Maraviroc, celastrol and azelastine alter Chlamydia trachomatis development in HeLa cells.

Introduction . Chlamydia trachomatis (Ct) is an obligate intracellular bacterium, causing a range of diseases in humans. Interactions between chlamydiae and antibiotics have been extensively studied in the past.Hypothesis/Gap statement: Chlamydial interactions with non-antibiotic drugs have received less attention and warrant further investigations. We hypothesized that selected cytokine inhibitors would alter Ct growth characteristics in HeLa cells.Aim. To investigate potential interactions between selected cytokine inhibitors and Ct development in vitro.Methodology. The CCR5 receptor antagonist maraviroc (Mara; clinically used as HIV treatment), the triterpenoid celastrol (Cel; used in traditional Chinese medicine) and the histamine H1 receptor antagonist azelastine (Az; clinically used to treat allergic rhinitis and conjunctivitis) were used in a genital in vitro model of Ct serovar E infecting human adenocarcinoma cells (HeLa).Results. Initial analyses revealed no cytotoxicity of Mara up to 20 µM, Cel up to 1 µM and Az up to 20 µM. Mara exposure (1, 5, 10 and 20 µM) elicited a reduction of chlamydial inclusion numbers, while 10 µM reduced chlamydial infectivity. Cel 1 µM, as well as 10 and 20 µM Az, reduced chlamydial inclusion size, number and infectivity. Morphological immunofluorescence and ultrastructural analysis indicated that exposure to 20 µM Az disrupted chlamydial inclusion structure. Immunofluorescence evaluation of Cel-incubated inclusions showed reduced inclusion sizes whilst Mara incubation had no effect on inclusion morphology. Recovery assays demonstrated incomplete recovery of chlamydial infectivity and formation of structures resembling typical chlamydial inclusions upon Az removal.Conclusion. These observations indicate that distinct mechanisms might be involved in potential interactions of the drugs evaluated herein and highlight the need for continued investigation of the interaction of commonly used drugs with Chlamydia and its host.

Subtle changes in host cell density cause a serious error in monitoring of the intracellular growth of Chlamydia trachomatis in a low-oxygen environment: Proposal for a standardized culture method.

We monitored Chlamydia trachomatis growth in HeLa cells cultured with either DMEM or RPMI medium containing 10% FCS under 2% or 21% O2 conditions for 2 days. Bacterial numbers, host cell numbers, and fibrosis-related gene expression in the host cells were estimated by an inclusion forming unit assay, a cell counting assay, and a PCR array, respectively. In contrast to RPMI, bacterial growth under low oxygen conditions in DMEM rapidly decreased with increasing host cell density. The addition of supplements (glucose, glutamine, vitamin B12, D-biotin, non-essential amino acids, glutathione) to the media had no effect. The growth of host cells in DMEM under low oxygen conditions rapidly decreased, although the cells remained healthy morphologically. Furthermore, the downregulation of 17 genes was observed under low oxygen in DMEM. Whereas no effect on bacterial growth was observed when culturing in RPMI medium at low oxygen, and the downregulation of three genes (CTGF, SERPINE1, JUN) was observed following bacterial infection compared with the uninfected control cells. Thus, our findings indicate the need for carefully selected culture conditions when performing experiments with C. trachomatis under low-oxygen environments, and RPMI (rather than DMEM) is recommended when a low host cell density is to be used, proposing the major modification of cell culturing method of C. trachomatis in a low-oxygen environment.

Antichlamydial sterol from the Red Sea sponge Callyspongia aff. implexa.

Marine sponges are rich sources of natural products exhibiting diverse biological activities. Bioactivity-guided fractionation of the Red Sea sponge Callyspongia aff. implexa led to the isolation of two new compounds, 26,27-bisnorcholest-5,16-dien-23-yn-3ß,7α-diol, gelliusterol E (1) and C27-polyacetylene, callimplexen A (2), in addition to the known compound ß-sitosterol (3). The structures of the isolated compounds were determined by 1D- and 2D-NMR techniques as well as high-resolution tandem mass spectrometry and by comparison to the literature. The three compounds (1-3) were tested against Chlamydia trachomatis, an obligate intracellular gram-negative bacterium, which is the leading cause of ocular and genital infections worldwide. Only gelliusterol E (1) inhibited the formation and growth of chlamydial inclusions in a dose-dependent manner with an IC50 value of 2.3 µM.

