衣原体致病机制研究进展

衣原体是一类具有独特两相发育周期的专性胞内寄生菌,可引起人类多种疾病.衣原体致病主要依赖菌体脂多糖、膜蛋白、质粒编码蛋白、分泌的效应因子等,其或介导衣原体黏附和入侵宿主、引起炎症和病理损伤,或调控宿主细胞功能以利衣原体胞内生长,或抑制宿主免疫细胞以便衣原体逃逸免疫杀伤.这些发现在分子水平上阐述了衣原体的致病机制,为更好...     

衣原体Ⅲ型分泌系统效应蛋白研究进展

衣原体（Chlamydia）是一类进化来源尚未明确、发育周期独特、感染宿主广泛、致病表现复杂、介于细菌和病毒之间的革兰染色阴性微生物。当有传染性无代谢活性的原体（Elementary body,EB）粘附并进入真核宿主细胞后,分化为没有传染性但有代谢活性的网状体（Reticulate body,RB）,在寄生空泡内通过二分裂复制几代后,成为包涵体。网状体生长增殖持续18-36h后又可重新分化回原体。衣原体和很多病原体一样,把分泌各种毒力蛋白作为它们的致病机制,分泌的毒力蛋白称为效应蛋白。     

衣原体T3SS效应蛋白的研究进展

衣原体是一种专性细胞内寄生的革兰氏阴性菌,可在广泛的宿主中引起疾病。衣原体Ⅲ型分泌系统效应蛋白是指衣原体通过Ⅲ型分泌系统(TypeⅢsecretion system,T3SS)分泌的,与其存活及毒力相关的蛋白质。衣原体T3SS效应蛋白对于调控宿主细胞功能具有重要作用,如诱导肌动蛋白细胞骨架的重组,改变宿主细胞信号传导机制和抑制宿主细胞凋亡等。因此,研究衣原体T3SS效应蛋白对研究衣原体的致病机制以及研发衣原体疫苗具有重要意义。     

弓形虫棒状体蛋白与宿主细胞互作的研究进展

棒状体蛋白是弓形虫分泌于宿主细胞的主要效应因子, 定位于宿主细胞的不同部位, 并与其存在复杂的相互作 用。通过调控膜系及骨架结构, 该蛋白可影响宿主细胞的生物活性因子, 从而阻断其内在的防御机制, 使弓形虫成功实 现胞内入侵、 寄生及增殖。棒状体蛋白对宿主细胞的功能及作用方式反映了弓形虫的致病机制, 对探寻治疗弓形虫病的 药物靶点及安全有效的疫苗候选分子意义重大。本文就近年来弓形虫棒状体蛋白与宿主细胞间相互作用的研究进展进 行综述。     

沙眼衣原体III型分泌系统效应蛋白研究进展

沙眼衣原体(Chlamydia trachomatis,Ct)是专性细胞内致病菌,主要通过III型分泌系统(type III secretion system,T3SS)直接将效应蛋白分泌到宿主细胞。T3SS效应蛋白在Ct发育以及与宿主细胞相互作用中发挥重要作用,如调节宿主细胞肌动蛋白骨架、破坏免疫信号通路以及控制宿主细胞凋亡等。本文综述了沙眼衣原体 T3SS效应蛋白最新研究进展。     

肺炎衣原体假定蛋白Cpn0146的定位及其生物信息学分析

目的：对肺炎衣原体假定蛋白Cpn0146进行定位与生物信息学分析。方法：使用PCR方法从肺炎衣原体菌株AR39基因组中扩增Cpn0146,并将其插入到pGEX-6P2构建表达质粒pGEX-6p2-Cpn0146并转化大肠杆菌XL-Blue,IPTG诱导其表达融合蛋白GST-Cpn0146。纯化后的融合蛋白免疫小鼠制备特异性抗体,并应用间接免疫荧光法对肺炎衣原体的Cpn0146进行定位分析,最后利用软件Expasy和antheprot5.0对Cpn0146进行生物信息学分析。结果：成功构建了表达载体pGEX-6p2-Cpn0146和制备了抗Cpn0146的多克隆抗体。间接免疫荧光实验显示Cpn0146位于肺炎衣原体的包涵体膜上。生物信息学分析显示Cpn0146具有较好的亲水性与抗原性。结论：肺炎衣原体假定蛋白Cpn0146为一包涵体膜蛋白,其抗原性和亲水性较好。     

