抵抗女性生殖道沙眼衣原体感染的免疫新策略

沙眼衣原体是引起沙眼和泌尿生殖道感染的主要病原体。据世界卫生组织2015年统计,全球每年约有1.3亿沙眼衣原体感染新发病例。研究表明CD4^+Th1型细胞免疫应答在抵抗沙眼衣原体感染中发挥着重要作用。因此,研究者依照抗沙眼衣原体感染的免疫应答特点,构建出许多候选疫苗,但都没有成功地应用于临床。近年研究发现,生殖道黏膜组织不仅存在体液免疫和细胞免疫,还驻留着一些引人注目的免疫细胞,提示增强黏膜免疫可作为预防沙眼衣原体感染的潜在途径,是抵抗生殖道沙眼衣原体感染的免疫新策略。本文全面概述了黏膜免疫与女性生殖道沙眼衣原体感染的研究进展,并为今后研制沙眼衣原体疫苗提供一些建议。     

IgA antibodies to Chlamydia trachomatis and metabolic syndrome

Chlamydia pneumoniae may trigger atherogenesis. Chlamydia trachomatis (CT) can also induce endothelial activation. However, its role in metabolic syndrome (METS), a proatherogenic entity, has remained unexplored. In this study the frequencies of IgA and IgG anti-CT antibodies were evaluated by immunoenzymatic assay in METS patients and healthy controls. The survey included 238 individuals (148 with METS). The mean age was 59.7 years. IgA anti-CT antibodies were found significantly more frequently in METS patients (16.9%) than in controls (5.6%) (P= 0.015). The role of such IgA response in METS should be further investigated.     

Genomic factors related to tissue tropism in Chlamydia pneumoniae infection

Background

Chlamydia pneumoniae (Cpn) are obligate intracellular bacteria that cause acute infections of the upper and lower respiratory tract and have been implicated in chronic inflammatory diseases. Although of significant clinical relevance, complete genome sequences of only four clinical Cpn strains have been obtained. All of them were isolated from the respiratory tract and shared more than 99% sequence identity. Here we investigate genetic differences on the whole-genome level that are related to Cpn tissue tropism and pathogenicity.

Results

We have sequenced the genomes of 18 clinical isolates from different anatomical sites (e.g. lung, blood, coronary arteries) of diseased patients, and one animal isolate. In total 1,363 SNP loci and 184 InDels have been identified in the genomes of all clinical Cpn isolates. These are distributed throughout the whole chlamydial genome and enriched in highly variable regions. The genomes show clear evidence of recombination in at least one potential region but no phage insertions. The tyrP gene was always encoded as single copy in all vascular isolates. Phylogenetic reconstruction revealed distinct evolutionary lineages containing primarily non-respiratory Cpn isolates. In one of these, clinical isolates from coronary arteries and blood monocytes were closely grouped together. They could be distinguished from all other isolates by characteristic nsSNPs in genes involved in RB to EB transition, inclusion membrane formation, bacterial stress response and metabolism.

Conclusions

This study substantially expands the genomic data of Cpn and elucidates its evolutionary history. The translation of the observed Cpn genetic differences into biological functions and the prediction of novel pathogen-oriented diagnostic strategies have to be further explored.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1377-8) contains supplementary material, which is available to authorized users.     

Chlamydia pneumoniae binds to the lectin-like oxidized LDL receptor for infection of endothelial cells

The association of Chlamydia pneumoniae and atherosclerosis has been well documented. Recently, it has been demonstrated that C. pneumoniae up-regulates expression of the lectin-like ox-LDL receptor (LOX-1) in endothelial cells. Many of the pro-atherogenic effects of ox-LDL occur through its activation and uptake by LOX-1. This class E scavenger receptor contains a carbohydrate-recognition domain common to the C type lectin family. Previously, we have demonstrated that the major outer membrane protein of the chlamydiae is glycosylated and glycan removal abrogates infectivity of C. pneumoniae for endothelial cells. In this study, we investigated whether C. pneumoniae binds to LOX-1. The results show that 1) infection of endothelial cells by C. pneumoniae is inhibited by ligands that bind to the LOX-1 receptor, but not by ligands binding to other scavenger receptors; 2) anti-LOX-1 antibody inhibits C. pneumoniae infectivity, while antibodies against other scavenger receptors do not; 3) anti-LOX-1 antibody inhibits attachment of C. pneumoniae to endothelial cells; and 4) C. pneumoniae co-localizes with LOX-1. These effects were not observed for Chlamydia trachomatis. In conclusion, C. pneumoniae binds to the LOX-1 receptor, which is known to promote atherosclerosis.     

