绿脓杆菌外毒素A的研究进展

<正>绿脓杆菌广泛存在于土壤、水及各种动物体内,是一种常见条件致病菌。绿脓杆菌导致的感染占革兰氏阴性感染的10%,常继发于肿瘤,大面积烧伤、面积创伤及免疫抑制剂使用者。感染类型多种多样,有急、慢性局部感染,也有全身性感染。因为感染绿脓杆菌的患者往往免疫功能低下、临床有效的抗生素种类少,又缺乏有效的疫苗等因素给临床治疗带来了很大困难。     

绿脓杆菌外毒素A的结构、功能及其重组毒素

绿脓杆菌外毒素A有三个结构功能区.氨基端区(Ⅰ)通过关键位点Lys57结合靶细胞表面受体.中心区(Ⅱ)负责该毒素的跨膜转位功能,Arg276和Arg279是关键位点.在胞吞泡内此毒素于Arg279与Gly280间酶解成28 000和37 000两片段.羧基端区(Ⅲ)所在的37 000片段由其末端氨基酸序列REDLK介导到内质网再转位入胞浆通过Glu553位点结合NAD⁺使延伸因子-2受ADP-核糖基化而抑制细胞蛋白质合成导致细胞死亡.改造的毒素基因同识别蛋白基因融合成的重组毒素有应用前景.     

PAK株与PA103株绿脓杆菌外毒素结构基因间的差异及其对功能的影响

对ＰＡＫ株的绿脓杆菌外毒素结构基因进行全长核苷酸顺序测定时发现：来源于ＰＡ１０３株和ＰＡＫ株的ＰＥ基因一级结构间存在差异,其中Ｔｈｒ~（１７９）（ＡＣＣ）→Ａｌａ~（１７９）（ＧＣＣ）、Ｓｅｒ~（５１５）（ＡＧＣ）→Ｇｌｙ~（５１５）（ＧＧＣ）,并有１１个同义突变,但变异并不影响白细胞介素２－绿脓杆菌外毒素融合蛋白的ＡＤＰ－核糖基化活性及其细胞毒活性。     

抗绿脓杆菌外毒素 A 血浆的研制

试验制备了抗绿脓杆菌外毒素－Ａ血浆,使用马匹免疫抗原为精制ＰＡ－１０３菌株的外毒素－Ａ。抗血浆效价第一程采血达到了２０８８单位／ｍｌ,第三程采血达到５１８６单位／ｍｌ。     

绿脓杆菌外毒素A的最新研究进展

在免疫缺失、囊肿性纤维化、烧伤烫伤等患者的临床继发感染中,绿脓杆菌是引起感染的主要病原菌之一,其合成的细胞外毒性物质—绿脓杆菌外毒素A(PEA)被认为是引起感染的最关键内在机制。该文对中外学者们在PEA的结构、功能、提取和纯化方法、重组毒素、基因工程疫苗以及应用等方面的研究进行归纳总结,并对研究和应用中存在的问题加以分析,对预防和治疗绿脓菌感染、抗肿瘤、抗移植排斥和治疗自身免疫性疾病等具有重要而深远的意义。     

应用Vero细胞法测定重组减毒绿脓杆菌外毒素A的残余毒力

绿脓杆菌外毒素A(ETA)有ADP -核糖基转移酶的活性 ,在真核细胞中通过催化ADP -核糖基团从NAD+转移到EF 2 (延长因子 2 ) ,使蛋白质合成终止 ,致细胞死亡 ,其作用机制、机理与白喉毒素相同。本文应用Vero细胞法测定了绿脓杆菌外毒素A的毒性 ,发现Vero细胞对绿脓杆菌外毒素A的敏感度高达 3.0× 10 -5mg/ml,且精密度高 ,准确性好 ,是一种有效、可行的绿脓杆菌外毒素毒力的测定方法。对 3批经基因工程减毒后样品的残余毒力进行了测定 ,发现样品基本无细胞毒性 ,毒力比原毒素低 2× 10 15倍     

绿脓杆菌抗毒素精制方法与效价测定方法的研究

本文采用胃酶消化──硫酸铵盐析法对绿脓杆菌外毒素Ａ（ＰＥＡ）免疫马血浆进行了试制提纯,对该法酶处理及热变性中影响精制效果的几个主要因素进行了比较试验,同时对文中采用的４种效价测定方法进行了筛选。结果表明,胃酶消化法可用于ＰＥＡ免疫马血浆的精制;效价测定以生物学方法（细胞毒性中和试验、小鼠致死毒性中和试验）为佳。综合比较试验的结果,本文拟订了ＰＥＡ免疫马血浆精制的“修订法”并与“常规法”进行了精制比较。初步结果表明,修订法的精制效果优于常规法。     

Interaction of Pseudomonas aeruginosa Exotoxin A with the human Sec61 complex suppresses passive calcium efflux from the endoplasmic reticulum

According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca²⁺-calmodulin. In single channel measurements, the open Sec61 complex is Ca²⁺ permeable. It can be closed not only by interaction with BiP or Ca²⁺-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca²⁺ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.     

