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.     

Association and uptake studies of a Chlamydia trachomatis lymphogranuloma venereum strain by eukaryotic cells

We have studied association and uptake of Chlamydia trachomatis serovar L1 under various infection conditions. Chlamydiae attached to a greater extent to McCoy cells than to HeLa cells, although the number of inclusions formed in the latter was the same or higher. The amount of internalised chlamydiae was similar in the 2 cell types. Centrifugation of McCoy cell-attached chlamydiae did not affect the uptake of this serovar. However, if the inoculum was centrifuged to the cell surface and then incubated at 37°C, there was a pronounced increase in the relative amount of ingested chlamydiae in comparison with stationary infection. Chlamydiae were attached to and internalised insubconfluent McCoy cell monolayers as efficiently as in confluent layers. If the monolayers were sparse or very sparse, there was a great reduction of associated chlamydiae. Our results indicate that the host cell binding for chlamydiae vary with cell type, cell density, and mode of infection.     

Processing of McCoy cell cultures infected with Chlamydia trachomatis: sequential isolation of chlamydial elementary bodies and lipopolysaccharide

Abstract Triton X-100 (TX100) enhances the liberation of chlamydial elementary bodies (EB) from host cells and dissolves the host cell membrane. In the presence of TX100 only differential centrifugation is needed to isolate reasonably pure EBs. The remaining high-speed supernatant still contains a large part of the chlamydial lipopolysaccharide (LPS), which can be isolated with the standard phenol-chloroform-petroleum ether extraction.     

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.     

Reversibility of heat shock in Chlamydia trachomatis

The heat shock effect on chlamydia development was studied. We report here that the reversibility of the heat shock response did not depend on the stage of chlamydial morphogenesis at which transfer to high temperature occurred, and the infectivity of the particles produced was not affected significantly, so long as the heat shock exposure was not prolonged. Exposure to heat shock for more than 9 h resulted in stagnation of the growth cycle, appearance of aberrant reticulate body particles and loss of infectivity. SDS-PAGE analysis of proteins synthesized under prolonged heat shock showed increased relative abundance of heat shock proteins in common with other procaryotic organisms.     

Surface components of HeLa cells that inhibit cytadherence of Chlamydia trachomatis

Abstract Isolated HeLa plasma membrane (PM) preparations and extracts containing either cell-surface proteins or lipids were examined for inhibition of adherence of radiolabeled Chlamydia trachomatis serovar E elementary bodies to glutaraldehydefixed HeLa monolayers. A dose-dependent adherence-inhibitory activity could be demonstrated with the PM. A urea extract as well as lipids from HeLa cells also inhibited chlamydial cytadherence. The inhibitory activity of the PM was trypsin-sensitive. It was absent when the urea extract was prepared from trypsin-treated HeLa cells. The urea extract was subjected to electrophoresis and protein blotting using a native gel system. Probing with radiolabeled chlamydial cytadhesin showed a single protein present in the urea extract that could represent a HeLa cell protein receptor for the chlamydiae.     

简易的沙眼衣原体扩增及纯化方法的建立

目的建立扩增培养和纯化沙眼衣原体的简化方法.方法采用HeLa细胞培养扩增衣原体,并应用一步法将沙眼衣原体纯化回收.结果获得了具有高感染活性的沙眼衣原体.结论该方法与常规方法相比更简便、快速,同时减少污染机会、不需要超声和超速离心机等设备.     

青海藏区B型沙眼衣原体的分离培养与鉴定

【目的】对青海藏区沙眼患者标本进行沙眼衣原体分离培养与鉴定。【方法】分别采集患者的单眼结膜和结膜囊拭子标本至1 mL样本保护培养基中。取50μL样本采用离心法感染BGM细胞,37°C培养72 h,连续传代3次,相差显微镜观察衣原体包涵体。对临床样本和分离株分别进行主要外膜蛋白基因ompA序列分析。【结果】共采集了45例活动性沙眼患者的115份临床样本,其中54份样本为ompA PCR阳性,15份样本为沙眼衣原体培养阳性。ompA分析发现,青海藏区沙眼衣原体有3个不同的同源ompA变异株,均为基因B型,都包含有一个泌尿生殖道型沙眼衣原体特有密码子。分离株QH111L和QH111R分别来自编号111患者的左眼和右眼样本,它们ompA基因的可变区有一个非同义碱基差异。该碱基变异仅存在于111号患者的左眼样本中,说明QH111L可能是新出现的ompA突变体。【结论】青海藏区的眼型沙眼衣原体流行株为基因B型,至少存在3个不同的ompA变异株。从青海藏区分离培养了15株眼型沙眼衣原体,发现同一患者的左右眼样本中的沙眼衣原体有不同ompA。本研究为研制沙眼疫苗和诊断试剂奠定了基础,也将有助于理解沙眼的进化和传播。     

