共患传染病。目前对于猪链球菌病的预防主要依靠灭活疫苗,而灭活疫苗对于同源菌的攻击保护率仅在70%左右,对异源菌的攻击保护率更低。目前对于猪链球菌2型基因工程疫苗的研究主要有两个方面,一是对某些毒力因子缺失的基因缺失疫苗的研究,由于尚未发现猪链球菌2型的标志性毒力因子,因此这方面的研究尚不太多;二是对基因重组亚单位疫苗的研究。随着分子生物学技术的发展,许多学者对猪链球菌2型的具有免疫原性的一些蛋白片段进行分析、重组、表达,以期制造出可以对猪链球2型进行有效预防的基因工程疫苗。由于猪链球菌2型的毒力因子复杂,对其毒力因子及其基因组成的研究尚不彻底,随着研究的深入,如在基因工程疫苗方面取得突破性进展,对于猪链球菌2型引起的链球菌病的预防与控制必将有着重要意义。     

猪链球菌2型Orf207基因缺失菌株的构建及其生物学特性检测

【背景】猪链球菌2型(streptococcus suis type 2, SS2)可引起人、猪的脑膜炎、关节炎及败血症等,不仅给养猪业带来巨大的经济损失,同时严重威胁公共卫生安全。本团队前期通过噬菌体展示文库技术发现Orf207编码蛋白可能参与SS2诱导的脑膜炎发生,然而其在SS2致病过程中的具体作用尚不清楚。【目的】探究Orf207基因对SS2致病性的影响。【方法】采用温敏性自杀质粒介导的同源重组系统,构建SC19 Orf207基因缺失菌株ΔOrf207及其回补菌株CΔOrf207,系统比较缺失菌株与野生株间在生长特性、形态、组织定殖能力、毒力情况、细胞黏附与侵袭及抗巨噬细胞吞噬能力等生物学特性方面的差异。【结果】与野生株相比,缺失菌株链长变短,生长速度略慢;而且Orf207缺失显著增加了小鼠的存活率,降低了细菌在血液、心脏、肝脏、脾脏、肺脏、肾脏、脑组织的定殖能力和对肺组织的病理损伤并显著减弱SS2对HeLa细胞的黏附与侵袭能力及抗巨噬细胞吞噬能力。【结论】Orf207基因可以显著降低SS2对宿主的致病能力,本研究结果不仅丰富了SS2的致病机制,也为SS2疫苗等研发提供了新靶点。     

衣原体与宿主细胞相互作用研究进展

衣原体是专性细胞内寄生、有独特发育周期的原核细胞型微生物,为了建立有益于病原体复制的细胞内环境,衣原体依赖于它们操纵宿主细胞内环境的能力,并且进化成了与宿主细胞相互作用的复杂机制,本文从感染衣原体后宿主细胞变化、免疫反应及蛋白质组改变等几个方面简要综述了衣原体与宿主细胞相互作用的最新进展,为进一步研究衣原体致病机制提供参考。     

2型猪链球菌疫苗候选分子的研究进展

猪链球菌是一种全球性严重人兽共患病病原体,因为缺乏有效疫苗,使感染难以控制。目前疫苗研究主要集中在血清2型,因其流行范围最广。猪链球菌疫苗研究的方法包括构建基因表达文库、免疫蛋白质组学方法、反向疫苗学方法和其它传统方法。本文对目前为止所识别和评价的猪链球菌2型疫苗候选分子进行综述,包括全菌疫苗、英膜多糖、蛋白抗原。其中很多疫苗候选分子对小鼠或者猪有保护效果,而要获得针对更多血清型的通用疫苗则需要更多努力。     

致病性猪链球菌2型溶血素基因的克隆与表达

目的：克隆溶血素基因,为评价溶血素的毒力与抗原活性,构建猪链球菌疫苗提供依据。方法：从致病性猪链球菌II型四川资阳分离株基因组中分离溶血素基因,经中间T载体,将其克隆至改造的pGEX-6p1中,最后通过蛋白质电泳和溶血实验检验溶血素的表达及活性。结果：SDS-PAGE结果表明,重组溶血素在5h表达水平最高,10h次之,15h最弱;溶血实验重组菌周围形成明显的透明溶血环。结论：溶血素为分泌性早期表达,具有正常的β溶血活性。     

Two Spx Regulators Modulate Stress Tolerance and Virulence in Streptococcus suis Serotype 2

Streptococcus suis serotype 2 is an important zoonotic pathogen causing severe infections in pigs and humans. The pathogenesis of S. suis 2 infections, however, is still poorly understood. Spx proteins are a group of global regulators involved in stress tolerance and virulence. In this study, we characterized two orthologs of the Spx regulator, SpxA1 and SpxA2 in S. suis 2. Two mutant strains (ΔspxA1 and ΔspxA2) lacking the spx genes were constructed. The ΔspxA1 and ΔspxA2 mutants displayed different phenotypes. ΔspxA1 exhibited impaired growth in the presence of hydrogen peroxide, while ΔspxA2 exhibited impaired growth in the presence of SDS and NaCl. Both mutants were defective in medium lacking newborn bovine serum. Using a murine infection model, we demonstrated that the abilities of the mutant strains to colonize the tissues were significantly reduced compared to that of the wild-type strain. The mutant strains also showed a decreased level of survival in pig blood. Microarray analysis revealed a global regulatory role for SpxA1 and SpxA2. Furthermore, we demonstrated for the first time that Spx is involved in triggering the host inflammatory response. Collectively, our data suggest that SpxA1 and SpxA2 are global regulators that are implicated in stress tolerance and virulence in S. suis 2.     