Interaction of Pseudomonas aeruginosa Exotoxin A with the human Sec61 complex suppresses passive calcium efflux from the endoplasmic reticulum

According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca²⁺-calmodulin. In single channel measurements, the open Sec61 complex is Ca²⁺ permeable. It can be closed not only by interaction with BiP or Ca²⁺-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca²⁺ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.     

Characterization of Competitive Inhibitors for the Transferase Activity of Pseudomonas aeruginosa Exotoxin A

A series of small, nonpolar compounds were tested for their ability to inhibit the ADP-ribosyl transferase activity of Pseudomonas aeruginosa exotoxin A. The IC 50 values for the compounds tested ranged from 87 nM to 484 μM for NAP and CMP12, respectively. It was demonstrated that NAP was a competitive inhibitor of the ADPRT reaction for the NAD + substrate with a K i of 45 ± 5 nM, which was in good agreement with the dissociation constant determined independently (K D =56 ± 6 nM). The IC 50 value for NAP was 87 ± 12 nM, which strongly correlated with the K i and K D values. Furthermore, NAP was shown to noncovalently associate with the exotoxin A active site using exhaustive dialysis, NMR, and electrospray mass spectrometry. Finally, a computer molecular model using the X-ray structure of the substrate-bound toxin was generated with NAP bound to the active site of exotoxin A at the nicotinamide-binding site. This model is consistent with the X-ray structure of the catalytic domain of poly-ADP-ribose polymerase complexed with 4-amino-naphthalimide (Compound 4) that was included in this study.     

Analysis of immunization with DNA encoding Pseudomonas aeruginosa exotoxin A

The promising arena of DNA-based vaccines has led us to investigate possible candidates for immunization against bacterial pathogens. One such target is the opportunistic pathogen Pseudomonas aeruginosa which produces exotoxin A (PE), a well-characterized virulence factor encoded by the toxA gene. In its native protein form, PE is highly cytotoxic for susceptible eukaryotic cells through ADP-ribosylation of elongation factor-2 following internalization and processing of the toxin. To study the biologic and immunological effects of PE following in situ expression, we have constructed eukaryotic plasmid expression vectors containing either the wild-type or a mutated, non-cytotoxic toxA gene. In vitro analysis by transfection of UM449 cells suggests that expression of the wild-type toxA gene is lethal for transfected cells whereas transfection with a mutated toxA gene results in the production of inactive PE which can be readily detected by immunoblot analysis of cell lysates. To investigate the effects resulting from the intracellular expression of potentially cytotoxic gene products in DNA vaccine constructs, we immunized mice with both the wild-type and mutant toxA plasmid constructs and analyzed the resulting humoral and cellular immune responses. Immunization with the mutated toxA gene results in production of neutralizing antibodies against native PE and potentiates a T(H)1-type response, whereas only a minimal humoral response can be detected in mice immunized with wild-type toxA. DNA-based vaccination with the non-cytotoxic toxA(mut) gene confers complete protection against challenge with the wild-type PE. Therefore, genetic immunization with genes encoding potentially cytotoxic gene products raises concern with regard to the selection of feasible gene targets for DNA vaccine development.     

流感病毒M2蛋白胞外区与铜绿假单胞菌外毒素A融合蛋白的表达及其免疫原性研究

本研究构建了表达甲型流感病毒M2蛋白胞外区与铜绿假单胞菌外毒素A(PEA)融合蛋白的原核表达载体,根据铜绿假单胞菌外毒素A(PEA)核苷酸序列设计突变PCR引物并实施突变PCR,以获得PEA基因编码区第553位氨基酸密码子缺失的突变PEA(ntPE),从而产生无毒性的PEA突变基因,然后用合成的M2e编码区替换ntPE基因中的非必需区Ib,产生ntPE-M2e嵌合基因。将该嵌合基因导入pET表达载体以构建原核表达载体,将表达产物胶回收后与弗氏不完全佐剂联合皮下免疫BALB/c小鼠,终免两周后用5个LD50流感病毒A/PR/34/8株进行攻击。取动物血清作ELISA并取脾脏作ELISPOT试验结果表明,免疫组可以诱导小鼠产生抗M2e特异性抗体反应和细胞免疫反应并能够抑制病毒在肺内的复制。本研究为甲型流感病毒广谱疫苗的进一步研发打下了基础。     

GPR107, a G-protein-coupled Receptor Essential for Intoxication by Pseudomonas aeruginosa Exotoxin A,Localizes to the Golgi and Is Cleaved by Furin

A number of toxins, including exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation, and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan G-protein-coupled receptor. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin, and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport.