Chlamydia trachomatis persistence: an update

Chlamydial persistence is a reversible state generated during conditions deleterious to growth. In persistence, Chlamydia trachomatis remains viable but atypical, with an enlarged, aberrant form and quiescent metabolism. It favours chronic chlamydiosis, leading to serious sequelae. Although the mechanism of persistence formation is still unknown, more reliable molecular approaches tend to confirm that its occurs in vivo, even lasting 3 years. One approach consists of identifying unprocessed rRNA found only in viable Chlamydia, when infection is not apparent. Another approach, referring to the fact that immunity is type-specific, consists of showing by genotyping that multiple recurrences are due to the same genovar. At the molecular level, persistence is characterized by increased expression of ct755, one of the three heat shock protein (hsp60)-coding genes. In addition, chromosomal replication occurs continuously, and cell division is blocked possibly due to the repression of genes such as ftsW and amiA. At the immunological level, persistence reveals the failure of host-defence mechanisms because of reduced or suppressed pro-inflammatory or cytotoxic responses.     

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.     

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.     

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.     

In vitro tetracycline susceptibility of Chlamydia trachomatis in clinical specimens

A novel approach to determine the tetracycline susceptibility of Chlamydia trachomatis directly from specimens without cell culture propagation and adaptation has been explored. Out of a total of 1290 genital specimens from a sexually transmitted disease clinic, 211 (16.4%) were positive for C. trachomatis. A tetracycline concentration of 0.032 g/ml completely inhibited the appearance of inclusions in all of the 211 positive specimens. Of the positive specimens, 120 (56.9%) and 18 (8.5%) respectively showed the presence of 1 to 9 and 100 or more inclusions per microtiter well in antibiotic free medium. Other antibiotics are being tested in the same manner.A part of this paper was presented at the Canadian Public Health Association, STD meeting in Ottawa, Canada, 28–29 October 1985     

PCR amplification and cycle DNA sequencing analysis of the Chlamydia trachomatis elongation factor Tu gene

Based on the elongation factor Tu (EF-Tu) gene of Chlamydia trachomatis, a pair of oligonucleotide primers CTUFU and CTUFD, were designed to amplify a specific target fragment of 931bp. The PCR assay could detect C. trachomatis in cervical smear specimens obtained from sex workers undergoing routine examination in an STD clinic. Distinct target bands were also amplified from at least 10ng of positive control DNA samples from cultured cells infected with C. trachomatis. PCR with these primers could differentiate C. trachomatis from eight non-chlamydial bacterial species. Further verification could be obtained from the non-digestion of C. trachomatis PCR products by MspA1I restriction endonuclease, in contrast to the digestion of the non-specific PCR products of Klebsiella and Bacillus. Direct cycle DNA sequencing of 450bp of the PCR products of four C. trachomatis isolates revealed complete identity of one isolate with the known sequence of serovar F, while the other three isolates harboured three phenotypically silent point mutations at codons 96, 305 and 312 of the EF-Tu gene. The sequence analyses confirm the authenticity of the target bands, reiterate the conservation and role of the EF-Tu gene in protein biosynthesis, and indicate the utility of the primers for the rapid detection of C. trachomatis.     

A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate

The major outer membrane protein (MOMP) of Chlamydia trachomatis is a highly antigenic and hydrophobic transmembrane protein. Our attempts to express the full-length protein in a soluble form in Escherichia coli and in transgenic plants failed. A chimeric gene construct of C. trachomatis serovar E MOMP was designed in order to increase solubility of the MOMP protein but with retained antigenicity. The designed construct was successfully expressed in E. coli, in Arabidopsis thaliana, and in Daucus carota. The chimeric MOMP expressed in and purified from E. coli was used as antigen for production of antibodies in rabbits. The anti-chimeric MOMP antibodies recognized the corresponding protein in both E. coli and in transgenic plants, as well as in inactivated C. trachomatis elementary bodies. Transgenic Arabidopsis and carrots were characterized for the number of MOMP chimeric genetic inserts and for protein expression. Stable integration of the transgene and the corresponding protein expression were demonstrated in Arabidopsis plants over at least six generations. Transgenic carrots showed a high level of expression of the chimeric MOMP – up to 3% of TSP.     