Productive Chlamydia trachomatis lymphogranuloma venereum 434 infection in cells with augmented or inactivated autophagic activities

Autophagy, a eukaryotic cellular activity leading to the degradation of cellular components, serves as a defense mechanism against facultative intracellular bacteria as well as a growth niche for the obligate intracellular bacterium Coxiella burnetii . We here demonstrate that the obligate intracellular bacterial pathogen Chlamydia trachomatis lymphogranuloma venereum strongly induced autophagy in the middle of the chlamydial developmental cycle (24 h after infection), a time point with maximal level of chlamydial replication, but not during the early stages with low overall chlamydial metabolism (before 8 h). No autophagy induction was evident in cells exposed to heat- and UV-inactivated elementary bodies (EBs, the infectious form of Chlamydia ) or to inocula from which EBs had been removed before inoculation. Blocking chlamydial development with chloramphenicol also prevented autophagy induction in cells infected with infectious EBs. It appears that autophagy is activated primarily in response to the metabolic stress consequent to chlamydial replication. However, autophagy-defective ATG5^−/− cells supported chlamydial development as efficiently as autophagy-proficient ATG5^+/+ cells.     

Endothelial Chlamydia pneumoniae infection promotes oxidation of LDL

The bacterium Chlamydia pneumoniae chronically infects atheromatous lesions and is linked to atherosclerosis by modifying inflammation, proliferation, and the lipid metabolism of blood monocytes. As continuous LDL modification in the vascular intima is crucial for atherogenesis we investigated the impact of endothelial infection on LDL oxidation. HUVEC were infected with a vascular C. pneumoniae strain. Supernatants of infected cells but not cell lysates increased lipid peroxidation products (6.44 vs 6.14 nmol/ml, p<0.05) as determined by thiobarbituric acid reacting substances assay. Moreover, supernatants rendered human LDL more susceptible to oxidation as shown in a copper-ion catalysed LDL oxidation assay by a 16% reduction of LDL resistance against pro-oxidative stimuli (p<0.05). Chlamydial infection of vascular endothelial cells releases acellular components that convert LDL to its proatherogenic form and reduce its resistance against oxidation. Foci of chronic endothelial chlamydial infection may thus continuously contribute to the dysregulated lipid metabolism that promotes atherogenesis.     

Differences in the envelope proteins ofChlamydia pneumoniae,Chlamydia trachomatis,andChlamydia psittaci shown by two-dimensional gel electrophoresis

Analysis by two-dimensional gel electrophoresis of theN-laurylsarkosinate(Sarkosyl)-insoluble envelope complexes ofl-^[35]S-cysteine-iabeled elementary bodies ofChlamydia pneumoniae strain IOL-207,Chlamydia trachomatis serovar LGV2, D, and F, andChlamydia psittaci strain 6BC showed differences in the molecular charges of chlamydial outer membrane proteins. The apparent isoelectric point (pI) of the major outer membrane protein ofC. pneumoniae strain IOL-207 was 6.4, whereas the pI of the major outer membrane protein of theC. trachomatis andC. psittaci strains differed little from one another, ranging from 5.3 to 5.5. The 60-kDa cysteinerich protein ofC. pneumoniae was the only 60-kDa chlamydial protein with a pI value (5.9) more acidic than that of the corresponding major outer membrane protein. As a general rule, the charges of both the 60-kDa and the lowmolecular-mass (12–15 kDa) cysteine-rich proteins were widely variable, depending on the strain. However, in cach individual strain, the variation of the charge of the 60-kDa protein had a compensatory change in the lowmolecular-mass cysteine-rich protein.     

