Chlamydia trachomatis in cell culture

Summary 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. This research was supported by a grant from the National Eye Institute (EI-00812-08), and by the Arabian American Oil Company. The paper is dedicated to the memory of Francis B. Gordon, who pioneered research methods for the cultivation of trachoma and inclusion conjunctivitis (TRIC) agents in cell culture. Dr. Gordon patiently studied tables and photographs which accompany this text when he visited our laboratory on the day prior to his sailing to England on the ill-fated voyage in which he and Mrs. Gordon perished (October 1973).     

Pathogenicity of Chlamydia psittaci after serial passage in chicken embryos

The virulence in pregnant ewes of a single strain of $\text{Chlamydia psittaci}$ was tested after the following treatments: (a) 110 serial passages in chicken embryos (CE) incubated at 37 C, and (b) 7 CE passages at 37 C followed by 10 CE passages at 40 C. Neither treatment caused significant reduction in abortions (P > 0.3) nor incidence of ewes shedding chlamydia in placentas or vaginal discharges (P > 0.5) compared to ewes inoculated with the original virulent strain.     

Characterization of estrogen-responsive epithelial cell lines and their infectivity by genital Chlamydia trachomatis

Chlamydial attachment and infectivity in vitro and ascending disease and sequelae in vivo have been reported to be enhanced/modulated by estrogen. Endometrial carcinoma cell lines Ishikawa and HEC-1B and the breast cancer lines MCF-7 and HCC-1806 were examined for Chlamydia trachomatis E infectivity. Estrogen receptor (ER) presence was confirmed by Western blot and qRT-PCR analyses. FACS analysis was used to determine the percent of plasma membrane-localized ERs (mERs), and their activity was tested by estrogen binding and competitive estrogen antagonists assays. Chlamydiae grew in all cell lines with HEC (90%) > MCF-7 (57%)>Ishikawa (51%) > HCC-1806 (20%). The cell line ER isoform composition was re-defined as: ERalpha + ERbeta + for MCF-7, HCC-1806 and Ishikawa; and ERbeta only for HEC-1B. HeLa cells were also tested and found to express ERbeta, but not ERalpha. A small percentage of both ERs were surface-exposed and functionally active. The endometrium-predominant ERbeta isoform was found in all cell lines, including those most representative of the common sites of C. trachomatis infection. Thus, the role of chlamydial attachment/infectivity will now be analyzed in ERbeta+and-isogenic HEC-1B cells.     

沙眼衣原体质粒蛋白研究进展

沙眼衣原体除含有高度保守的基因组外,也含一个7．5kb的隐蔽性质粒,隐蔽性质粒具有8个开放阅读（ORF1-8）,编码8种质粒蛋白pgpl-8。质粒蛋白在沙眼衣原体致病过程中发挥重要的作用,尤其是新近发现沙眼衣原体的唯一一种分泌到胞浆中的分泌性蛋白pgp3和对毒力相关基因具有转录调节功能的pgp4。对就沙衣原体的质粒蛋白研究现状进行了综述。     

The 18-kDa lectin-binding protein of Chlamydia trachomatis is different from the 18-kDa histone-like protein

Abstract Five proteins of Chlamydia trachomatis at the 18000 (18-kDa) molecular mass region were resolved by two-dimensional electrophoresis. Three proteins at 18.2 kDa, p I 6.9, 18.0 kDa, p I 6.3, and 17.9 kDa, p I 6.4 were shown to bind lectin. A fourth protein of 18.0 kDa at p I 10 was the histone-like protein. The fifth protein at 17.9 kDa, p I 7.0 was not characterized.     

Lysosome repair enables host cell survival and bacterial persistence following Chlamydia trachomatis infection

Chlamydiae are obligate intracellular bacteria that replicate within the confines of a membrane-bound vacuole termed the inclusion. The final event in the infectious process is the disruption of the inclusion membrane and release of a multitude of infectious elementary bodies, each capable of eliciting a new infection. Strains of the trachoma biovar of Chlamydia trachomatis are released from the host cell without concomitant host cell death. In this study, analysis of events associated with chlamydial egress revealed that the integrity of the host cell plasma membrane was compromised prior to the inclusion membrane. This disruption was accompanied by the appearance of LAMP-1 at the infected cell surface, implicating lysosome repair of plasma membrane lesions in response to infection. Analysis of the effects of calcium chelators and actin stabilizing agents, indicated calcium-induced actin depolymerization as a requisite to lysosome-plasma membrane fusion and host cell survival. A consequence of this lysosome-mediated repair process, was the retention of residual bacteria within the surviving host cell, providing a unique mechanism for intracellular persistence of C. trachomatis.     