肺炎衣原体Cpn0147基因的克隆、表达与定位及其重组蛋白的生物信息学分析

目的重组表达肺炎衣原体Cpn0147基因,并对表达的内源性蛋白进行细胞内定位,对重组蛋白进行生物信息学分析。方法使用PCR方法从肺炎衣原体菌株AR39基因组中扩增第0147段开放读码区基因,使用限制性内切酶BamHⅠ和NotⅠ酶切目的片段和载体PGEX-6P2,T4连接酶连接后转化入大肠埃希菌感受态XL-Blue,并诱导表达融合蛋白GST-Cpn0147。用融合蛋白免疫小鼠,制备抗体,应用IFA方法对衣原体感染细胞内Cpn0147基因表达的内源性蛋白进行定位。利用软件Expasy对重组蛋白进行生物信息学分析。结果克隆出肺炎衣原体基因Cpn0147,全长450 bp,编码蛋白分子质量单位为14.727 ku,表达的融合蛋白GST-Cpn0147分子质量单位约为43 ku;用制备的抗体做IFA,显示现该蛋白定位于肺炎衣原体包涵体膜上。生物信息学分析该蛋白含2个抗原决定簇,具有良好的抗原性和亲水性。结论肺炎衣原体Cpn0147基因编码蛋白为一包涵体膜蛋白,抗原性和亲水性较好。     

弓形虫微线体蛋白MIC3的研究进展

弓形虫（Toxoplasma gondii）的微线体（mi-croneme）散布于虫体前端棒状体周围,是一种具有分泌功能的细胞器,其分泌的微线体蛋白（mi-croneme proteins MICs）与虫体对宿主细胞的识别和结合密切相关,在虫体入侵宿主细胞早期发挥重要作用。     

弓形虫棒状体蛋白ROP16的研究进展

棒状体蛋白16（ROP16）是弓形虫棒状体蛋白家族成员, 其蛋白结构存在丝氨酸、 苏氨酸激酶区, 是弓形虫入侵 过程中的重要毒力因子。ROP16可分泌到宿主细胞核, 能磷酸化宿主细胞的转录活化因子STAT3/6, 干扰宿主细胞信号 通路传导, 在弓形虫入侵宿主细胞过程中发挥着重要作用。本文对弓形虫ROP16的发现、 功能、 免疫保护性等方面进行 综述。     

寄生虫分泌/膜蛋白的生物信息学特征及研究策略

分泌/膜蛋白足寄生虫与宿主相互作用的主要分子,由于其直接暴露于宿主免疫系统及体内药物,因此是寄生虫诊断试剂、疫苗及药物作用靶标主要的候选分子,对其系统深入的研究有助于阐明寄生虫与宿主相瓦作用的分子机制.该文概述了寄生虫分泌/膜蛋白的牛物信息学特征及研究策略,并用生物信息学方法对部分寄生虫一些重婴的诊断及疫苗候选分子进行了分泌/膜篮白特征的分析.     

Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole

The acquisition of host-derived lipids is essential for the pathogenesis of the obligate intracellular bacteria Chlamydia trachomatis. Current models of chlamydial lipid acquisition center on the fusion of Golgi-derived exocytic vesicles and endosomal multivesicular bodies with the bacteria-containing parasitophorous vacuole ("inclusion"). In this study, we describe a mechanism of lipid acquisition and organelle subversion by C. trachomatis. We show by live cell fluorescence microscopy and electron microscopy that lipid droplets (LDs), neutral lipid storage organelles, are translocated from the host cytoplasm into the inclusion lumen. LDs dock at the surface of the inclusion, penetrate the inclusion membrane and intimately associate with reticulate Bodies, the replicative form of Chlamydia. The inclusion membrane protein IncA, but not other inclusion membrane proteins, cofractionated with LDs and accumulated in the inclusion lumen. Therefore, we postulate that the translocation of LDs may occur at IncA-enriched subdomains of the inclusion membrane. Finally, the chlamydial protein Lda3 may participate in the cooption of these organelles by linking cytoplasmic LDs to inclusion membranes and promoting the removal of the LD protective coat protein, adipocyte differentiation related protein (ADRP). The wholesale transport of LDs into the lumen of a parasitophorous vacuole represents a unique mechanism of organelle sequestration and subversion by a bacterial pathogen.     

CD8+ T cells recognize an inclusion membrane-associated protein from the vacuolar pathogen Chlamydia trachomatis

During infection with Chlamydia trachomatis, CD8(+) T cells are primed, even though the bacteria remain confined to a host cell vacuole throughout their developmental cycle. Because CD8(+) T cells recognize antigens processed from cytosolic proteins, the Chlamydia antigens recognized by these CD8(+) T cells very likely have access to the host cell cytoplasm during infection. The identity of these C. trachomatis proteins has remained elusive, even though their localization suggests they may play important roles in the biology of the organism. Here we use a retroviral expression system to identify Cap1, a 31-kDa protein from C. trachomatis recognized by protective CD8(+) T cells. Cap1 contains no strong homology to any known protein. Immunofluorescence microscopy by using Cap1-specific antibody demonstrates that this protein is localized to the vacuolar membrane. Cap1 is virtually identical among the human C. trachomatis serovars, suggesting that a vaccine incorporating Cap1 might enable the vaccine to protect against all C. trachomatis serovars. The identification of proteins such as Cap1 that associate with the inclusion membrane will be required to fully understand the interaction of C. trachomatis with its host cell.     

A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis

The intracellular pathogen Chlamydia trachomatis possesses a type III secretion (TTS) system believed to deliver a series of effector proteins into the inclusion membrane (Inc-proteins) as well as into the host cytosol with perceived consequences for the pathogenicity of this common venereal pathogen. Recently, small molecules were shown to block the TTS system of Yersinia pseudotuberculosis. Here, we show that one of these compounds, INP0400, inhibits intracellular replication and infectivity of C. trachomatis at micromolar concentrations resulting in small inclusion bodies frequently containing only one or a few reticulate bodies (RBs). INP0400, at high concentration, given at the time of infection, partially blocked entry of elementary bodies into host cells. Early treatment inhibited the localization of the mammalian protein 14-3-3beta to the inclusions, indicative of absence of the early induced TTS effector IncG from the inclusion membrane. Treatment with INP0400 during chlamydial mid-cycle prevented secretion of the TTS effector IncA and homotypic vesicular fusions mediated by this protein. INP0400 given during the late phase resulted in the detachment of RBs from the inclusion membrane concomitant with an inhibition of RB to elementary body conversion causing a marked decrease in infectivity.     

Chlamydia-induced arthritis

PURPOSE OF REVIEW: Chlamydia-induced arthritis is the most frequent form of reactive arthritis in Western countries. This article gives an overview of the recent findings with respect to diagnosis, pathogenesis, and therapy of the disease. RECENT FINDINGS: Recent advances in the modification and standardization of polymerase chain reaction techniques give promise to identify Chlamydia more frequently from joint samples. Based on the sequenced chlamydial genome, considerable progress has been achieved in the understanding of the Chlamydia-host cell interaction, indicating that persistence is an alternate state of the bacteria used by Chlamydia to escape the immune system of the host rather than a general stress response. Furthermore, Chlamydia has the ability to reprogram the host cell by chlamydial effector proteins, which are transported from the inclusion into the host cell cytoplasm. The role of HLA-B27 is discussed in view of the pathogenesis of the disease. HLA-B27 should be considered a risk factor for chronic and/or axial disease rather than a true susceptibility factor for the development of Chlamydia-induced arthritis. No progress has been made in terms of causative therapy aiming at eradication of the bacteria. Tumor necrosis factor-alpha blocking agents may represent a new option in cases that are refractory to therapy. SUMMARY: Molecular biology not only has improved the ability to detect Chlamydia in the joint for diagnostic purposes but also has extended the current understanding of the pathogenesis of the disease. In contrast to this progress, causative therapy of Chlamydia-induced arthritis is still an unfulfilled need.     