Identification of an in vivo CD4+ T cell-mediated response to polymorphic membrane proteins of Chlamydia pneumoniae during experimental infection

Chlamydia pneumoniae is an obligate intracellular bacterium that causes upper and lower respiratory tract infection in humans. C. pneumoniae harbors the polymorphic membrane protein (Pmp) family with 21 different proteins with a molecular mass around 100 kDa. The Pmps are species-specific, abundant and, together with major outer membrane protein and outer membrane protein 2, the dominant proteins in the C. pneumoniae outer membrane complex. Nevertheless, it is unknown whether Pmps are recognized by the cell-mediated immune response. To address this issue, C57BL/6J mice were infected intranasally with C. pneumoniae and the immune response to primary infection was investigated. We demonstrate, as expected, that the primary response is of the Th1 type by IgG2a- and IgG1-specific sELISA (Medac) on serum. In vivo-primed spleen lymphocytes were found to be reactive to Pmp8, Pmp20 and Pmp21 in an interferon-gamma ELISpot assay. The responses were shown to be mediated by CD4(+) T cells. To our knowledge, this is the first identification of antigens recognized by CD4(+) T cells during murine C. pneumoniae infection.     

生殖道解脲支原体和沙眼衣原体的感染与输卵管妊娠关系探究

目的 探讨分析生殖道解脲支原体和沙眼衣原体的感染与输卵管妊娠的关系。方法 选取新疆医科大学第二附属医院2018年1月—6月妇科住院经腹腔镜手术证实输卵管妊娠并行患侧输卵管切除的患者作为研究组,同期腹腔镜下行妇科良性疾病输卵管或附件切除术者为对照组,分别采集两组宫颈管分泌物、输卵管组织进行解脲支原体培养以及沙眼衣原体检测,并作宫颈分泌物解脲支原体药敏试验。结果 输卵管妊娠组的宫颈分泌物及输卵管组织中解脲衣原体检出率（34.3%、30.2%）及沙眼衣原体的检出率（18.6%、16.2%）明显高于对照组宫颈分泌物及输卵管中解脲衣原体检出率（16.2%、6.9%）及沙眼衣原体的检出率（4.6%、2.3%）,对照组与实验组两组比较差异具有统计学意义（χ2=3.909、4.074、7.679、4.962,Ps＜0.05）。本研究22例宫颈解脲支原体感染中,解脲支原体对交沙霉素、强力霉素、美满霉素、阿奇霉素、克拉霉素的敏感率均高于80%。结论 输卵管妊娠与生殖道解脲支原体及沙眼衣原体感染有密切关系,临床工作中应尽早筛查和诊断,并根据药敏试验进行预防及合理治疗用药,减少输卵管妊娠的发生。     

Trifluoperazine inhibits the infectivity of Chlamydia trachomatis for McCoy cells

Abstract Trifluoperazine (TFP), an inhibitor of the Ca²⁺-binding protein calmodulin, was used to study the infectivity of Chlamydia trachomatis for McCoy cells. TFP inhibited the number of chlamydial inclusions and the chlamydia-dependent amino acid incorporation when added within 9 h after inoculation with chlamydiae. However, TFP did not affect the attachment of chlamydiae to the cells or the protease-removable fraction of cell-bound chlamydiae.
These results suggest that an early step in the intracellular development of chlamydiae, partly coinciding with the elementary body-reticulate body conversion, is sensitive to TFP and that clathrin coats are not crucial in the ingestion of chlamydiae by McCoy cells.     

Typing Chlamydia trachomatis: from egg yolk to nanotechnology

A historical review is provided of the various methods used for half a century to differentiate and type Chlamydia trachomatis strains. Typing of C. trachomatis is an important tool for revealing transmission patterns in sexual networks, and enabling association with clinical manifestations and pathogenicity. Serotyping using the major outer membrane protein (MOMP) has been the mainstay of epidemiological work for several decades. However, the development of nucleic acid amplification techniques (NAAT) and easy access to sequencing have shifted the focus from MOMP serotypes to omp1 genotypes. However, insufficient epidemiological resolution is achieved by characterization of both MOMP and omp1 . This calls for new high-resolution genotyping methods applying for example a multilocus variable number tandem repeat assay (MLVA) or multilocus sequence typing (MLST). The futuristic nanotechnology already seems at hand to further simplify and automate the high-resolution genotyping method based on NAAT and sequencing of various targets in the C. trachomatis genome. Thereby, a high throughput can be achieved and more epidemiological information can be obtained. However, it is important to realize that culture of C. trachomatis may still be needed to detect and characterize new variants of C. trachomatis .     