Inhibitory activity of the isoflavone biochanin A on intracellular bacteria of genus Chlamydia and initial development of a buccal formulation.

Given the established role of Chlamydia spp. as causative agents of both acute and chronic diseases, search for new antimicrobial agents against these intracellular bacteria is required to promote human health. Isoflavones are naturally occurring phytoestrogens, antioxidants and efflux pump inhibitors, but their therapeutic use is limited by poor water-solubility and intense first-pass metabolism. Here, we report on effects of isoflavones against C. pneumoniae and C. trachomatis and describe buccal permeability and initial formulation development for biochanin A. Biochanin A was the most potent Chlamydia growth inhibitor among the studied isoflavones, with an IC50 = 12 µM on C. pneumoniae inclusion counts and 6.5 µM on infectious progeny production, both determined by immunofluorescent staining of infected epithelial cell cultures. Encouraged by the permeation of biochanin A across porcine buccal mucosa without detectable metabolism, oromucosal film formulations were designed and prepared by a solvent casting method. The film formulations showed improved dissolution rate of biochanin A compared to powder or a physical mixture, presumably due to the solubilizing effect of hydrophilic additives and presence of biochanin A in amorphous state. In summary, biochanin A is a potent inhibitor of Chlamydia spp., and the in vitro dissolution results support the use of a buccal formulation to potentially improve its bioavailability in antichlamydial or other pharmaceutical applications.

Activation of epidermal growth factor receptor is required for Chlamydia trachomatis development.

BACKGROUND: Chlamydia trachomatis (C. trachomatis) is a clinically significant human pathogen and one of the leading causative agents of sexually transmitted diseases. As obligate intracellular bacteria, C. trachomatis has evolved strategies to redirect the host's signaling and resources for its own survival and propagation. Despite the clinical notoriety of Chlamydia infections, the molecular interactions between C. trachomatis and its host cell proteins remain elusive. RESULTS: In this study, we focused on the involvement of the host cell epidermal growth factor receptor (EGFR) in C. trachomatis attachment and development. A combination of molecular approaches, pharmacological agents and cell lines were used to demonstrate distinct functional requirements of EGFR in C. trachomatis infection. We show that C. trachomatis increases the phosphorylation of EGFR and of its downstream effectors PLCγ1, Akt and STAT5. While both EGFR and platelet-derived growth factor receptor-ß (PDGFRß) are partially involved in bacterial attachment to the host cell surface, it is only the knockdown of EGFR and not PDGFRß that affects the formation of C. trachomatis inclusions in the host cells. Inhibition of EGFR results in small immature inclusions, and prevents C. trachomatis-induced intracellular calcium mobilization and the assembly of the characteristic F-actin ring at the inclusion periphery. By using complementary approaches, we demonstrate that the coordinated regulation of both calcium mobilization and F-actin assembly by EGFR are necessary for maturation of chlamydial inclusion within the host cells. A particularly important finding of this study is the co-localization of EGFR with the F-actin at the periphery of C. trachomatis inclusion where it may function to nucleate the assembly of signaling protein complexes for cytoskeletal remodeling required for C. trachomatis development. CONCLUSION: Cumulatively, the data reported here connect the function of EGFR to C. trachomatis attachment and development in the host cells, and this could lead to new venues for targeting C. trachomatis infections and associated diseases.

Herpes simplex virus co-infection-induced Chlamydia trachomatis persistence is not mediated by any known persistence inducer or anti-chlamydial pathway.