Golgi-dependent transport of cholesterol to the Chlamydia trachomatis inclusion

Cholesterol, a lipid not normally found in prokaryotes, was identified in purified Chlamydia trachomatis elementary bodies and in the chlamydial parasitophorous vacuole (inclusion) membrane of infected HeLa cells. Chlamydiae obtained eukaryotic host cell cholesterol both from de novo synthesis or low-density lipoprotein. Acquisition of either de novo-synthesized cholesterol or low-density lipoprotein-derived cholesterol was microtubule-dependent and brefeldin A-sensitive, indicating a requirement for the Golgi apparatus. Transport also required chlamydial protein synthesis, indicative of a pathogen-directed process. The cholesterol trafficking pathway appears to coincide with a previously characterized delivery of sphingomyelin to the inclusion in that similar pharmacological treatments inhibited transport of both sphingomyelin and cholesterol. These results support the hypothesis that sphingomyelin and cholesterol may be cotransported via a Golgi-dependent pathway and that the chlamydial inclusion receives cholesterol preferentially from a brefeldin A-sensitive pathway of cholesterol trafficking from the Golgi apparatus to the plasma membrane.     

Mechanisms of host cell exit by the intracellular bacterium Chlamydia

The mechanisms that mediate the release of intracellular bacteria from cells are poorly understood, particularly for those that live within a cellular vacuole. The release pathway of the obligate intracellular bacterium Chlamydia from cells is unknown. Using a GFP-based approach to visualize chlamydial inclusions within cells by live fluorescence videomicroscopy, we identified that Chlamydia release occurred by two mutually exclusive pathways. The first, lysis, consisted of an ordered sequence of membrane permeabilizations: inclusion, nucleus and plasma membrane rupture. Treatment with protease inhibitors abolished inclusion lysis. Intracellular calcium signaling was shown to be important for plasma membrane breakdown. The second release pathway was a packaged release mechanism, called extrusion. This slow process resulted in a pinching of the inclusion, protrusion out of the cell within a cell membrane compartment, and ultimately detachment from the cell. Treatment of Chlamydia-infected cells with specific pharmacological inhibitors of cellular factors demonstrated that extrusion required actin polymerization, neuronal Wiskott-Aldrich syndrome protein, myosin II and Rho GTPase. The participation of Rho was unique in that it functioned late in extrusion. The dual nature of release characterized for Chlamydia has not been observed as a strategy for intracellular bacteria.     

Lipooligosaccharide is required for the generation of infectious elementary bodies in Chlamydia trachomatis

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are the main lipid components of bacterial outer membranes and are essential for cell viability in most Gram-negative bacteria. Here we show that small molecule inhibitors of LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase], the enzyme that catalyzes the first committed step in the biosynthesis of lipid A, block the synthesis of LOS in the obligate intracellular bacterial pathogen Chlamydia trachomatis. In the absence of LOS, Chlamydia remains viable and establishes a pathogenic vacuole ("inclusion") that supports robust bacterial replication. However, bacteria grown under these conditions were no longer infectious. In the presence of LpxC inhibitors, replicative reticulate bodies accumulated in enlarged inclusions but failed to express selected late-stage proteins and transition to elementary bodies, a Chlamydia developmental form that is required for invasion of mammalian cells. These findings suggest the presence of an outer membrane quality control system that regulates Chlamydia developmental transition to infectious elementary bodies and highlights the potential application of LpxC inhibitors as unique class of antichlamydial agents.