Rapid homogeneous PCR assay for the detection of Chlamydia trachomatis in urine samples

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease and a major public health problem worldwide. Fast and sensitive point-of-care diagnostics including non-invasive sample collection would be of value for the prevention of C. trachomatis transmission. The aim of this study was to develop a fast, reliable, non-invasive and easy-to-use homogenous PCR assay for the detection of C. trachomatis. Bacteria were concentrated from urine by a simple and fast centrifugation-based urine pretreatment method. Novel automated GenomEra technology was utilized for the rapid closed-tube PCR including time-resolved fluorometric detection of the target using lanthanide chelate labeled probes. We have developed a rapid C. trachomatis assay which provides qualitative results in 1 h with diagnostic sensitivity and specificity of 98.7% and 97.3%, respectively. The novel assay can be performed with minimal laboratory expertise and without sophisticated DNA-extraction devices and has performance comparable to current gold standard assays.     

Fatty acid profiles of Chlamydia using capillary gas chromatography

Fatty acid profiles of purified elementary bodies of Chlamydia trachomatis (CT) serotypes D, G and L3 were investigated by gas liquid chromatography (GLC) utilizing three fused silica capillary columns of different polarities. CT serotype C and C. psittaci (CP) strain DD34 were investigated using one column only due to the lack of adequate quantities of purified material. Significantly similar fatty acid profiles were observed in the serotypes examined. However, based on the percentage ratio of 13-methyl tetradecanoate (i-15:0) to 12-methyl tetradecanoate (a-15:0), serotypes D and L3, with ratios of 0.18 and 0.19, respectively, could be differentiated from serotypes C and G with ratios of 1.3 and 1.5, respectively. CP demonstrated a ratio of 0.4, thus differentiating it from the CT serotypes examined. Fatty acids i-15:0 and a-15:0 were absent in uninfected McCoy cells. Results were significantly comparable in all three capillary columns. This study suggests that GLC could be used for identification and differentiation of Chlamydia serotypes.This paper was presented, in part, at the 55th Conjoint Meeting on Infectious Diseases (CAC-MID) on November 24, 1987, Ottawa, Canada.     

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.     

Chlamydia and mycoplasma infections during pregnancy and their relationships to orofacial cleft

Serum antibodies to Mycoplasma pneumoniae and Chlamydia trachomatis have been studied in a group of newborns with orofacial cleft (OC) and their mothers (n = 59) as compared to a control group of healthy newborns and their mothers (n = 40) assayed by ELISA and Western blot analysis. In the first group, IgG antibodies to M. pneumoniae were found by ELISA in 12 newborns with OC and 22 mothers, while IgA antibodies were detected only in 5 and 11 cases, respectively. IgM antibodies indicating an acute infection were found in 2 mothers only. IgG antibodies to C. trachomatis were found in 2 newborns with OC and 4 mothers. In the control group, IgG antibodies to M. pneumoniae were found in 3 newborns and 7 mothers. IgG antibodies to C trachomatis were observed in 1 newborn and 1 mother, while IgM antibodies to C trachomatis were present in 1 mother only. Immunoblot analysis revealed in newborns with OC and their mothers C. trachomatis-specific bands associated with MOMP 1, 29 kDa, 45 kDa, and heat shock proteins (HSP) 60 and 70. Based on these results we suggest that the risk associated with the exposure to M. pneumoniae and/or C. trachomatis is so far unknown and further study is needed for its elucidation.     

Life cycle and morphology ofMelampsora yezoensis onSalix serissaefolia

Uredinial and telial states of aMelampsora species occurring on the leaves ofSalix serissaefolia were for the first time recorded. Field observations and inoculation experiments showed that the spermogonial and aecial states of the fungus were formed onChelidonium majus var.asiaticum andCorydalis incisa. The fungus was identified asMelampsora yezoensis based on the morphological observation of all the spore states. Urediniospores were able to infectS. erocarpa andS. pierotii as well asS. serissaefolia andS. jessoensis. Salix serissaefolia, S. eriocarpa, andS. pierotii are new uredinial and telial hosts ofM. yezoensis, andC. majus var.asiaticum andC. incisa are new spermogonial and aecial hosts of the fungus. Contribution No. 124, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry University of Tsukuba.     

