Adherence of multiple serovars of Chlamydia trachomatis to a common receptor on HeLa and McCoy cells is mediated by thermolabile protein(s)

Several aspects of the adherence of purified elementary bodies (EB) of Chlamydia trachomatis to HeLa and to McCoy cells were examined using different techniques, including an ELISA. Serovar-specific, biotinylated monoclonal antibodies were used to detect cell-bound chlamydiae. In addition, purified chlamydiae were biotinylated and their adherence properties were studied. The assays were done at 4 degrees C to exclude the energy-dependent internalization of the cell-bound EB and host-cell membrane recycling that occur at 37 degrees C. Saturation kinetics were routinely observed at 4 degrees C, and the rate of adherence remained linear for approximately 60 min. Lineweaver-Burk analysis of the kinetics data showed that adherence of any one serovar was competitively inhibited by other serovars of C. trachomatis. This competition for the same receptor on the two alternative hosts, HeLa and McCoy, was also seen when the adherence assays were done at 37 degrees C in the presence of sodium azide, an energy poison that inhibits endocytosis of cell-bound chlamydiae. Chlamydiae exposed to 56 degrees C for 5 min, or treated with low doses of trypsin, failed to exhibit competitive inhibition, having suffered considerable loss of the ability to adhere to host-cells. These data suggest that heat- and trypsin-labile chlamydial moieties participate in the adherence reaction, and that oculo-genital serovars of C. trachomatis, including that of lymphogranuloma venereum, attach to the same receptor on the host-cell membrane.     

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.     

Antigenic analysis of Chlamydiae by two-dimensional immunoelectrophoresis. II. A trachoma-LGV-specific antigen.

Two-dimensional immunoelectrophoresis was utilized to study precipitins in hyperimmune rabbit serum made against chlamydiae and from patients with chlamydial infections. An antigen of Triton X-100-solubilized L2/434/Bu organisms with an electrophoretic mobility of 0.65 relative to bovine serum albumin at pH 8.6 was excised from the agarose gel of electrophorograms as antigen-antibody complexes and used to immunize rabbits. A monospecific antiserum to antigen 0.65 was obtained that reacted with Trachoma-LGV strains L2/434/Bu, B/TW-5/OT, and K/UW-31/Cx, but not with the mouse pneumonitis (Nigg) strain or the psittacosis strain meningopneumonitis (Cal-10). The Trachoma-LGV specificity of antigen 0.65 was further shown by indirect immunofluorescence straining with the monospecific antiserum of chlamydial inclusions in infected HeLa cells. Precipitins with a specificity for antigen 0.65 were indentified in 15 of 18 sera from patients with diagnosed Chlamydia trachomatis infections LGV, trachoma, nongonococcal urethritis, and nongonococcal cervicitis by using monospecific antiserum to antigen 0.65 in the peak suppression test. Thus, antigen 0.65 appears to be a Trachoma-LGV-specific antigen that has considerable promise for serodiagnosis.     

Factors affecting the adhesion and uptake of an oculo-genital strain of Chlamydia trachomatis to McCoy cells

Abstract We have studied adhesion and uptake of C. trachomatis serovar E in McCoy cells under various infection conditions. Adhesion and uptake of chlamydiae was completed about 3 h after the initiation of stationary infection at 37°C, but ingestion of cell membrane-attached organisms was finished within 0.5 h at 37°C. Reincubated chlamydiae, not attached after 3 h at 37°C, attached readily to fresh McCoy cell monolayers, but to a lesser extent than the original inoculum. Our results indicate that the lack of further attachment after 3 h incubation at 37°C under stationary infection conditions has complex causes, involving both host cell and parasite. Centrifugation did not affect the uptake of chlamydiae already bound to the cell membrane, suggesting that the uptake phase of C. trachomatis serovar E by McCoy cells is unaffected by centrifugation.     

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。     

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.     

Extensive heterogeneity of the protein composition of Chlamydia trachomatis following serial passage in two different cell lines

To determine if the host-modulated adherence characteristics of the intracellular bacterial pathogen Chlamydia trachomatis were due to the acquisition of altered surface-exposed proteins, highly purified chlamydiae grown in two different host cells were analysed. Two serovars, L1 and E, were grown for multiple passages in both HeLa and McCoy host cells. Numerous protein differences in the chlamydial elementary bodies (EB) of each serovar grown in the two different hosts were detected by two-dimensional (2-D) gel electrophoresis and fluorography of radioactively labelled proteins. At least four to six serial passages in the alternative host were necessary before the changes were apparent. Iodination of suspensions of purified chlamydiae and 2-D electrophoresis revealed several surface proteins that were determined by the host cells in which the bacteria had replicated. These iodinated chlamydial proteins were removed by treatment of the iodinated EB with trypsin, indicating their location at the bacterial surface. Two of the major constituents of the outer-membrane complex, the cysteine- and methionine-rich 60 kDa and 40 kDa proteins, remained unchanged in both molecular mass and charge during the host adaptation. Several chlamydial proteins capable of binding iodinated host membrane preparations also exhibited host-dependent alterations. Immunoblotting experiments with a rabbit and a human polyclonal sera indicated that distinct host-specified chlamydial proteins were reactive with the two sera.     