Mapping of transcripts encoded by the plasmid in Chlamydia trachomatis

Mapping of the plasmid-encoded RNA of the intracellular parasite, Chlamydia trachomatis revealed that the upstream control elements are different from those of other Gram-negative bacteria. A tetranucleotide, AYAA was found near the -10 position, in 5 out of 8 upstream sequences described so far. The plasmid also has a developmentally regulated promoter. The chlamydial upstream elements do not function as promoters in E. coli and vice versa. An E. coli promoter-like sequence has been found to occur fortuitously upstream from the plasmid-encoded dnaB gene. Such sequences may be evolutionary relics.     

Primary and secondary immune responses of mucosal and peripheral lymphocytes during Chlamydia trachomatis infection

Chlamydia trachomatis infection is followed by the development of antigen-specific cell-mediated immunity, which is detectable as a positive lymphocyte proliferation response to the chlamydial major outer membrane protein (MOMP) antigen. To date, however, there have been no studies on the mucosal immune responses to chlamydial antigens. This study aimed to study the primary and secondary immune responses of cervical lymphocytes in response to the chlamydial antigen. Median proliferative responses were found to be significantly (P<0.05) higher in patients with chlamydial infections than in controls. The chlamydial MOMP induced significantly higher IL-6 and IL-10 and lower interferon-gamma (IFN-gamma) secretion in cervical lymphocytes of Chlamydia-positive women, resulting in a T helper 2 response. On stimulation of peripheral blood mononuclear cells (PBMC) obtained from Chlamydia-positive women with the chlamydial antigen, the median levels of IL-10, IL-12 and IFN-gamma were higher than in controls, but the differences were not significant. Our study suggests that the mucosal immune responses towards Chlamydia trachomatis are different from those of PBMCs and are more helpful in understanding the cytokine responses in the female genital tract during chlamydial infection.     

肺炎支原体和沙眼衣原体致儿童急性下呼吸道感染的研究

本文旨在研究儿童社区获得性急性下呼吸道感染(ALRTI)中肺炎支原体(MP)和沙眼衣原体(CT)的感染特征。采用实时荧光定量聚合酶链反应(PCR)检测2006年10月～2008年2月因ALRTI收入复旦大学附属儿科医院的患儿呼吸道标本中MP和CT的DNA,并分析2种病原体感染患儿的临床特征与实验室检查结果。在1312份深部鼻咽分泌物标本中,MP和CT检出率分别为7.85%(103/1312)和2.97%(39/1312)。MP在5岁以上患儿中的检出率为33.33%(30/90),而CT在3个月以内患儿中的检出率为6.28%(31/494)。MP感染后易出现40℃以上高热,较少发生发绀与重症呼吸道感染;白细胞计数常无明显升高,但C反应蛋白升高较多见。CT感染后40℃以上高热和重症呼吸道感染少见,C反应蛋白升高也较少见。结果提示,在5岁以上儿童的社区获得性ALRTI中,MP是重要的病原体;而在3个月以内儿童中,CT为常见病原体之一。     

Chlamydia trachomatis pulmonary infection induces greater inflammatory pathology in immunoglobulin A deficient mice

