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.     

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.     

Positive cooperativity in the adherence between elementary bodies of Chlamydia trachomatis strain UW-31 and HeLa cells

Abstract The adherence of purified elementary bodies of Chlamydia trachomatis strain UW-31 to monolayer cultures of HeLa 229 cells exhibited kinetic evidence of positive cooperativity. An abrupt increase in the rate of adherence occurred as chlamydial dose was increased. Only freshly isolated chlamydiae showed this behavior. In the presence of the lectin wheat germ agglutinin, the stimulated adherence showed typical Michaelis-Menten kinetics. The results suggest that chlamydiae may promote their own binding to the host-cell surface, and the lectin, when cell-bound, may provide additional chlamydiae-binding sites.     

Induction of DNA strand scissions in HeLa cells by human polymorphonuclear leucocytes activated by Chlamydia trachomatis elementary bodies

Incubation of human polymorphonuclear leucocytes (HPMN) with Chlamydia trachomatis elementary bodies (EB) or phorbol 12-myristate 13-acetate (PMA) resulted in the production of superoxide anions (.O2-) and hydrogen peroxide (H2O2). Exposure of HeLa cells to EB- or PMA-activated HPMN and to EB alone, for 2 h, resulted in the formation of DNA strand scissions (nicks) in the HeLa cells. The nicks were visualized by incorporation of biotin 11-dUTP with its detection by streptavidin-peroxidase, and quantified by using [3H]dCTP in the in situ nuclear nick-translation reaction. Catalase, and to a lesser extent superoxide dismutase, reduced the amount of nicks induced by the EB- or PMA-activated HPMN. The possible relationship between the activity of PMN in chlamydial infections and the development of chronic diseases is discussed.     

Detection of type-specific antibody to lymphogranuloma venereum serotypes ofChlamydia trachomatis with a microneutralization test

Ten-day monospecific mouse antisera prepared against serotypes L1, L2, and L3 ofChlamydia trachomatis were titrated against homologous and heterologous antigens in the microimmunofluorescence (micro-IF) test and by a new microneutralization method. While broad crossreactivity between serotypes was detected in the micro-IF test, the same antisera demonstrated serotype specific neutralizing activity. The microneutralization test could be used for serotyping new chlamydial isolates when equivocal results have been obtained by the microimmunofluorescence method.     

Chlamydia trachomatis elementary bodies possess proteins which bind to eucaryotic cell membranes.

Chlamydia trachomatis proteins were electrophoresed and then transferred to nitrocellulose paper to detect chlamydial proteins which bind to eucaryotic cell membranes. Resolved polypeptides of C. trachomatis serovars J and L2 were reacted with iodinated HeLa cell membranes and autoradiographed. Infectious elementary bodies of both serovars possess 31,000- and 18,000-dalton proteins which bind to HeLa cells. In contrast, noninfectious reticulate bodies do not possess eucaryotic cell-binding proteins. Both proteins are antigenic when reacted with hyperimmune rabbit antisera in immunoblots and antisera raised against the 31,000- and 18,000-dalton proteins are inhibitory to chlamydia-host cell association. In addition, these antisera exhibit neutralizing activity. Our data suggest that these putative chlamydial adhesins play a key role in the early steps of chlamydia-host cell interaction and that antibody directed against them may be protective.     

Flow cytometric analysis of Chlamydia trachomatis interaction with L cells

Immunofluorescent staining and flow cytometric analysis have been investigated as means of studying the early stages of in vitro infection of Chlamydia trachomatis. The lymphogranuloma venereum strain of C. trachomatis was grown in vitro in L cells, fixed in p-formaldehyde, stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody to the chlamydial major outer membrane protein, and analyzed flow cytometrically. Infected cells stained 50-100 times more intensely than uninfected cells, and they could easily be discriminated by flow analysis. The number of infected cells and the fluorescence intensity of individual cells were proportional to the multiplicity of infection. The attachment of purified elementary bodies to L cells could be analyzed by immunofluorescence and flow cytometry. Cells exposed to 0.26 inclusion-forming units/cell could be discriminated from an unexposed population. Flow analysis of purified elementary bodies was possible after fluorescent staining with the aid of a laser-based cytometer and gating on low volume.     

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.     

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.     

