Application of a C. trachomatis expression system to identify C. pneumoniae proteins translocated into host cells.

Chlamydia pneumoniae is a Gram-negative, obligate intracellular pathogen that causes community-acquired respiratory infections. C. pneumoniae uses a cell contact-dependent type-III secretion (T3S) system to translocate pathogen effector proteins that manipulate host cellular functions. While several C. pneumoniae T3S effectors have been proposed, few have been experimentally confirmed in Chlamydia In this study, we expressed 382 C. pneumoniae genes in C. trachomatis as fusion proteins to an epitope tag derived from glycogen synthase kinase 3ß (GSK3ß) which is the target of phosphorylation by mammalian kinases. Based on the detection of the tagged C. pneumoniae protein with anti-phospho GSK3ß antibodies, we identified 49 novel C. pneumoniae-specific proteins that are translocated by C. trachomatis into the host cytoplasm and thus likely play a role as modifiers of host cellular functions. In this manner, we identified and characterized a new C. pneumoniae effector CPj0678 that recruits the host cell protein PACSIN2 to the plasma membrane. These findings indicate that C. trachomatis provides a powerful screening system to detect candidate effector proteins encoded by other pathogenic and endosymbiotic Chlamydia species.Importance Chlamydia injects numerous effector proteins into host cells to manipulate cellular functions important for bacterial survival. Based on findings in C. trachomatis, it has been proposed that between 5-10% of the C. pneumoniae genome, a related respiratory pathogen, encodes secreted effectors. However only a few C. pneumoniae effectors have been identified and experimentally validated. With the development of methods for the stable genetic transformation of C. trachomatis, it is now possible to use C. trachomatis shuttle plasmids to express and explore the function of proteins from other Chlamydia in a more relevant bacterial system. In this study, we experimentally determined that 49 C. pneumoniae-specific proteins are translocated into the host cytoplasm by Chlamydia secretion systems, and identify a novel effector required to recruit the host factor PACSIN2 to the plasma membrane during infection.

Effect of a Moderate Carbohydrate-Restricted Diet on DPP-4 Inhibitor Action among Individuals with Type 2 Diabetes Mellitus: A 6-Month Intervention Study.

To decrease body weight and insulin resistance, a calorie-restricted diet-with minimal caloric intake required for daily activities-is the primary treatment strategy for patients with type 2 diabetes (T2D) in Japan. However, many patients cannot continue with this diet for long, because calorie restriction is difficult and nutritional balance is hard to understand. Carbohydrate-restricted diets are easier for patients than conventional calorie-restricted diet. In this study we aimed to elucidate the effects of a moderate carbohydrate-restricted diet on glucose metabolism and renal function in patients with T2D on dipeptidyl peptidase-4 (DPP-4) inhibitors. Nineteen outpatients with T2D continued on a moderate carbohydrate-restricted diet (targeting 50% of calories) for 6 mo. Meanwhile, 10 other outpatients with T2D on DPP-4 inhibitors had the conventional calorie-restricted diet using the food exchange table. No change in prescription drugs occurred for both groups during the study period. After the intervention, the carbohydrate content in dietary intake was lowered significantly from 56.8±8.3 to 46.8±10.1%, while the lipid concentration, primarily n-6 polyunsaturated fatty acids, was significantly increased. There was no significant change in protein intake. Hemoglobin A1c (HbA1c) fell from 7.22±0.74% to 6.95±0.72% (mean±SD). Furthermore, salt intake decreased significantly from 6.8±2.5 g prior to the intervention, to 5.7±1.9 g after the intervention. The estimated glomerular filtration rates (eGFR) decreased slightly, while serum creatinine levels did not change. These findings suggest that a moderate carbohydrate-restricted diet (50%) is effective in patients with T2D, without affecting kidney function.

Pretreatment monocyte counts and neutrophil counts predict the risk for febrile neutropenia in patients undergoing TPF chemotherapy for head and neck squamous cell carcinoma.

