glycogen and Chlamydia-Infections

Chlamydia trachomatis is an obligate intracellular epitheliotropic bacterial pathogen of humans. Infection of the eye can result in trachoma, the leading cause of preventable blindness in the world. The pathophysiology of blinding trachoma is driven by multiple episodes of reinfection of conjunctival epithelial cells, producing an intense chronic inflammatory response resulting in submucosal tissue remodeling and scarring. Recent reports have shown that infection with trachoma organisms lacking the cryptic chlamydial plasmid is highly attenuated in macaque eyes, a relevant experimental model of human trachoma infection. To better understand the molecular basis of plasmid-mediated infection attenuation and the potential modulation of host immunity, we conducted transcriptional profiling of human epithelial cells infected with C. trachomatis plasmid-bearing (A2497) and plasmid-deficient (A2497P(-)) organisms. Infection of human epithelial cells with either strain increased the expression of host genes coding for proinflammatory (granulocyte-macrophage colony-stimulating factor [GM-CSF], macrophage colony-stimulating factor [MCSF], interleukin-6 [IL-6], IL-8, IL-1α, CXCL1, CXCL2, CXCL3, intercellular adhesion molecule 1 [ICAM1]), chemoattraction (CCL20, CCL5, CXCL10), immune suppression (PD-L1, NFKB1B, TNFAIP3, CGB), apoptosis (CASP9, FAS, IL-24), and cell growth and fibrosis (EGR1 and IL-20) proteins. Statistically significant increases in the levels of expression of many of these genes were found in A2497-infected cells compared to the levels of expression in A2497P(-)-infected cells. Our findings suggest that the chlamydial plasmid plays a focal role in the host cell inflammatory response to infection and immune avoidance. These results provide new insights into the role of the chlamydial plasmid as a chlamydial virulence factor and its contributions to trachoma pathogenesis.

Topics: Bacterial Proteins; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Chlamydia Infections; Chlamydia trachomatis; Cytokines; Epithelial Cells; Gene Expression Profiling; Glycogen; HeLa Cells; Humans; Inflammation; Interleukins; Plasmids; Trachoma; Virulence Factors

The development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis provides the basis for the detailed investigation of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In this study we constructed a plasmid vector with CDS6 deleted (pCDS6KO) from the original Escherichia coli/C. trachomatis shuttle vector pGFP::SW2. pCDS6KO was transformed into a clinical isolate of C. trachomatis from Sweden that is plasmid-free (C. trachomatis SWFP-). Penicillin-resistant transformants expressing the green fluorescent protein were selected. These transformants did not stain with iodine, indicating that this property is regulated by CDS6 or its gene product. In addition, mature inclusions of C. trachomatis SWFP- transformed by pCDS6KO displayed an identical morphological phenotype to the untransformed plasmid-free recipient host. In this phenotype the morphology of inclusions was altered with the chlamydiae lining the periphery of the inclusion leaving a 'hole' in the centre. These green fluorescent inclusions appear 'doughnut-shaped' with an empty centre when examined under blue light, giving rise to a characteristic 'black hole' phenotype. Our study demonstrates the power of the new genetic system for investigating chlamydial gene function using gene deletion technology.

Topics: Bacterial Proteins; Chlamydia Infections; Chlamydia trachomatis; Cloning, Molecular; Escherichia coli; Gene Deletion; Gene Transfer Techniques; Genetic Vectors; Genitalia; Glycogen; Phenotype; Plasmids; Transformation, Bacterial

We previously demonstrated that plasmid-deficient Chlamydia muridarum retains the ability to infect the murine genital tract but does not elicit oviduct pathology because it fails to activate Toll-like receptor 2 (TLR2). We derived a plasmid-cured derivative of the human genital isolate Chlamydia trachomatis D/UW-3/Cx, strain CTD153, which also fails to activate TLR2, indicating this virulence phenotype is associated with plasmid loss in both C. trachomatis and C. muridarum. As observed with plasmid-deficient C. muridarum, CTD153 displayed impaired accumulation of glycogen within inclusions. Transcriptional profiling of the plasmid-deficient strains by using custom microarrays identified a conserved group of chromosomal loci, the expression of which was similarly controlled in plasmid-deficient C. muridarum strains CM972 and CM3.1 and plasmid-deficient C. trachomatis CTD153. However, although expression of glycogen synthase, encoded by glgA, was greatly reduced in CTD153, it was unaltered in plasmid-deficient C. muridarum strains. Thus, additional plasmid-associated factors are required for glycogen accumulation by this chlamydial species. Furthermore, in C. trachomatis, glgA and other plasmid-responsive chromosomal loci (PRCLs) were transcriptionally responsive to glucose limitation, indicating that additional regulatory elements may be involved in the coordinated expression of these candidate virulence effectors. Glucose-limited C. trachomatis displayed reduced TLR2 stimulation in an in vitro assay. During human chlamydial infection, glucose limitation may decrease chlamydial virulence through its effects on plasmid-responsive chromosomal genes.