Chlamydia trachomatis is an intracellular bacterial pathogen that primarily infects via mucosal surfaces. Using mice with a targeted disruption in IgA gene expression (IgA(-/-) mice), we have studied the contribution of IgA, the principal mucosal antibody isotype, in primary immune defenses against pulmonary C. trachomatis infection. Bacterial burden was comparable between IgA(-/-) and IgA(+/+) animals following C. trachomatis challenge. Serum and pulmonary anti-Chlamydia antibody levels were higher in IgA(-/-) animals, with the exception of IgA. Lung sections of challenged IgA(-/-) mice showed more extensive immunopathology than corresponding IgA(+/+) animals. Real-time PCR analysis demonstrated significantly greater IFN-gamma and TGF-beta mRNA expression in IgA(-/-) as compared to IgA(+/+) animals. Together, these results suggest that IgA may not be necessary for clearance of primary C. trachomatis infection. However, IgA(-/-) mice displayed exaggerated lung histopathology and altered cytokine production, indicating an important role for IgA in regulating C. trachomatis induced pulmonary inflammation and maintenance of mucosal homeostasis.     

Association of caveolin with Chlamydia trachomatis inclusions at early and late stages of infection

The mechanism by which the intracellular bacterial pathogen Chlamydia trachomatis enters eukaryotic cells is poorly understood. There are conflicting reports of entry occurring by clathrin-dependent and clathrin-independent processes. We report here that C. trachomatis serovar K enters HEp-2 and HeLa 229 epithelial cells and J-774A.1 mouse macrophage/monocyte cells via caveolin-containing sphingolipid and cholesterol-enriched raft microdomains in the host cell plasma membranes. First, filipin and nystatin, drugs that specifically disrupt raft function by cholesterol chelation, each impaired entry of C. trachomatis serovar K. In control experiments, filipin did not impair entry of the same organism by an antibody-mediated opsonic process, nor did it impair entry of BSA-coated microspheres. Second, the chlamydia-containing endocytic vesicles specifically reacted with antisera against the caveolae marker protein caveolin. These vesicles are known to become the inclusions in which parasite replication occurs. They avoid fusion with lysosomes and instead traffic to the Golgi region, where they intercept Golgi-derived vesicles that recycle sphingolipids and cholesterol to the plasma membrane. We also report that late-stage C. trachomatis inclusions continue to display high levels of caveolin, which they likely acquire from the exocytic Golgi vesicles. We suggest that the atypical raft-mediated entry process may have important consequences for the host-pathogen interaction well after entry has occurred. These consequences include enabling the chlamydial vesicle to avoid acidification and fusion with lysosomes, to traffic to the Golgi region, and to intercept sphingolipid-containing vesicles from the Golgi.     

Comparative PCR-based restriction fragment length polymorphism analysis of the plasmid gene orf3 of Chlamydia trachomatis and Chlamydia psittaci

The BfaI digestion of PCR-based restriction fragment length polymorphism analysis of the plasmid orf3 of Chlamydia trachomatis and Chlamydia psittaci provided evidence for two distinct restriction patterns, respectively. The nucleotide sequences of orf3 genes confirmed these differences. Serum antibodies against recombinant C. psittaci protein (pgp3) encoded by orf3 were detected both in pigeons with C. psittaci infection and in a human patient with psittacosis.     

沙眼衣原体包涵体膜蛋白CT225与宿主波形蛋白存在相互作用

【背景】衣原体独特的发育周期是在包涵体内完成的,大约7%–10%的基因编码包涵体膜蛋白,由此可见包涵体膜蛋白可能在其发育和致病过程中发挥重要作用。然而,其具体功能仍有待深入研究。【目的】筛选包涵体膜蛋白CT225的互作分子,以期进一步了解其可能的生物学功能。【方法】首先表达融合蛋白GST-CT225,用亲和层析法从HeLa细胞裂解液中筛选CT225的互作分子,所得蛋白进行质谱分析。确定候选蛋白,然后通过免疫共沉淀方法(Co-Immunoprecipitation,CO-IP)、谷胱甘肽巯基转移酶(Glutathione S-Transferase,GST)下拉/沉降实验和亚细胞定位等方法进行验证。【结果】参考质谱分析得分,通过实验初步验证得出波形蛋白(Vimentin,VIM)为与CT225相互作用的蛋白。【结论】CT225与HeLa细胞的波形蛋白Vimentin互相作用,提示其功能可能与维持细胞骨架完整性、膜运输和脂质转运等有关。