BACKGROUND: Febrile neutropenia (FN) is the most serious hematologic toxicity of systemic chemotherapy. However, accurate prediction of FN development has been difficult because the risk varies largely depending on the chemotherapy regimen and various individual factors. METHODS: We retrospectively analyzed diverse clinical factors including pretreatment hematological parameters to clarify the reliable predictors of FN development during chemotherapy with a docetaxel, cisplatin, and fluorouracil (TPF) regimen in patients with head and neck squamous cell carcinoma. RESULTS: Among the 50 patients, grade ≥3 neutropenia, grade 4 neutropenia, and FN developed in 36 (72%), 21 (42%), and 12 (24%) patients, respectively. Multivariate logistic regression revealed that a pretreatment absolute monocyte count (AMC) <370/mm3 is an independent predictor of TPF chemotherapy-induced FN (odds ratio=6.000, p=0.017). The predictive performance of the model combining AMC and absolute neutrophil count (ANC), in which the high-risk group was defined as having an AMC <370/mm3 and/or ANC <3500/mm3, was superior (area under the curve [AUC]=0.745) to that of the model with a cutoff for AMC alone (AUC=0.679). CONCLUSIONS: On the basis of our results, we recommend primary prophylactic use of granulocyte colony-stimulating factor and/or antibiotics selectively for patients predicted to be at high risk for TPF chemotherapy-induced FN.

Pyoderma gangrenosum on the nose.

Pyoderma gangrenosum is a rare ulcerative condition associated with various systemic diseases. Lesions on the lower extremities and the trunk are common, but lesions on the nose are rare. Here we report a case of pyoderma gangrenosum on the nose. A 33-year-old woman presented with fever, nasal obstruction, and painful swelling on the nasal bridge. Physical examination revealed swellings on the nasal septal mucosa bilaterally. Computed tomography showed a septal abscess and a subcutaneous abscess on the nasal bridge. The lesions worsened despite treatment with intravenous antibiotics and abscess drainage. Meanwhile, the patient also complained of bloody stools and was diagnosed with ulcerative colitis. Therefore, pyoderma gangrenosum on the nose was suspected, and was diagnosed by exclusion of other diseases. Treatment with systemic corticosteroids was started and the nasal lesions improved rapidly. However, saddle nose deformity occurred. A review of the literature reveals that pyoderma gangrenosum on the nose can cause ulcerations, septal abscess, and sinusitis. Further, there is a high likelihood of nasal complications, including saddle nose deformity, septal perforation, and skin defects. Pyoderma gangrenosum should be included in the differential diagnosis when nasal ulceration, abscesses, and sinusitis do not improve with antibiotics and drainage.

Ehrlichia chaffeensis uses its surface protein EtpE to bind GPI-anchored protein DNase X and trigger entry into mammalian cells.

Ehrlichia chaffeensis, an obligatory intracellular rickettsial pathogen, enters and replicates in monocytes/macrophages and several non-phagocytic cells. E. chaffeensis entry into mammalian cells is essential not only for causing the emerging zoonosis, human monocytic ehrlichiosis, but also for its survival. It remains unclear if E. chaffeensis has evolved a specific surface protein that functions as an 'invasin' to mediate its entry. We report a novel entry triggering protein of Ehrlichia, EtpE that functions as an invasin. EtpE is an outer membrane protein and an antibody against EtpE (the C-terminal fragment, EtpE-C) greatly inhibited E. chaffeensis binding, entry and infection of both phagocytes and non-phagocytes. EtpE-C-immunization of mice significantly inhibited E. chaffeensis infection. EtpE-C-coated latex beads, used to investigate whether EtpE-C can mediate cell invasion, entered both phagocytes and non-phagocytes and the entry was blocked by compounds that block E. chaffeensis entry. None of these compounds blocked uptake of non-coated beads by phagocytes. Yeast two-hybrid screening revealed that DNase X, a glycosylphosphatidyl inositol-anchored mammalian cell-surface protein binds EtpE-C. This was confirmed by far-Western blotting, affinity pull-down, co-immunoprecipitation, immunofluorescence labeling, and live-cell image analysis. EtpE-C-coated beads entered bone marrow-derived macrophages (BMDMs) from wild-type mice, whereas they neither bound nor entered BMDMs from DNase X(-/-) mice. Antibody against DNase X or DNase X knock-down by small interfering RNA impaired E. chaffeensis binding, entry, and infection. E. chaffeensis entry and infection rates of BMDMs from DNase X(-/-) mice and bacterial load in the peripheral blood in experimentally infected DNase X(-/-) mice, were significantly lower than those from wild-type mice. Thus this obligatory intracellular pathogen evolved a unique protein EtpE that binds DNase X to enter and infect eukaryotic cells. This study is the first to demonstrate the invasin and its mammalian receptor, and their in vivo relevance in any ehrlichial species.