慢性阴道炎患者生殖道解脲脲原体和沙眼衣原体感染分析

目的通过荧光定量聚合酶链反应(PCR)技术检测慢性阴道炎患者阴道内解脲脲原体(Uu)和沙眼衣原体(Ct)感染状况,用以指导临床正确治疗。方法应用荧光定量聚合酶链反应对380例慢性阴道炎患者的阴道分泌物进行解脲脲原体和沙眼衣原体PCR检测。结果解脲脲原体阳性率为42.9%,其阳性标本的平均拷贝数为3.4×105copies/ml,沙眼衣原体阳性率为16.6%,其阳性标本的平均拷贝数为5.8×104copies/ml,解脲脲原体和沙眼衣原体合并感染的阳性率为12.6%。结论PCR技术具有简便、快捷、准确的优点,是目前快速诊断慢性阴道炎患者Uu、Ct感染状况的可靠诊断方法之一。     

Determination of the number of tuf genes in Chlamydia trachomatis and Neisseria gonorrhoeae

Restriction endonuclease fragments of DNA from Neisseria gonorrhoeae and Chlamydia trachomatis (mouse pneumonitis biovar) were hybridized to probes from the N-terminal and C-terminal portions of the Escherichia coli tufA gene. In common with other Gram-negative bacteria, the genome of N. gonorrhoeae was found to contain two homologous sequences (presumptive tuf genes). The C. trachomatis genome contained a single tuf sequence.     

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 .     

泌尿生殖道沙眼衣原体omp1基因多态性研究

从中国不同城市收集疑为沙眼衣原体(Ct)感染的泌尿生殖道标本323份,巢式PCR扩增Ctomp1基因片段(包括4个变异区),测定其中96份阳性标本omp1基因序列,根据同源性分型并分析其多态位点;根据氨基酸序列,用Mega 3软件构建进化树,分析临床株与相应参考株之间的亲缘关系。从96份沙眼衣原体阳性标本中,检出28种基因变体,其中E型最常见;同时发现Ct E、F型omp1基因高度保守,而其它基因型都显示一定的变异性。进化树分析发现,各临床株与相应参考株之间遗传距离较近。实验结果表明沙眼衣原体omp1基因呈现较大的多态性,可为其疫苗的研制及感染的防治提供重要的实验依据。     

沙眼衣原体血清型L2体外培养影响因素的研究

研究沙眼衣原体（Chlamydia trachomatis,Ct）血清型LGV L2在体外培养的繁殖规律及其影响因素,确定血清型LGV L2在体外培养的最佳生长发育条件。用沙眼衣原体血清型LGV L2分别感染HEp-2细胞、HeLa229细胞、HepG-2细胞、SGC-7901细胞和Vero细胞,在荧光显微镜下计数包涵体形成单位（inclusion fig-ure unity,IFU）和观察培养不同时间后包涵体的形态,比较不同细胞对血清型LGV L2的敏感性及血清型LGVL2在不同细胞内的生长情况。同时分别设DEAE葡聚糖处理组与未处理组,含放线菌酮培养组与不含放线菌酮培养组,比较培养12、24、36和48 h后血清型LGV L2包涵体形态、IFU和Real-Time PCR定量检测血清型LGV L2的核酸量,判断DEAE葡聚糖和放线菌酮对沙眼衣原体血清型LGV L2生长的影响。在感染20 h后,显微镜下观察发现HEp-2、Vero、HepG-2、HeLa和SGC-7901细胞均不同程度肿胀,5种细胞内均可见包涵体,大约40~48 h后包涵体占据整个胞浆。IFU计数和Real-Time PCR结果显示5种细胞中HeLa细胞感染率最高,HepG-2细胞感染率最低,血清型LGV L2在HeLa细胞中生长速度最快。荧光显微镜下计数IFU,发现DE-AE葡聚糖预处理组和对照组中血清型LGV L2的感染率和生长发育没有明显区别,而含放线菌酮培养组中各细胞内血清型LGV L2生长速度较对照组快,Real-Time PCR检测结果显示放线菌酮组各细胞内血清型LGVL2核酸量较对照组高。血清型LGV L2在HeLa细胞中的感染率最高,DEAE葡聚糖对血清型LGV L2的感染没有明显影响,而体外培养时添加放线菌酮有利于血清型LGV L2的生长发育。     