Genome Sequence of the Swine Pathogen Streptococcus suis Serotype 2 Strain S735

Streptococcus suis is a major swine pathogen responsible for significant, worldwide economic losses in the swine industry, in addition to being an emerging zoonotic agent. Strains of serotype 2 are the most commonly associated with infections causing meningitis, endocarditis, and septicemia. Here we present the genome sequence of S. suis serotype 2 strain S735.     

Sequence Analysis of a Small Cryptic Plasmid Isolated from Streptococcus suis Serotype 2

A small cryptic plasmid designated pSSU1 was isolated from Streptococcus suis serotype 2 strain DAT1. The complete sequence of pSSU1 was 4975 bp and contained six major open reading frames (ORFs). ORF1 and ORF2 encode for proteins highly homologous to CopG and RepB of the pMV158 family, respectively. ORF5 encodes for a protein highly homologous to Mob of pMV158. ORF4 encodes for a protein highly homologous to orf3 of pVA380-1 of S. ferus, but its function is unknown. There was no similarity between ORF3 and ORF6 and other protein sequences. In this plasmid, the ORF1 (CopG protein) was preceded by two multiples of direct repeat and the conserved nucleotides that could be the double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The ORF5 (Mob protein) was followed by a potential hairpin loop that could be the single-strand origin (SSO) of RCR mechanism. The sequence, which was complementary to the leader region of Rep mRNA, was homologous to the countertranscribed RNA (ctRNA) of pLS1. Moreover, a 5-amino acid conserved sequence was found in C terminal of Rep and putative Rep proteins of several pMV158 family plasmids. These observations suggest that this plasmid replicates by use of the rolling circle mechanism. Received: 26 June 1999 / Accepted: 10 August 1999     

Characterization and Proteome Analysis of Inosine 5-Monophosphate Dehydrogenase in Epidemic Streptococcus suis Serotype 2

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes severe disease symptoms in pigs and humans. In the present study, we found one isogenic mutant lacking inosine 5-monophosphate dehydrogenase (IMPDH) ΔZY05719 was attenuated in pigs compared with the wild-type SS2 strain ZY05719. Comparative proteome analysis of the secreted proteins expression profiles between ZY05719 and ΔZY05719 allowed us to identify Triosephosphate isomerase (TPI) and glyceraldehyde phosphate dehydrogenase (GAPDH), which were down expressed in the absence of the IMPDH. Both of them are glycolytic enzymes participating in the glycolytic pathway. Compared with ZY05719, ΔZY05719 lost the ability of utilize mannose, which might relate to down expression of TPI and GAPDH. In addition, GAPDH is a well-known factor that involved in adhesion to host cells, and we demonstrated ability of adhesion to HEp-2 and PK15 by ΔZY05719 was significantly weakened, in contrast to ZY05719. The adhesion to host cells is the crucial step to cause infection for pathogen, and the reduction adhesion of ΔZY05719, to some extent illustrates the attenuated virulence of ΔZY05719.     

猪链球菌2型菌毛骨架蛋白编码基因SSU2101敲除突变株的构建及其生物学功能

利用同源重组基因敲除方法构建猪链球菌2型(Streptococcus suis serotype2,S.suis2)中国强毒株05ZYH33菌毛骨架蛋白(Backbone protein,BP)编码基因SSU2101敲除突变株。采用引物特异性PCR分析、Southern杂交及RT-PCR等方法鉴定,证实成功构建了BP基因缺失突变株。生物学特性显示,突变株的菌落形态、溶血活性以及染色特性方面与野生株之间均无明显差异。小鼠致病性试验结果显示,突变株的毒力比野生株显著减弱。研究结果提示菌毛在S.suis2感染致病过程中起重要作用,为系统研究S.suis2菌毛分子装配机制及其生物学功能奠定了基础。     

Characterization of the Pivotal Carbon Metabolism of Streptococcus suis Serotype 2 under ex Vivo and Chemically Defined in Vitro Conditions by Isotopologue Profiling

Streptococcus suis is a neglected zoonotic pathogen that has to adapt to the nutritional requirements in the different host niches encountered during infection and establishment of invasive diseases. To dissect the central metabolic activity of S. suis under different conditions of nutrient availability, we performed labeling experiments starting from [¹³C]glucose specimens and analyzed the resulting isotopologue patterns in amino acids of S. suis grown under in vitro and ex vivo conditions. In combination with classical growth experiments, we found that S. suis is auxotrophic for Arg, Gln/Glu, His, Leu, and Trp in chemically defined medium. De novo biosynthesis was shown for Ala, Asp, Ser, and Thr at high rates and for Gly, Lys, Phe, Tyr, and Val at moderate or low rates, respectively. Glucose degradation occurred mainly by glycolysis and to a minor extent by the pentose phosphate pathway. Furthermore, the exclusive formation of oxaloacetate by phosphoenolpyruvate (PEP) carboxylation became evident from the patterns in de novo synthesized amino acids. Labeling experiments with S. suis grown ex vivo in blood or cerebrospinal fluid reflected the metabolic adaptation to these host niches with different nutrient availability; however, similar key metabolic activities were identified under these conditions. This points at the robustness of the core metabolic pathways in S. suis during the infection process. The crucial role of PEP carboxylation for growth of S. suis in the host was supported by experiments with a PEP carboxylase-deficient mutant strain in blood and cerebrospinal fluid.