Mutations of FLVCR1 in posterior column ataxia and retinitis pigmentosa result in the loss of heme export activity.

The feline leukemia virus subgroup C receptor 1 (FLVCR1) is a heme exporter that maintains the intracellular heme concentration. FLVCR1 was previously assumed to be involved in Diamond-Blackfan anemia, and it was recently reported that mutations in the FLVCR1 gene are found in patients with posterior column ataxia and retinitis pigmentosa (PCARP). Four mutations in FLVCR1 (Asn121Asp, Cys192Arg, Ala241Thr, and Gly493Arg) are located within putative transmembrane domains; however, the effects of FLVCR1 mutations on PCARP are unclear. In this study, we analyzed the function of FLVCR1 mutants by using a fluorescent heme analog as a transporter substrate, and found that all 4 FLVCR1 mutants lost their heme export activity. To investigate the mechanism responsible for this loss of activity, we determined the subcellular localization of FLVCR1 mutants. FLVCR1 mutants did not localize to the plasma membrane and were observed in intracellular structures, including lysosomes. We hypothesize that the loss of function of FLVCR1 mutants is caused by their mislocation. We examined the half-life of FLVCR1 in cells, which was >16h for wild-type FLVCR1 compared with 2-4h for the mutants. Based on these results, we propose that FLVCR1 mutants failed to fold properly in the ER, were rapidly degraded in the lysosomes, and therefore, could not export heme out of cells. Thus, accumulation of heme in FLVCR1-mutant cells could cause cellular toxicity.

Genomic screening for Chlamydophila pneumoniae-specific antigens using serum samples from patients with primary infection.

Chlamydophila pneumoniae, an obligate intracellular human pathogen, causes respiratory tract infections. The most common techniques used for the serological diagnosis of C. pneumoniae infections are microimmunofluorescence tests and commercial serological ELISA tests; these are based on the detection of antibodies against whole chlamydial elementary bodies and lipopolysaccharide/outer membrane protein, respectively. Identification of more specific and highly immunodominant antigens is essential for the development of new serodiagnostic assays. To identify novel specific antigens from C. pneumoniae, we screened 455 genes with unknown function in the genome of C. pneumoniae J138. Extracts of Saccharomyces cerevisiae cells expressing GFP-tagged C. pneumoniae proteins were subjected to Western blot analysis using serum samples from C. pneumoniae-infected patients as the primary antibodies. From this comprehensive analysis, 58 clones expressing C. pneumoniae open reading frames, including hypothetical proteins, were identified as antigens. These results have provided useful information for the development of new serological tools for the diagnosis for C. pneumoniae infections and for the development of vaccines in future.

Insights into the CtrA regulon in development of stress resistance in obligatory intracellular pathogen Ehrlichia chaffeensis.

Ehrlichia chaffeensis is an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Ehrlichiae have a biphasic developmental cycle consisting of dense-cored cells (DCs) and reticulate cells (RCs). Isolated DCs are more stress resistant and infectious than RCs. Here, we report that a response regulator, CtrA was upregulated in human monocytes at the late growth stage when DCs develop. E. chaffeensis CtrA bound to the promoters of late-stage transcribed genes: ctrA, ompA (peptidoglycan-associated lipoprotein), bolA (stress-induced morphogen) and surE (stationary-phase survival protein), which contain CtrA-binding motifs, and transactivated ompA, surE and bolA promoter-lacZ fusions in Escherichia coli. OmpA was predominantly expressed in DCs. E. chaffeensis binding to and subsequent infection of monocytes were inhibited by anti-OmpA IgG. E. chaffeensis BolA bound to the promoters of genes encoding outer surface proteins TRP120 and ECH_1038, which were expressed in DCs, and transactivated trp120 and ECH_1038 promoter-lacZ fusions. E. chaffeensis bolA complemented a stress-sensitive E. coli bolA mutant. E. coli expressing E. chaffeensis SurE exhibited increased resistance to osmotic stress. Our results suggest that E. chaffeensis CtrA plays a role in co-ordinating development of the stress resistance for passage from the present to the next host cells through its regulon.