Interaction between human polymorphonuclear leucocytes and Chlamydia trachomatis elementary bodies: electron microscopy and chemiluminescent response

Incubation of human polymorphonuclear leucocytes (HPMN) with highly purified Chlamydia trachomatis serotype L2/434/Bu elementary bodies (EB), in the presence and absence of specific antibody, resulted in a 10(3)-fold reduction of viable count after 24 h incubation. Electron microscopy observations indicated activation of the HPMN by the EB. Attachment of the EB to the HPMN cell membrane, formation of a cytoplasmic cup and EB-containing vacuoles were observed. In addition, two types of phagocytic vacuoles were observed after 30 min incubation; in one type, a single EB was tightly surrounded by the vacuolar membrane, while the other type was enlarged and held one or more intact EB or degenerated EB or both. A fuzzy coat was observed on EB located in the HPMN vacuoles only in the presence of specific antibody. Empty vacuoles containing degenerated EB were observed in the HPMN after 24 h incubation. HPMN exposed to EB of C. trachomatis produced a marked chemiluminescent response with a peak 14 times greater than the peak value of the control. A second stimulation with phorbol 12-myristate 13-acetate and zymosan was achieved. The chemiluminescent peak value in the presence of heat-treated EB (56 degrees C, 20 min) was 50% of that obtained in the presence of untreated EB. The significance of the chemiluminescent response in the killing mechanism of C. trachomatis EB by HPMN is discussed.     

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。     

Chlamydia trachomatis infection prevents front–rear polarity of migrating HeLa cells

Chlamydiae are obligate intracellular bacterial pathogens that cause trachoma, sexually transmitted diseases and respiratory infections in humans. Fragmentation of the host cell Golgi apparatus (GA) is essential for chlamydial development, whereas the consequences for host cell functions, including cell migration are not well understood. We could show that Chlamydia trachomatis‐infected cells display decelerated migration and fail to repopulate monolayer scratch wounds. Furthermore, infected cells lost the ability to reorient the fragmented GA or the microtubule organization centre (MTOC) after a migratory stimulus. Silencing of golgin‐84 phenocopied this defect in the absence of the infection. Interestingly, GA stabilization via knockdown of Rab6A and Rab11A improved its reorientation in infected cells and it was fully rescued after inhibition of Golgi fragmentation with WEHD‐fmk. These results show that C. trachomatis infection perturbs host cell migration on multiple levels, including the alignment of GA and MTOC.     

The effect of ions on the adhesion and internalization of Chlamydia trachomatis by HeLa cells

The adhesion and internalization of Chlamydia trachomatis by HeLa cells was unaffected by removal of K+, Mg2+, or glucose from the incubation medium, slightly reduced by removal of Na+, and significantly reduced by omission of Ca2+, Sr2+, Mg2+, and Mn2+ could replace Ca2+ in the adhesion but only Sr2+ supported internalization, and La3+, Co2+, Fe3+, Ba2+, and Zn2+ all reduced internalization more than adhesion. During initial infection there was no measurable difference in the uptake or release of 45Ca2+ or 86Rb+ between infected and noninfected HeLa monolayers. Infection was not prevented by pretreatment of the monolayers with the calcium channel blockers, verapamil, D600, and nitrendipine, or the calmodulin inhibitors, TMB-8 or trifluperazine. The results suggest that divalent cations are not essential for chlamydial infection but that the process of internalization is facilitated by the presence of cations, particularly Na+ and Ca2+.     

沙眼衣原体基因CT703在HeLa细胞中的表达

本文旨在探讨沙眼衣原体( Chlamydia trachomatis, Ct) L2 血清型感染HeLa细胞后, 在急性感染和持续感染状态下基因CT703 mRNA 表达水平的变化。透射电子显微镜下确定Ct 急性感染和持续感染模型的建立; 吉姆萨染色显示, 细胞内包涵体体积增大且出现异常增大的网状体, 反转录-聚合酶链反应( RT-PCR) 检测显示, 持续感染状态下Ct 基因CT703 mRNA表达水平显著低于急性感染状态, 推测这可能是Ct 在细胞内持续感染的形成机制之一。     

Reactivity of elementary and reticulate bodies of Chlamydia trachomatis LGV2 with monoclonal antibodies specific for the major outer membrane protein

The reactivity of the major outer membrane protein (MOMP) of Chlamydia trachomatis (LGV2 serotype) with 15 monoclonal antibodies was studied during the course of developmental cycle by immunoblotting and immunofluorescence. The monoclonal antibodies reacted in immunoblots with the MOMP of both elementary bodies (EBs) and reticulate bodies (RBs). Using an immunofluorescence test with LGV2-infected cell cultures, the 15 monoclonal antibodies could be divided into 5 groups, according to the time of appearance of their reactivity with the cell culture.     