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.     

Interaction of Chlamydia trachomatis with human genital epithelium in culture

Primary cultures of human endometrial and ectocervical epithelial cells were examined as a new model system to study genital infection by Chlamydia trachomatis. Initial studies demonstrated that these cells were indeed susceptible to chlamydial infection. Inocula, adjusted to produce inclusions in 50 to 80% of equivalent numbers of standard McCoy cells, resulted in infection rates of approximately 15 to 30% for the columnar cells of the endometrium and 5 to 10% for the squamous cells of the ectocervix. Exposure of cultures to DEAE-dextran and centrifugation-assisted inoculation, manipulations reported to enhance infection of HeLa and McCoy cells, did not alter the number of inclusion-positive genital cells. Addition of cycloheximide to the post-inoculation culture medium slightly increased numbers of inclusion-bearing cells while growth of genital cells in hormone-supplemented medium resulted in a variable effect on inclusion development and a significant reduction in the association of radiolabelled organisms with these cells. The basis for the different levels of infection in McCoy versus genital cell cultures was revealed by immunofluorescence analysis of chlamydial association with host cells immediately after inoculation. Chlamydiae failed to adhere to many cells in the genital cell cultures while adherence to McCoy cells was uniform. In addition, the association of radiolabelled C. trachomatis was significantly lower with genital cells than with McCoy cells. Finally, culture conditions were defined which markedly inhibited inclusion development without an immediate loss of chlamydial growth potential. This investigation indicates that primary genital cell cultures are susceptible to chlamydial infection and will be valuable for studies on the nature of C. trachomatis interactions with natural human target cells.     

Growth of Chlamydia trachomatis in McCoy Cells Treated with Cytochalasin B

When the fungal metabolite cytochalasin B was added to McCoy cells, multinucleated giant cells developed. Monolayers of these cells proved as efficient as irradiated cells for the growth of three different serotypes of Chlamydia trachomatis and for the primary isolation of chlamydiae from clinical specimens obtained from patients attending a venereal disease clinic. Cytochalasin treatment of McCoy cells provides a convenient alternative to irradiation and should be of value in the isolation of chlamydiae from the eye and genital tract.     

Chlamydia trachomatis in cell culture. II. Susceptibility of seven established mammalian cell types in vitro. Adaptation of trachoma organisms to McCoy and BHK-21 cells

Trachoma organisms of serotype B were grown serially in irradiated cells (McCoy, BHK-21, Microbiological Associates, and BHK-21, Lister) and tested for infectivity in monolayers of five mammalian cell lines (BHK-21, CHO, HeLa S3, McCoy and OWMK) and two diploid strains (ST/BTL and WI-38). All cell types had low susceptibility to chlamydial infection but the number of inclusions increased when the inoculum was centrifuged onto the monolayers, or when the cells were irradiated. Infection was higher in non-irradiated CHO than in irradiated CHO in three out of a total of six experiments. Inclusion number was increased 300 times in HeLa S3 and up to three times in the other cell types after treatment with diethylaminoethyl-dextran (DEAE-D). Serial passage of Chlamydia trachomatis serotype B (strain Har-36) in CO60 McCoy and CO60 BHK-21 Lister resulted in partial adaptation of the strain to the host cell. The phenomenon of adaptation of serotype B to McCoy compensated for the lower susceptibility of this cell revealed when McCoy cells were inoculated with trachoma elementary bodies grown in BHK-21 Lister or in chick embryo yolk sac. Trachoma organisms of immunotypes A, B and C prepared in yolk sac produced more inclusion-forming units per ml in CO60 BHK-21 Lister than in CO60 McCoy.     

Low-nutrient induction of abnormal chlamydial development: A novel component of chlamydial pathogenesis?

Abstract The intracellular development of chlamydiae in McCoy cells incubated in Eagle's minimal essential medium lacking all 13 amino acids was examined both by fluorescence and electron microscopy and by infectivity titration. Aberrant development occurred in almost all inclusions of strains of Chlamydia trachomatis and C. psittaci with the production of abnormal forms which differed in size, shape and internal structure from normal reticulate and elementary body forms. Detailed analysis of the response of C. trachomatis L2 strain 434 to graded reductions in amino acid level showed that infectivity was reduced and morphological abnormality increased as amino acid concentrations were lowered from 33 to 0% of amino acids present in minimal essential medium. Reversion of inclusions to normal and reappearance of infectious forms occured on restoration of amino acids and further incubation. It is suggested that aberrant development may account for the presence in vivo of non-cultivable chlamydiae and that such development can arise via tryptophan deprivation mediated by local release of interferon gamma.     