Identification of an iron-responsive protein that is antigenic in patients with Chlamydia trachomatis genital infections

Chlamydia trachomatis is an important cause of immune-mediated damage to the reproductive tract of infected patients. Certain chlamydial antigens and host genetic factors have been identified as contributing to immunopathological events, but a comprehensive understanding of specific components involved in destructive vs. protective immune responses to chlamydial infections is far from clear. In this study, it is shown that C. trachomatis-infected patients generate antibodies against an iron-responsive chlamydial protein, YtgA. The identity of YtgA was confirmed by mass spectrometry following two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. This finding underscores a necessity to examine patient sera samples to identify chlamydial antigens that are likely encountered and important to the immune response during human infections.     

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.     

Chlamydia trachomatis targets mitochondrial dynamics to promote intracellular survival and proliferation

Chlamydia trachomatis is an obligate intracellular bacterium that scavenges host metabolic products for its replication. Mitochondria are the power plants of eukaryotic cells and provide most of the cellular ATP via oxidative phosphorylation. Several intracellular pathogens target mitochondria as part of their obligatory cellular reprogramming. This study was designed to analyse the mitochondrial morphological changes in response to C. trachomatis infection in HeLa cells. Mitochondrial elongation and fragmentation were found at the early stages and late stages of C. trachomatis infection, respectively. C. trachomatis infection‐induced mitochondrial elongation was associated with the increase of mitochondrial respiratory activity, ATP production, and intracellular growth of C. trachomatis. Silencing mitochondrial fusion mediator proteins abrogated the C. trachomatis infection‐induced elevation in the oxygen consumption rate and attenuated chlamydial proliferation. Mechanistically, C. trachomatis induced the elevation of intracellular cAMP at the early phase of infection, followed by the phosphorylation of fission‐inactive serine residue 637 (S637) of Drp1, resulting in mitochondrial elongation. Accordingly, treatment with adenylate cyclase inhibitor diminished mitochondrial elongation and bacterial growth in infected cells. Collectively, these results strongly indicate that C. trachomatis promotes its intracellular growth by targeting mitochondrial dynamics to regulate ATP synthesis via inhibition of the fission mediator Drp1.     

慢性盆腔炎患者支原体、衣原体感染状况分析

目的了解慢性盆腔炎患者支原体与沙眼衣原体的感染状况并对支原体的药敏试验结果进行分析,为临床防治提供依据。方法选取2011年1月至2013年12月来院就诊的慢性盆腔炎患者680例,无菌采集宫颈分泌物,培养法检测解脲支原体和人型支原体,用免疫层析法检测沙眼衣原体。结果共检出解脲支原体（Uu）290例（42.6%）,人型支原体（Mh）53例（7.8%）,两者混合感染（Uu＋Mh）76例（11.2%）,沙眼衣原体（Ct）感染58例（8.5%）。Uu感染在20~40岁年龄段阳性率最高（P〈0.05）,Mh、Uu＋Mh和Ct在各年龄段阳性率差异无统计学意义（P〉0.05）。药物敏感染性结果显示,支原体对交沙霉素、强力霉素和美满霉素的耐药率较低,对环丙沙星、左氧氟沙星和加替沙星的耐药率较高。结论解脲支原体和沙眼衣原体为慢性盆腔炎的重要致病因素,临床应重视宫颈解脲支原体和沙眼衣原体的检测。防治解脲支原体和沙眼衣原体感染,可能有益于预防慢性盆腔炎的发生。     

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.     

Further characterization of an outer membrane protein of Chlamydia trachomatis with cytadherence properties

To further characterize the chlamydial cytadhesin (CCA), we have examined it for saturability of binding to HeLa cells that were grown as monolayers and in suspension. The CCA exhibited specific cytadherence properties of binding to HeLa cells that appeared to be saturable. The CCA showed a substantial decrease in binding to trypsin-treated HeLa cells in suspension. This finding, together with the fact that the CCA itself is known to be trypsin-sensitive, suggested a protein-protein type of interaction between CCA and HeLa cells. Periodate treatment of the CCA did not result in significant reduction in cytadherence, which implies that sugar moieties were probably not involved in CCA binding to HeLa cells. Whilst attempts to produce antibodies to the CCA in rabbits was unsuccessful, the CCA reacted with antibodies in a human serum known to contain high titer antibodies to Chlamydia trachomatis, suggesting it can be immunogenic, and is possibly expressed during human infection.