Ultrastructural studies on the intracellular fate of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) and Chlamydia trachomatis (strain lymphogranuloma venereum 434): modulation of intracellular events and relationship with endocytic mechanism

Previous observations on the highly infectious LGV strain 434 of Chlamydia trachomatis and the guinea pig inclusion conjunctivitis (GPIC) strain of C. psittaci (which requires centrifugation of inocula with host cell monolayers for maximum infectivity) indicated that infectivity differences were expressed, not at entry, but at an intracellular stage affecting multiplication. Centrifugation increased the potential of internalized chlamydiae to undergo productive infection. Here, analysis of the intracellular fate of chlamydiae by ultrastructural methods indicates that strain GPIC exhibits two patterns of behaviour depending on the mode of inoculation. Strain GPIC showed limited entry, with 47% of intracellular organisms becoming associated with thorotrast-labelled lysosomes, following static incubation with monolayers. In contrast, with centrifugation, entry was not limited and association with lysosomes was reduced to 12%; strain 434 behaved similarly but independently of the mode of inoculation. The different results for strain GPIC correlated with distinct entry mechanisms. Entry during static incubation was unimpaired either by treatment with cytochalasin D or by temperature reduction to 20 degrees C, suggesting that it was pinocytic. Entry during centrifugation was markedly impaired by both treatments, suggesting that it was phagocytic. The data lead to two novel conclusions: first, that chlamydiae can apparently enter cells by both pinocytic and phagocytic mechanisms; second, that the entry mechanism influences intracellular fate. It is suggested that entry mechanism is linked to selection of the vesicle membrane forming around the internalizing chlamydiae. This, in turn, may influence both intracellular translocation and subsequent inhibition or promotion of multiplication of the internalized parasite.     

Staining of Chlamydia trachomatis elementary bodies: a suitable method for identifying infected human monocytes by flow cytometry

Persistence of Chlamydia trachomatis (C. trachomatis) in the joint is the most frequent cause of reactive arthritis following urogenital tract infection. The resulting changes of host cell antigen- and cytokine-expression are not precisely understood. We developed and evaluated a direct cytometric approach to visualize in vitro C. trachomatis-infected monocytes. Infectious elementary bodies (EBs) of C. trachomatis serovar K were labelled by incubation with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). Afterwards, human peripheral blood monocytes were cultured with the CFSE-labelled EBs and analysed by flow cytometry. Real-time polymerase chain reaction (PCR) was used to demonstrate intracellular uptake and viability of CFSE-labelled C. trachomatis by the determination of gene expression. Labelling EBs with CFSE may become a valuable tool for studying the interaction between C. trachomatis and the host cell.     

Control mechanisms governing the infectivity of Chlamydia trachomatis for HeLa cells: mechanisms of endocytosis

The mechanism by which Chlamydia trachomatis is endocytosed by host cells is unclear. Studies of the kinetics of chlamydial attachment and uptake in the susceptible HeLa 229 cell line showed that chlamydial endocytosis was rapid and saturable but limited by the slow rate of chlamydial attachment. To overcome this limitation and to investigate the mechanism of endocytosis, chlamydiae were centrifuged onto the host cell surface in the cold to promote attachment. Endocytosis of the adherent chlamydiae was initiated synchronously by rapid warming to 36 degrees C. Electron micrographs of chlamydial uptake 5 min after onset showed that chlamydial ingestion involves movement of the host cell membrane, leading to interiorization in tight, endocytic vacuoles which were not clathrin coated. Chlamydial ingestion was not inhibited by monodansylcadaverine or amantadine, inhibitors of receptor-mediated endocytosis and chlamydiae failed to displace [3H]sucrose from micropinocytic vesicles. Chlamydial endocytosis was markedly inhibited by cytochalasin D, an inhibitor of host cell microfilament function, and by vincristine or vinblastine, inhibitors of host cell microtubules. Hyperimmune rabbit antibody prevented the ingestion of adherent chlamydiae, suggesting that endocytosis requires the circumferential binding of chlamydial and host cell surface ligands. These findings were incompatible with the suggestion that chlamydiae enter cells by taking advantage of the classic mechanism of receptor-mediated endocytosis into clathrin-coated vesicles, used by the host cell for the internalization of beta-lipoprotein and other macromolecules, but were consistent with the hypothesis that chlamydiae enter cells by a microfilament-dependent zipper mechanism.