Computational analysis of the polymorphic membrane protein superfamily of Chlamydia trachomatis and Chlamydia pneumoniae

Whole sequence genome analysis is invaluable in providing complete profiles of related proteins and gene families. The genome sequences of the obligate intracellular bacteria Chlamydia trachomatis and Chlamydia pneumoniae both encode proteins with similarity to several 90-kDa Chlamydia psittaci proteins. These proteins are members of a large superfamily, C. trachomatis with 9 members and C. pneumoniae with 21 members. All polymorphic membrane protein (Pmp) are heterogeneous, both in amino acid sequence and in predicted size. Most proteins have apparent signal peptide leader sequences and hence are predicted to be localized to the outer membrane. The unifying features of all proteins are the conserved amino acid motifs GGAI and FXXN repeated in the N-terminal half of each protein. In both genomes, the pmp genes are clustered at various locations on the chromosome. Phylogenetic analysis suggests six related families, each with at least one C. trachomatis and one C. pneumoniae orthologue. One of these families has seen prolific expansion in C. pneumoniae, resulting in 13 protein paralogues. The maintenance of orthologues from each species suggests specific functions for the proteins in chlamydial biology.     

沙眼衣原体质粒蛋白pgp3的研究进展

沙眼衣原体分泌性蛋白在沙眼衣原体致病过程中起重要作用,质粒编码的蛋白pgp3(即pORF5)是迄今为止发现的唯一由沙眼衣原体质粒编码的分泌性蛋白。pgp3在沙眼衣原体感染早期即可表达,在感染人群中具有很强的免疫原性,且人抗体对pgp3的识别具有高度的结构依赖性,对该蛋白的研究将有助于进一步了解衣原体质粒编码蛋白的作用及衣原体致病机制,以寻找更好的衣原体诊断方法和防治措施。本文就沙眼衣原体质粒编码蛋白pgp3的生物学性质及其致病机制作一简要综述。     

Diversity of the Chlamydia trachomatis common plasmid in biovars with different pathogenicity

The 7.5-kb plasmid of Chlamydia trachomatis (CT) is believed to encode essential genes and might have a role in CT pathogenicity. Accordingly, analysis of plasmid-linked mutations in isolates from biovars with different pathogenic properties should help in identifying which plasmid-encoded genes, if any, may be involved in modulating virulence. For this purpose, the plasmid present in a low-virulence isolate (trachoma biovar, serotype D) was cloned and sequenced. Nucleotide changes were experimentally checked against the sequence of the plasmid variant from the highly virulent strain L2/434/Bu (LGV biovar). By aligning our data with two published sequences of different trachoma and LGV variants a general consensus structure was determined, comprising eight major open reading frames (ORF) and a number of points where there is consensus only between isolates of the same biovar (biovar-specific mutations). The degree of variation between different isolates is less than 1%. In particular, comparison of serotype-D and -L2 plasmids shows mutations which are generally silent or lead to few (one to four), often conservative, amino acid changes in ORFs 1, 2, 4, 5, 6, and 7. The protein encoded by ORF8 is completely conserved. In contrast, the polypeptide variants encoded by ORF3 show nine amino acid changes, seven of which are due to biovar-specific mutations.     

Phenotypic heterogeneity in lymphoblastic cell lines

Four T-cell and two B-cell lines from patients with lymphoblastic leukemia were examined with a panel of monoclonal antibodies for a variety of lineage and differentiation stage-associated antigens during growth in liquid suspension. In five of the lines, markers normally associated with the granulopoietic lineage were found and the level of expression of these markers varied during culture. The sixth line, MOLT-3, was examined in more detail. Using clonal selection it was found that phenotypic heterogeneity could readily be obtained and that subclones could be acquired that expressed a wide range of markers, typically in reproducible kinetic patterns, that were not detected on the parent clone. Previous results were confirmed showing that treatment with the drug 5-azacytidine (5-aza) prior to selection promoted the expression of the granulopoietic lineage associated antigen 80H.5 on MOLT-3 subclones; however, treatment with 5-aza appeared to inhibit substantially the expression of other novel markers by subclones following a second selection compared to untreated controls. It appears that the expression of markers normally associated with other lineages on leukemic lymphoblasts (lineage infidelity) may be characteristic of such lines and that phenotypically variant subclones expressing lineage infidelity can readily be obtained by clonal selection.     