Ehrlichia chaffeensis induces monocyte inflammatory responses through MyD88, ERK, and NF-κB but not through TRIF, interleukin-1 receptor 1 (IL-1R1)/IL-18R1, or toll-like receptors.

Human monocytic ehrlichiosis, an influenza-like illness accompanied by signs of hepatitis, is caused by infection of monocytes/macrophages with a lipopolysaccharide-deficient bacterium, Ehrlichia chaffeensis. The E. chaffeensis strain Wakulla induces diffuse hepatitis with neutrophil infiltration in mice with severe combined immunodeficiency, which is accompanied by strong CXCL2 (mouse functional homolog of interleukin-8 [IL-8]) and tumor necrosis factor alpha (TNF-α) expression in the liver. In this study, we found that expression of IL-1ß, CXCL2, and TNF-α was induced by strain Wakulla in mouse bone marrow-derived macrophages; this expression was dependent on MyD88, but not on TRIF, TLR2/4, IL-1R1/IL-18R1, or endosome acidification. When the human leukemia cell line THP-1 was exposed to E. chaffeensis, significant upregulation of IL-8, IL-1ß, and TNF-α mRNA and extracellular regulated kinase 2 (ERK2) activation were detected. U0126 (inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 [MEK1/2] upstream of ERK), manumycin A (Ras inhibitor), BAY43-9006 (Raf-1 inhibitor), and NS-50 (inhibitor of NF-κB nuclear translocation) inhibited the cytokine gene expression. A luciferase reporter assay using HEK293 cells, which lack Toll-like receptors (TLRs), showed activation of both the IL-8 promoter and NF-κB by E. chaffeensis. Activation of the IL-8 promoter in transfected HEK293 cells was inhibited by manumycin A, BAY43-9006, U0126, and transfection with a dominant-negative Ras mutant. These results indicate that the E. chaffeensis Wakulla strain can induce inflammatory responses through MyD88-dependent NF-κB and ERK pathways, without the involvement of TRIF and TLRs.

Four VirB6 paralogs and VirB9 are expressed and interact in Ehrlichia chaffeensis-containing vacuoles.

The type IV secretion system is an important virulence factor in several host cell-associated pathogens, as it delivers various bacterial macromolecules to target eukaryotic cells. Genes homologous to several virB genes and virD4 of Agrobacterium tumefaciens are found in an intravacuolar pathogen Ehrlichia chaffeensis, the tick-borne causative agent of human monocytic ehrlichiosis. In particular, despite its small genome size, E. chaffeensis has four tandem virB6 paralogs (virB6-1, -2, -3, and -4) that are 3- to 10-fold larger than A. tumefaciens virB6. The present study for the first time illustrates the relevance of the larger quadruple VirB6 paralogs by demonstrating the protein expression and interaction in E. chaffeensis. All four virB6 paralogs were cotranscribed in THP-1 human leukemia and ISE6 tick cell cultures. The four VirB6 proteins and VirB9 were expressed by E. chaffeensis in THP-1 cells, and amounts of these five proteins were similar in isolated E. chaffeensis-containing vacuoles and vacuole-free E. chaffeensis. In addition, an 80-kDa fragment of VirB6-2 was detected, which was strikingly more prevalent in E. chaffeensis-containing vacuoles than in vacuole-free E. chaffeensis. Coimmunoprecipitation analysis revealed VirB9 interaction with VirB6-1 and VirB6-2; VirB6-4 interaction with VirB6-1, VirB6-2, and VirB6-3; and VirB6-2 80-kDa fragment interaction with VirB6-3 and VirB6-4. The interaction of VirB9 and VirB6-2 was confirmed by far-Western blotting. The results suggest that E. chaffeensis VirB9, the quadruple VirB6 proteins, and the VirB6-2 80-kDa fragment form a unique molecular subassembly to cooperate in type IV secretion.

Liver transcriptome profiles associated with strain-specific Ehrlichia chaffeensis-induced hepatitis in SCID mice.