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.     

The flagellar developmental cycle in algae: flagellar transformation inCyanophora paradoxa (Glaucocystophyceae)

Summary Flagellar development during cell division was studied inCyanophora paradoxa using agarose-embedded cells, Nomarski optics and electronic flash photography. The cells bear two heterodynamic and differently oriented (anterior and posterior) flagella. Prior to cell division, cells produce two new anterior flagella while the parental anterior flagellum transforms into a posterior flagellum. The parental posterior flagellum remains a posterior flagellum throughout this and subsequent cell divisions. The development of a single flagellum thus extends through at least two cell cycles and flagellar heterogeneity is achieved by semiconservative distribution of the flagella during cell division. Based on these principles a universal numbering system for basal bodies and flagella of eukaryotic cells is proposed.     

Cloning and characterization of a secY homolog from Chlamydia trachomatis

Characterization of the genes involved in the process of protein translocation is important in understanding their structure-function relationships. However, little is known about the signals that govern chlamydial gene expression and translocation. We have cloned a 1.7 kb HindIII-PstI fragment containing the secY gene of Chlamydia trachomatis. The complete nucleotide sequence reveals three open reading frames. The amino acid sequence shows highest homology with Escherichia coli proteins L15, SecY and S13, corresponding to the spc- ribosomal protein operons. The product of the C. trachomatis secY gene is composed of 457 amino acids with a calculated molecular mass of 50 195 Daltons. Its amino acid sequence shows 27.4% and 35.7% identity to E. coli and Bacillus subtilis SecY proteins, respectively. The distribution of hydrophobic amino acids in the C. trachomatis secY gene product is suggestive of it being an integral membrane protein with ten transmembrane segments, the second, third and seventh membrane segments sharing > 45% identity with E. coli SceY. Our results suggest that despite evolutionary differences, eubacteria share a similar protein export apparatus.     

Synthesis of disulfide-bonded outer membrane proteins during the developmental cycle of Chlamydia psittaci and Chlamydia trachomatis.

The disulfide bond cross-linked major outer membrane protein (MOMP) of the extracellular elementary bodies (EBs) of Chlamydia psittaci was reduced to its monomeric form within 1 h of entry of EBs into host cells by a process which was inhibited by chloramphenicol, while monomeric forms of three cross-linked cysteine-rich proteins could not be detected in Sarkosyl outer membrane complexes at any time in either extracellular or intracellular forms of C. psittaci. Synthesis and incorporation of the MOMP into outer membrane complexes were detected early in the infection cycle (12 h postinfection), while synthesis and incorporation of the cysteine-rich proteins were not observed until reticulate bodies had begun to reorganize into EBs at 20 to 22 h postinfection. By 46 h postinfection, the intracellular population of C. psittaci consisted mainly of EBs, the outer membrane complexes of which were replete with monomeric MOMP and cross-linked cysteine-rich proteins. Upon lysis of infected cells at 46 h, the MOMP was rapidly cross-linked, and infectious EBs were released. The status of the MOMP of intracellular Chlamydia trachomatis was similar to the status of the MOMP of C. psittaci in that the MOMP was largely uncross-linked at 24 and 48 h postinfection, but formed interpeptide disulfide bonds when it was exposed to an extracellular environment late in the developmental cycle. In contrast to C. psittaci, only a fraction of the cross-linked MOMP of infecting EBs of C. trachomatis was reduced by 4 h postinfection, and reduction of the MOMP was not inhibited by chloramphenicol. Exposure of extracellular EBs of C. trachomatis and C. psittaci to dithiothreitol reduced the MOMP but failed to stimulate metabolic activities normally associated with reticulate bodies.     

Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora erythraea

In carbohydrate-based fermentations of Saccharopolyspora erythraea, a polar knockout of the methylmalonyl-CoA mutase (MCM) gene, mutB, improved erythromycin production an average of 126% (within the range of 102–153% for a 0.95 confidence interval). In oil-based fermentations, where erythromycin production by the wild-type strain averages 184% higher (141–236%, 0.95 CI) than in carbohydrate-based fermentations, the same polar knockout in mutB surprisingly reduced erythromycin production by 66% (53–76%, 0.95 CI). A metabolic model is proposed where in carbohydrate-based fermentations MCM acts as a drain on the methylmalonyl-CoA metabolite pool, and in oil-based fermentations, MCM acts in the reverse direction to fill the methylmalonyl-CoA pool. Therefore, the model explains, in part, how the well-known oil-based process improvement for erythromycin production operates at the biochemical level; furthermore, it illustrates how the mutB erythromycin strain improvement mutation operates at the genetic level in carbohydrate-based fermentations.     

A flow fluorimetric analysis of the cell cycle during growth and differentiation inDictyostelium discoideum

Summary We report a flow fluorimetric analysis of the DNA content of cells and nuclei from vegetative populations and various developmental stages of the cellular slime mouldDictyostelium discoideum using the dyes Hoechst 33258 and mithramycin. Nuclei from all of these populations showed an identical single DNA-content peak, indicating that most vegetative cells and most cells in all developmental stages are in one phase of the cell cycle. Our own data and findings in the literature indicate that this phase is G₂. On the other hand, we also found that various stages, subpopulations of cells at early stages and the different differentiated cell types in the slug stage differ in DNA content per cell. Any particular population typically has one major peak of DNA content, with a modal value that is characteristic for the cell type and for the developmental stage. These differences presumably reflect differences in mitochondrial DNA content per cell.     

Effect of photoperiod and temperature on ovarian cycle of the frogRana tigrina (Daud.)

The effect of varying photoperiod regimes (LD: 20,4; 4,20; 6,18; 18,6 and 12,12) on ovarian follicular development was analysed in the frogRana tigrina maintained at ambient and constant 30° ± l°C for 3 months. The experiments were conducted in early recrudescent and quiescent phases. The frogs were fed guppiesad libitum on alternate day. None of the photoperiod regimes had any effect on the ovaries or the fat bodies, whereas exposure to constant high temperature (regardless of photoperiod) during recrudescent phase induced production of greater number of eggs (∼ 18000 vs 13000 in controls) of ovulatory sizes (> 1400 μm) compared to the corresponding controls maintained at ambient temperature. Hence, ovarian mass also increased in these frogs. In the quiescent phase, high temperature merely enhanced growth of previtellogenic oocytes. In both the phases high temperature caused a reduction in the fat bodies over the respective controls, possibly due to increased metabolic activity. The above findings indicate that temperature plays a key role in the regulation of ovarian cycle ofRana tigrina and that the photoperiodic mechanisms may not govern the annual recrudescence of ovaries in the frog. The study also shows that the frog exhibits the phenomenon of “phenotypic plasticity” in its reproductive behaviour by producing significantly greater number of eggs in response to elevated temperature.     

Improved production of erythromycin A by expression of a heterologous gene encoding S-adenosylmethionine synthetase

An S-adenosylmethionine synthetase (SAM-s) gene from Streptomyces spectabilis was integrated along with vector DNA into the chromosome of a Saccharopolyspora erythraea E2. Elevated production of SAM was observed in the recombinant strain Saccharopolyspora erythraea E1. The results from the bioassay showed that the titer of erythromycin was increased from 920 IU ml⁻¹ by E₂ to approximately 2,000 IU ml⁻¹ by E₁. High performance liquid chromatography (HPLC) analysis revealed that there was a 132% increase in erythromycin A compared with the original strain, while the erythromycin B, the main impurity component in erythromycin, was decreased by 30%. The sporulation process was inhibited, while the SAM-s gene was expressed. The addition of the exogenous SAM also inhibited sporulation and promoted an increase in erythromycin titers. An erratum to this article can be found at