Culture in the rotating-wall vessel affects recombinant protein production capability of two insect cell lines in different manners

Summary The production of recombinant secreted alkaline phosphatase protein in virally infected insect cells was studied in shaker flask and high aspect rotating-wall vessel (HARV) culture. Two commonly used cell lines, Spodoptera frugiperda Sf-9 (Sf-9) and a nonaggregating isolate of the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) cell line, Trichoplusia ni Tn-5B1-4-NA (TN-5B1-4-NA), were used and monitored for 120-h postinfection. Different responses to culture in the HARV were seen in the two cell lines. While the Sf-9 cell line was able to produce slightly greater amounts of recombinant protein in the HARV than in shaker flask controls, the Tn-5B1-4-NA cell line produced significantly lesser amounts in the HARV than in the shaker flasks. Both cell lines exhibited longer life spans and longer periods of protein production in HARV culture than in shaker flask culture, presumably due to lower levels of shear encountered in the HARV. The important difference was in the protein production rate responses of the two cell lines. While the protein production rates of Sf-9 cells were comparable in both HARV and shaker flask cultures, the protein production rates of Tn-5B1-4-NA cells were much lower in HARV culture than in shaker flask cultures. The conclusion is drawn that cell line-specific adaptation to the HARV strongly influences recombinant protein production.     

NK T cell activation promotes Chlamydia trachomatis infection in vivo

We used two approaches to examine the role of NK T cells (NKT) in an intracellular bacterial (Chlamydia trachomatis mouse pneumonitis (C. muridarum)) infection. One is to use CD1 gene knockout (KO) mice, which lack NKT, and the other is to activate NKT using alpha-galactosylceramide (alpha-GalCer), a natural ligand of these cells. The data showed a promoting effect of NKT activation on Chlamydia lung infection. Specifically, CD1 KO mice exhibited significantly lower levels of body weight loss, less severe pathological change and lower chlamydial in vivo growth than wild-type mice. Immunological analysis showed that CD1 KO mice exhibited significantly lower C. muridarum-specific IL-4 and serum IgE Ab responses as well as more pronounced delayed-type hypersensitivity response compared with wild-type controls. In line with the finding in KO mice, the in vivo stimulation of NKT using alpha-GalCer enhanced chlamydial growth in vivo, which were correlated with reduced delayed-type hypersensitivity response and increased C. muridarum-driven IL-4/IgE production. Moreover, neutralization of IL-4 activity in the alpha-GalCer-treated BALB/c mice significantly reduced the promoting effect of alpha-GalCer treatment on chlamydial growth in vivo. These data provide in vivo evidence for the involvement of NKT in a bacterial pathogenesis and its role in promoting Th2 responses during infection.     

Diversity of Chlamydia trachomatis major outer membrane protein genes.

Genomic DNA libraries were constructed for Chlamydia trachomatis serovars B and C by using BamHI fragments, and recombinants that contained the major outer membrane protein (omp1) gene for each serovar were identified and sequenced. Comparisons between these gene sequences and the gene from serovar L2 demonstrated fewer base pair differences between serovars L2 and B than between L2 and C; this finding is consistent with the serologic and antigenic relationships among these serovars. The translated amino acid sequence for the major outer membrane proteins (MOMPs) contained the same number of amino acids for serovars L2 and B, whereas the serovar C MOMP contained three additional amino acids. The antigenic diversity of the chlamydial MOMP was reflected in four sequence-variable domains, and two of these domains were candidates for the putative type-specific antigenic determinant. The molecular basis of omp1 gene diversity among C. trachomatis serovars was observed to be clustered nucleotide substitutions for closely related serovars and insertions or deletions for distantly related serovars.     