Several inducers of chlamydial persistence have been described, including interferon-gamma (IFN-gamma), IFN-alpha, IFN-beta, and tumour necrosis factor-alpha (TNF-alpha) exposure, and iron, amino acid or glucose deprivation. A tissue-culture model of Chlamydia trachomatis/herpes simplex virus type-2 (HSV-2) co-infection indicates that viral co-infection stimulates the formation of persistent chlamydiae. This study was designed to ascertain whether co-infection-induced persistence is mediated by a previously characterized mechanism. Luminex assays indicate that IFN-gamma, IFN-alpha, and TNF-alpha are not released from co-infected cells. Semiquantitative RT-PCR studies demonstrate that IFN-beta, IFN-gamma, indoleamine 2,3-dioxygenase, lymphotoxin-alpha and inducible nitric oxide synthase are not expressed during co-infection. These data indicate that viral-induced persistence is not stimulated by any persistence-associated cytokine. Supplementation of co-infected cells with excess amino acids, iron-saturated holotransferrin, glucose or a combination of amino acids and iron does not restore chlamydial infectivity, demonstrating that HSV-2-induced persistence is not mediated by depletion of these nutrients. Finally, inclusions within co-infected cells continue to enlarge and incorporate C(6)-NBD-ceramide, indicating that HSV-2 co-infection does not inhibit vesicular transport to the developing inclusion. Collectively these data demonstrate that co-infection-induced persistence is not mediated by any currently characterized persistence inducer or anti-chlamydial pathway. Previous studies indicate that HSV-2 attachment and/or entry into the host cell is sufficient for stimulating chlamydial persistence, suggesting that viral attachment and/or entry may trigger a novel host pathway which restricts chlamydial development.

Long-term effects of natural amino acids on infection with Chlamydia trachomatis.

Supplementation of culture media with leucine, isoleucine, methionine, or phenylalanine was previously found to inhibit Chlamydia trachomatis growth in HEp-2 cells. Here, we investigated the long-term effects of these additives on C. trachomatis infection in the same cell model. Amino acid addition 30h post-infection (pi) effectively suppressed the generation of infectious progeny monitored for 10 days pi. With the exception of phenylalanine, amino acid treatment beginning at 2h pi for up to 15 days led to a complete lack of infectious progeny. Phenylalanine treatment resulted in residual minimal infectivity. In extended supplementation experiments, very small aberrant chlamydial inclusions formed, whose numbers decreased considerably over time, and the production of infectious chlamydiae could not be rescued even upon amino acid withdrawal. Interestingly, a state of chlamydial persistence was induced under these conditions, as 16S rRNA transcripts were detected throughout treatment. However, expression of several key chlamydial genes including omp1, groEL, omcB, and those functioning for chlamydial DNA replication and cytokinesis was generally very low or even undetected, particularly in monolayers treated with Leu, Ile, or Met. These data revealed a capacity of certain amino acids to eliminate infectious chlamydial progeny. Additionally, supplementation of certain amino acids resulted in the formation of a small persistent population. Extrapolating from these findings may help formulate an anti-chlamydial treatment based on nutritional elements.

Effects of sustained antibiotic bactericidal treatment on Chlamydia trachomatis-infected epithelial-like cells (HeLa) and monocyte-like cells (THP-1 and U-937).