Infection of humans with Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, can cause hepatitis of various levels of severity. When the three human isolates of E. chaffeensis, each belonging to a different genogroup, are inoculated into severe combined immunodeficiency mice, the order of severity of clinical signs and bacterial burden detected in the liver is as follows (from greatest to least severity and highest to lowest burden): strain Wakulla, followed by strain Liberty, followed by strain Arkansas. In this article, we used microarray analysis to define transcriptional profiles characteristic of the histopathological features in the mouse liver. Cytokine and chemokine profiles and their receptor profiles were strikingly different among the three strains of E. chaffeensis: gamma interferon, CCL5, CXCL1, CXCL2, CXCL7, CXCL9, interleukin 2 receptor gamma (IL2Rgamma), IL21R, CCR2, and CXCR6 were highly upregulated with strain Arkansas; and tumor necrosis factor (TNF), CCL2, CCL3, CCL5, CCL6, CCL12, CCL20, CXCL2, CXCL7, CXCL9, CXCL13, TNF receptor superfamily 9 (TNFRSF9), TNFRSF13beta, IL1R2, IL2Rgamma, IL20Rbeta, IL21R, CCR1, CCR2, and CXCR4 were highly upregulated with strain Wakulla. With strain Liberty, only CXCL13 was highly upregulated, and IL13Ralpha2 was downregulated. In livers infected with the Arkansas strain, monocytes/macrophages and NK cells were enriched in the granulomas and an increase in NK cell marker mRNAs was detected. Livers infected with the Wakulla strain displayed infiltration of significantly more neutrophils and an increase in neutrophil marker mRNAs. Genes commonly upregulated in liver tissue infected with the three strains are other host innate immune and inflammatory response genes, including those encoding several acute-phase proteins. Genes downregulated commonly are related to host physiologic functions. The results suggest that marked modulation of host cytokine and chemokine profiles by E. chaffeensis strains underlies the distinct host liver disease.

Genome-wide analysis of Chlamydophila pneumoniae gene expression at the late stage of infection.

Chlamydophila pneumoniae, an obligate intracellular eubacterium, changes its form from a vegetative reticulate body into an infectious elementary body during the late stage of its infection cycle. Comprehension of the molecular events in the morphological change is important to understand the switching mechanism between acute and chronic infection, which is deemed to relate to the pathogenesis of atherosclerosis. Herein, we have attempted to screen genes expressed in the late stage with a genome-wide DNA microarray, resulting in nomination of 17 genes as the late-stage genes. Fourteen of the 17 genes and six other genes predicted as late-stage genes were confirmed to be up-regulated in the late stage with a quantitative reverse transcriptase-polymerase chain reaction. These 20 late-stage genes were classified into two groups by clustering analysis: 'drastically induced' and 'moderately induced' genes. Out of eight drastically induced genes, four contain sigma(28) promoter-like sequences and the other four contain an upstream common sequence. It suggests that besides sigma(28), there are certain up-regulatory mechanisms at the late stage, which may be involved in the chlamydial morphological change and thus pathogenesis.

Virulence potential of Ehrlichia chaffeensis strains of distinct genome sequences.

Human monocytic ehrlichiosis, one of the most frequent life-threatening tick-borne zoonoses, is caused by Ehrlichia chaffeensis that lacks endotoxin and peptidoglycan. While sequence polymorphisms in several genes in E. chaffeensis strains have been reported, global genomic divergence and biological differences among strains are unknown. The objectives of the present study were to compare the genome sequences of strains of E. chaffeensis and to examine the virulence potentials of the strains with defined genome sequences. Genomic DNA was extracted from purified E. chaffeensis strains Wakulla and Liberty, and comparative genome hybridization was performed using a densely tiled microarray of 147,027 chromosome positions of the E. chaffeensis strain Arkansas genome. The results revealed that 4,663 and 5,325 positions in the chromosomes of strains Wakulla and Liberty, respectively, were different from those in the chromosome of strain Arkansas, including three common major polymorphic chromosomal regions. Of various functional categories, the differences were most concentrated in genes predicted to encode cell envelope proteins. Of all the open reading frames (ORFs), 21 omp-1 (p28 gene) paralogs, nine genes encoding hypothetical proteins, two genes encoding ankyrin repeat proteins, and hemE contained the most differences. Several highly polymorphic ORFs were confirmed by sequencing. When the E. chaffeensis strains were inoculated into severe combined immunodeficiency mice, the order of the severity of clinical signs and the bacterial burden detected in mice was Wakulla > Liberty > Arkansas. Severe diffuse inflammation and granulomatous inflammation were evident in the livers of mice infected with strains Wakulla and Arkansas, respectively, but not in the livers of mice infected with strain Liberty. These results revealed distinct virulence phenotypes of E. chaffeensis strains with defined genome sequences.