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.     

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.     

Initial characterization of a chlamydial receptor on mammalian cells

We have examined characteristics of the binding of eukaryotic cells to chlamydial elementary body (EB)-specific proteins. A wide variety of eukaryotic cell lines bound to representatives of both Chlamydia trachomatis lymphogranuloma venereum (LGV) and trachoma biovars and a C. psittaci strain meningopneumonitis (Mn) suggesting the presence of a common host cell receptor. Neither tunicamycin nor neuraminidase treatment of HeLa cells impaired binding to C. trachomatis EB, implying that host cell N-linked carbohydrate domains and sialic acid moieties, respectively, are not involved in attachment. However, trypsinized HeLa cells do not bind to EB, suggestive of a proteinaceous host cell receptor. The trypsin sensitivity of two EB-specific binding proteins Mr = 18,000 and 31,000) was also examined, and the finding that 125I-labeled HeLa cells bind both the 18,000 and 31,000-dalton proteins after chlamydial trypsinization corroborates our earlier observation that these EB binding proteins mediate attachment.     

Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia -containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule-associated Chlamydia vesicles also collocated with tyrosine-phosphorylated McCoy cell proteins. After migration, the Chlamydia -containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule-dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule-dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR-1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR-1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis .     

Detection and differentiation of chlamydiae by fluorescence in situ hybridization

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and PARACHLAMYDIA: The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.     

Analysis and detection of chlamydial DNA

Elementary bodies of lymphogranuloma venereum (LGV) strains of Chlamydia trachomatis contain, in addition to the genomic DNA, a 6.7 kb plasmid. The plasmid from serovar L2 (434-B) was cloned at the BamHI site of pBR327 into Escherichia coli and a restriction cleavage map of this pLGV125 recombinant plasmid determined. All 15 C. trachomatis serovars contained DNA sequences that hybridized with pLGV125. When total DNA from L2 elementary bodies was used as a probe in Southern blotting and spot hybridization, serovars L1, L2 and L3 exhibited significant homology. The detection level of homologous DNA was 100 pg and LGV DNA was detectable in infected cells when total L2 probe was used in the nucleic acid hybridization test. These DNA probes may be useful as investigative and diagnostic reagents for C. trachomatis.     

Influence of cysteine deprivation on chlamydial differentiation from reproductive to infective life-cycle forms

The effects of omission of individual amino acids from growth medium on the differentiation of Chlamydia trachomatis DK-20 (serotype E) during infection of cycloheximide-treated McCoy cells are described. As judged by inclusion body staining with acridine orange, omission of cysteine from the medium severely retarded differentiation of reproductive reticulate body (RB) to infective elementary body (EB) forms. The effect appeared specific to cysteine in that omission of other amino acids had little or no effect on differentiation once RBs appeared. On restoration of cysteine, culture infectivity increased and inclusions contained organisms which, by cytochemical and morphological criteria, were differentiating to infective forms, indicating that cysteine deprivation did not irreversibly inhibit differentiation. Impairment of RB to EB differentiation in cysteine-less medium was also observed for three strains of Chlamydia psittaci and 10 other strains of C. trachomatis. It is suggested that the effect arises via the biosynthetic requirement for cysteine for provision of three cysteine-rich proteins, whose synthesis and insertion into the outer membrane have previously been shown to accompany RB to EB differentiation of C. psittaci 6BC and C. trachomatis 434 (serotype L2). Synthesis of cysteine-rich outer membrane proteins during differentiation may thus be common to all chlamydiae.     

Binding, ingestion, and growth of Chlamydia trachomatis (L2 serovar) analyzed by flow cytometry

We have developed a method for quantitatively assessing binding, ingestion, and growth of Chlamydia trachomatis (L2 serovar) in several mammalian cell lines using fluorescence staining and flow cytometry. Cells were incubated with chlamydia at 4 degrees C to monitor binding; ingestion was determined by raising the temperature to 37 degrees C for 1-4 h and removing extracellular bacteria with pronase. Growth of bacteria was measured by assessing brightly stained intracellular inclusions. Fixation with methanol prior to fluorescent staining provided the most intense specific staining with minimal background, as well as preserving cell morphology. Our data reveal relatively slow ingestion of L2 by McCoy fibroblasts (maximum ingestion by 4 h) and a sizeable population of McCoy cells (30-40% of total cells) that ingest L2 but do not permit its growth under certain infectious conditions. It was possible to correlate specific histogram patterns on the flow cytometer with fluorescent microscope observations. This system provides a means of analyzing quantitative interactions between chlamydia and individual host cells.