Chlamydia trachomatis is a human pathogen that causes multiple diseases worldwide. Despite appropriate therapy with existing antichlamydial antibiotics, chronic exacerbated diseases often occur and lead to serious sequelae. Since C. trachomatis has been found to enter a persistent state after exposure to deleterious conditions, the role of persistence in the failure of chlamydial antibiotherapy is questioned. HeLa, THP-1 and U-937 cells were infected with 10(4)C. trachomatis serovar L2 infectious particles. Three days later the infected cells were treated with minimal bactericidal concentrations of doxycycline (DOX), erythromycin (ERY) or tetracycline (TET) for 24 days or 30 days. Antibiotic efficacy was assessed by measuring chlamydial inclusions and infectious particles, by investigating the resumption of chlamydial growth after antibiotic removal and by testing Chlamydia viability using reverse transcriptase polymerase chain reaction targeting unprocessed 16S rRNA, processed 16S rRNA and Omp-1 mRNA. Treatment of infected HeLa cells with the usual antichlamydial antibiotics suppressed chlamydial active growth. The infection remained unapparent. However, 24 days post treatment the bacterium was found to be viable, as proved by continued expression of unprocessed and processed 16S rRNA and Omp-1 mRNA. This inactive unapparent chlamydial state is not infectious, suggesting Chlamydia persistence. Chlamydia trachomatis also developed persistence both in permissive THP-1 and non-permissive U-937 cells. Unlike in HeLa cells, persistent chlamydial infection in THP-1 and U-937 cells was resolved after 30 days of DOX treatment. Of interest, we noticed that only THP-1 and U-937 cells that were persistently infected following their interaction with infected HeLa cells remained capable of transmitting active infection to HeLa cells. These findings suggest that DOX, TET and ERY, usually administered to combat chlamydial diseases, fail to resolve persistent infection occurring during treatment in non-immune HeLa cells. However, in immune THP-1 and U-937 cells, the persistent infection is resolved by therapy with DOX. Epithelial cells could be the reservoir of persistent chlamydial particles.

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.

Biosynthesized tea polyphenols inactivate Chlamydia trachomatis in vitro.

Biosynthesized tea polyphenols showed antichlamydial activity against Chlamydia trachomatis D/UW-3/Cx and L2/434/Bu using cell culture. The most active compounds were (-)-epigallocatechin gallate and (-)-epicatechin gallate, followed by (-)-epicatechin (EC). (+)-Epicatechin and (-)-epigallocatechin were intermediate. EC was the least toxic. These results warrant evaluation of tea polyphenols as topical antichlamydial agents.

In vitro inhibitory effects of tea polyphenols on the proliferation of Chlamydia trachomatis and Chlamydia pneumoniae.

In vitro inhibitory effects of tea polyphenols on Chlamydia trachomatis and C. pneumoniae were investigated. A product of tea polyphenols, Polyphenon 70S was used. Chlamydial strains used were C. trachomatis D/UW-3/Cx and L(2)/434/Bu, and C. pneumoniae AR-39 and AC-43 strains. HeLa229 cells and HL cells were used for cultivation of C. trachomatis and C. pneumoniae, respectively. In the post-inoculation method, no inclusions of C. trachomatis were observed at 0.5 mg/ml of Polyphenon 70S. However, the toxicity of Polyphenon 70S was noted in HeLa229 cells and HL cells at a concentration of 0.25 mg/ml. In the pre-inoculation method, no toxic effects of Polyphenon 70S on the cells were noted. Complete inhibition of C. trachomatis D and L(2) was noted at concentrations of 1.6 and 0.4 mg/ml, respectively. With C. pneumoniae strains, the end points were 0.8 and 1.6 mg/ml for AR-39 and AC-43, respectively. Our findings encouraged the application of tea polyphenols for topical usage.

The in vitro antimicrobial capacity of human colostrum against Chlamydia trachomatis.

We sought to assess the antimicrobial capacity of human colostrum against Chlamydia trachomatis. a common agent of ophthalmia neonatorum. Colostrum was collected from 13 post-partum females and tested in an in vitro assay of chlamydial growth inhibition using HeLa 229 cells as the host cell line. All samples significantly inhibited chlamydial growth in a dose-response manner. The percent inhibition ranged from 45.3 to 99.0 (mean=88.1+/-4.1). The chlamydial growth inhibition activity of colostrum was found to be: heat- and freezing-resistant: more concentrated in colostrum than breast milk; was not attributable to interferon or antibody activity; and, could not be attributed to host cell cytotoxicity. Additionally, chlamydial growth inhibition occurred in < or = 15 min and was effective only when colostrum was incubated with chlamydiae prior to addition to HeLa 229 monolayers. Lastly, centrifugal fractionation of the colostrum yielded similar activity in the lipid pellicle and in the lipid-free supernatant. These results indicate that topically applied colostrum may have efficacy in the prophylaxis of ophthalmia neonatorum of chlamydial etiology in the absence of conventional modalities.