Chlamydial SET domain protein functions as a histone methyltransferase.

SET domain genes have been identified in numbers of bacterial genomes based on similarity to SET domains of eukaryotic histone methyltransferases. Herein, a Chlamydophila pneumoniae SET domain gene was clarified to be coincidently expressed with hctA and hctB genes encoding chlamydial histone H1-like proteins, Hc1 and Hc2, respectively. The SET domain protein (cpnSET) is localized in chlamydial cells and interacts with Hc1 and Hc2 through the C-terminal SET domain. As expected from conservation of catalytic sites in cpnSET, it functions as a protein methyltransferase to murine histone H3 and Hc1. However, little is known about protein methylation in the molecular pathogenesis of chlamydial infection. cpnSET may play an important role in chlamydial cell maturation due to modification of chlamydial histone H1-like proteins.

Identification of genes linked to gefitinib treatment in prostate cancer cell lines with or without resistance to androgen: a clue to application of gefitinib to hormone-resistant prostate cancer.

Understanding the molecular action of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, might allow us to perform more effective therapies for hormone-independent advanced prostate cancer. A DNA microarray study was undertaken to comprehensively analyze the alteration of levels of 1,081 genes after gefitinib treatment in androgen-independent PC3 and DU145 cells and androgen-dependent LNCaP cells. The proliferation of PC3, DU145 and LNCaP cells was significantly inhibited by 50.2%, 83.8% and 55.2%, respectively, 6 days after 10 microM gefitinib administration. Of the above 1,081 genes, we identified 23, 13 and 33 genes with significantly different expression in PC3, DU145 and LNCaP cells, respectively, 24 h after 10 microM-gefitinib exposure. Among the identified genes, only Quiescin Q6, a negative cell cycle regulator, was increased after gefitinib treatment in all three cell lines regardless of gefitinib sensitivity. Except for Quiescin Q6, there were no overlapping genes between PC3 and DU145 cells. However, levels of several oncogenes or proliferation-related genes were changed after gefitinib treatment in the 2 androgen-independent cell lines. We also identified 7 unique genes [glycyl-tRNA synthetase, interferon, alpha-inducible protein, stratifin, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1, dual specificity phosphatase 9, guanine nucleotide binding protein (G protein) beta polypeptide 2, neural retina leucine zipper] whose levels were altered exclusively after gefitinib administration in gefitinib-resistant PC3 and LNCaP cells, but not in DU145 cells, suggesting that these 7 genes could be targets for overcoming gefitinib resistance. Collectively, our molecular profiling data will serve as a framework for understanding the molecular action of gefitinib for prostate cancer.

Serotonin and melatonin, neurohormones for homeostasis, as novel inhibitors of infections by the intracellular parasite chlamydia.

OBJECTIVES: Chlamydiae are obligate intracellular bacteria, causing a variety of diseases, i.e. pneumonia, sexually transmitted disease, conjunctivitis and zoonosis. Tryptophan depletion by interferon-gamma (IFN-gamma) is the most important host defence system against chlamydial infection. Thus chlamydial tryptophan metabolism is thought to play key roles for IFN-gamma resistance, persistent infection and host/tissue tropisms. We tested tryptophan derivatives for activity against chlamydia-infected cells. METHODS: Rates of chlamydial infection and sizes of the inclusions were evaluated by in vitro infection using three Chlamydiaceae species, Chlamydia trachomatis, Chlamydophila pneumoniae and Chlamydophila felis, which show significant divergence of tryptophan synthesis genes and different susceptibilities to IFN-gamma. RESULTS: Melatonin and serotonin, which are recognized as neural hormones for maintenance of organism homeostasis, reduced chlamydial infection but not other bacterial growth tested here. Unlike IFN-gamma, melatonin limited infection of all three chlamydiae and the effects were not recovered by tryptophan supplementation. Melatonin treatment only of host cells could diminish infection and the infection reduction was neutralized by a pertussis toxin, an inhibitor of G proteins. Ligands of melatonin and serotonin receptors also hampered infection. CONCLUSIONS: Inhibition mechanisms of chlamydial infection by melatonin and serotonin appear to be different from those of IFN-gamma and involve specific G-protein-coupled receptors. Melatonin is deemed to inhibit early progression of the chlamydial development cycle, such as establishment of intracellular infection and/or conversion from elementary body to reticulate body. Utilization of melatonin, serotonin or their derivatives may be advantageous for harmless prevention of chlamydial infection.