Topics: Animals; Cell Line; Chlamydia Infections; Chlamydia muridarum; Chlamydia trachomatis; Chromosomes, Bacterial; Gene Expression; Gene Expression Regulation, Bacterial; Genetic Loci; Glucose; Glycogen; Glycogen Synthase; Humans; Inclusion Bodies; Mice; Oligonucleotide Array Sequence Analysis; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; Toll-Like Receptor 2; Virulence

Using a new plaque cloning technique, we obtained unique Chlamydia trachomatis strains which were confirmed to be free of the 7.5-kb common plasmid and glycogen in inclusions. The in vitro susceptibility of these strains to various chemotherapeutic agents was tested by comparison with their parent strains and clinical isolates possessing the common plasmid. No difference was detected for any of the agents tested, indicating that the 7.5-kb common plasmid is unrelated to the drug resistance of C. trachomatis.

Topics: Anti-Bacterial Agents; Anti-Infective Agents; Chlamydia Infections; Chlamydia trachomatis; Clarithromycin; Drug Resistance, Microbial; Erythromycin; Fluoroquinolones; Glycogen; Humans; Male; Microbial Sensitivity Tests; Minocycline; Ofloxacin; Plasmids; Urethritis

Chlamydia trachomatis can synthesize glycogen at various stages in its developmental cycle. The glycogen content of female genital epithelial cells was detected by anthrone, and the results were compared with those from PCR. A total of 320 cervical samples were examined. Of 92 specimens that were positive by PCR, 78 were positive and 14 were negative by the glycogen assay. Of 228 specimens that were negative by PCR, 220 were negative and 8 were positive by the glycogen assay. The sensitivity and specificity of the glycogen assay obtained from these data were 84.8% (78 of 92) and 96.5% (220 of 228), respectively. Use of the glycogen assay to detect the glycogen content in genital epithelial cells may be helpful in the diagnosis of C. trachomatis infection. This is an easy, fast, and inexpensive assay and can be done in less-sophisticated labs.

Topics: Cervix Uteri; Chlamydia Infections; Chlamydia trachomatis; Female; Genital Diseases, Female; Glycogen; Humans; Polymerase Chain Reaction; Sensitivity and Specificity

The association of mitochondria with the inclusions of five strains of Chlamydia psittaci, two strains of C. trachomatis and one strain of C. pneumoniae was examined by electron microscopy. The results demonstrated that mitochondrial association occurred only with the C. psittaci inclusions, but not with the C. trachomatis and C. pneumoniae inclusions. Multiple inclusions formed by infection with a single strain could fuse with each other during their expansion, but fusion never occurred between the C. trachomatis and C. psittaci inclusions as a result of dual infection with these different species.

Topics: Cell Nucleus; Cells, Cultured; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Chlamydophila pneumoniae; Chlamydophila psittaci; Glycogen; HeLa Cells; Host-Parasite Interactions; Humans; Inclusion Bodies; Mitochondria; Models, Biological

Topics: Adult; Anti-Bacterial Agents; Antibodies, Bacterial; Antigens, Bacterial; Child; Chlamydia Infections; Chlamydia trachomatis; Chlamydophila psittaci; Conjunctivitis, Inclusion; Female; Glycogen; Humans; Infant, Newborn; Trachoma

Topics: Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Chlamydophila psittaci; Glycogen; Inclusion Bodies; Lymphogranuloma Venereum; Pathology; Penicillins; Psittacosis; Research; Tissue Culture Techniques; Trachoma