沙眼衣原体GlgA蛋白表达和分泌的调控机制

【背景】沙眼衣原体(Chlamydia trachomatis,Ct)的分泌蛋白在Ct与宿主细胞的相互作用、感染发育周期及致病过程中发挥着至关重要的作用。GlgA蛋白是课题组前期研究发现的一种新的Ct分泌蛋白,其表达和分泌的具体机制及作用还不清楚。【目的】寻找调控CtGlgA蛋白表达和分泌的分子机制,为后续Ct致病机制研究提供实验基础和新思路。【方法】采用Signal P 4.1软件对GlgA蛋白N端进行信号肽预测分析,并用细菌分泌蛋白特异性阻断剂C16和C1化合物分别或同时处理Ct感染的He La细胞,观察阻断Ⅱ型、Ⅲ型分泌途径对GlgA蛋白分泌的影响;经新生霉素处理、噬斑筛选及穿梭质粒转染技术,构建Ct质粒缺失株和缺失互补株,并鉴定质粒编码基因在两种菌株的缺失及表达情况;间接免疫荧光法观察质粒缺失对GlgA表达和分泌的影响。【结果】GlgA蛋白N端无信号肽序列,细菌Ⅱ型、Ⅲ型分泌途径特异性阻断剂C16和C1化合物不能阻断GlgA的胞浆分泌;Ct质粒缺失株CTD1的质粒编码基因pgp7丢失,且质粒编码蛋白Pgp3及基因组编码蛋白GlgA的表达和分泌现象均消失;Ct缺失互补株CTD1-pGFP::SW2重新获得pgp7基因,并恢复Pgp3蛋白和GlgA的表达和分泌。【结论】初步证实Ct糖原合酶GlgA蛋白的表达和分泌不依赖细菌Ⅱ型和Ⅲ型分泌途径,而且与衣原体质粒密切相关。     

Chlamydia trachomatis and pneumonia in infants: report of two cases.

In two cases of pneumonia associated with Chlamydia trachomatis in infants the symptoms began in the second week of life and the illness was severest at 4 weeks of age. Both infants were afebrile. One had a history of conjunctivitis. Both presented with a characteristic staccato cough and tachypnea but little evidence of peripheral airway obstruction. Chest roentgenograms showed interstitial and alveolar pulmonary infiltration in hyperexpanded lungs. The serum IgM concentrations were markedly elevated. C. trachomatis was cultured from specimens from both infants and one mother, and titres of antibody to the organism were substantially elevated in one infant and one mother.     

Heat-induced modulation of lamin B content in two different cell lines

Previous work in our laboratory indicates that the nuclear matrix protein lamin B is a "prompt" heat shock protein, which increases significantly when human U-1 melanoma and HeLa cells are exposed to 45.5 degrees C for 5-40 min. Using Western blotting, we found that the lamin B content in U-1 and HeLa cells increased to a greater extent during post-heat incubation at 37 degrees C than during the heat dose itself. When HeLa cells were heated at 45.5 degrees C for 30 min, and then incubated at 37 degrees C for up to 7 h, lamin B content was increased significantly (1.69-fold maximum increase at 3 h) compared to unincubated heated cells. Also, thermotolerant HeLa cells showed a greater increase (up to 1.72-fold) in lamin B content during subsequent heating compared to nontolerant cells. The increase in lamin B content in thermotolerant cells, or when heated cells were incubated at 37 degrees C, was also observed in U-1 cells. HeLa cells heated in the presence of glycerol (a heat protector) showed a 1.21-1.72-fold increase in lamin B content compared to cells heated for 10-30 min without glycerol. In contrast, lamin B content decreased 1.23-1.85-fold when cells were heated for 10-30 min in the presence of procaine (a heat sensitizer) compared to cells heated without procaine. These data suggest that lamin B may play an important role in the heat shock response, and that modulation of lamin B content by heat sensitizers or protectors may play a role in regulation of heat sensitivity.     

Thiamine requirement of two different cultured cell lines of soybean

When supplied with 6-benzyladenine (0.5–5 mg/liter) insteadof thiamine, thiamine-requiring soybean cells (strain TU) couldgrow successively. The effect of cytokinin was much more remarkableat a relatively higher concentration of 2,4-dichlorophenoxyaceticacid (4 mg/liter) than at a lower concentration (0.5 mg/liter).Among calli initiated from soybean hypocotyls on a medium withoutthiamine, the thiamine-nonrequiring variant (strain G) was obtainedincidentally. As this cell line became green in light, it couldbe visually separated from the other necrotic tissues. StrainG cells could grow successively not only without thiamine butalso without phytohormones, auxin and cytokinin. This cell linehad relatively higher amounts of chlorophyll and thiamine, andgrew in rigid, large cell aggregates which differed from cellaggregates of the strain TU cell line. The thiamine requirementof plant cultured cells seems to be associated with the degreeof dedifferentiation of the cells rather than the kind of plant.In general, the higher the degree of redifferentiation of thecells, the higher is their thiamine level and the less theyrequire externally supplied thiamine. ¹Present address: Section of Phytochemical Research, Eisai Co.,Ltd., Kawashima, Gifu 483, Japan. (Received December 15, 1978; )