Aspirin inhibits Chlamydia pneumoniae-induced NF-kappa B activation, cyclo-oxygenase-2 expression and prostaglandin E2 synthesis and attenuates chlamydial growth.

Infection with Chlamydia pneumoniae has been implicated as a potential risk factor for atherosclerosis. This study was designed to investigate the mechanisms of the anti-chlamydial activity of aspirin. A reporter gene assay for NF-kappa B activity, immunoblot analysis for cyclo-oxygenase (COX)-2 and radioimmunoassay for prostaglandin E(2) (PGE(2)) were performed. Following infection of HEp-2 cells with C. pneumoniae, NF-kappa B was activated, COX-2 was induced and PGE(2) was elevated. Aspirin inhibited NF-kappa B activation at a concentration of 0.1 mM, partially inhibited COX-2 induction and blocked PGE(2) synthesis completely. In addition, high doses of aspirin (1 and 2 mM) inhibited chlamydial growth in HEp-2 cells, decreasing the number and size of inclusion bodies; this effect could be overcome by adding tryptophan to the culture. Indomethacin also blocked the synthesis of PGE(2), but had no effect on COX-2 expression or chlamydial growth. These results indicate that aspirin not only has an anti-inflammatory activity through prevention of NF-kappa B activation but also has anti-chlamydial activity at high doses, possibly through depletion of tryptophan in HEp-2 cells.

In silico inference of inclusion membrane protein family in obligate intracellular parasites chlamydiae.

Chlamydiae are obligate intracellular pathogens that proliferate only inside a vacuole, called an inclusion. Chlamydial Inc proteins are known to be a major component of the inclusion membrane, but little is known about the gene number and function. The Inc proteins share very low sequence similarity but a similar hydropathy profile among them. Using the hydropathy profile, we computationally searched the open reading frames (ORFs) having a similar profile and predicted 90 and 36 ORFs (Inc-like ORFs) as candidates for Inc proteins in Chlamydia pneumoniae J138 and Chlamydia trachomatis serovar D, respectively. On the other hand, only a few Inc-like ORFs were found in organisms other than chlamydiae, suggesting that the Inc-like ORFs are specific to chlamydiae. Comparative genome analysis also revealed that the Inc-like ORFs have multiplied and diverged as paralogues and orthologues in the chlamydial genomes, and that some Inc-like ORFs lacked the N-terminal portion or encoded the split form. The data suggest that these gene products constitute a large protein family and may play an important role in chlamydial infection, growth and survival in the host cell.

Direct atomic force microscopy visualization of integration host factor-induced DNA bending structure of the promoter regulatory region on the Pseudomonas TOL plasmid.

Atomic force microscopy (AFM) was used to analyze DNA bending induced by integration host factor (IHF). The direct AFM visualization of IHF-DNA complexes on the OP1 promoter regulatory regions on the Pseudomonas TOL plasmid showed that there was no intrinsic DNA bend in the OP1 promoter region, but a sharp DNA bend was induced by binding of IHF to the region between the upstream regulatory sequence and the promoter sequence. The DNA bending angles were distributed with a mean bend angle of 123 degrees. The IHF-DNA complexes were shown to bend at the IHF binding site giving rise to an asymmetric structure. These results provide direct evidence that IHF is required functionally for activation of OP1 transcription and support the DNA-loop model that the sharp DNA bend induced by binding of IHF facilitates the contact between RNA polymerase bound by the promoter sequence and XylR protein attached to the upstream sequence in the OP1 promoter.