RESUMO
Irises are perennial plants, representing a large genus with hundreds of species. While cultivated extensively for their ornamental value, commercial interest in irises lies in the secondary metabolites present in their rhizomes. The Dalmatian Iris (Iris pallida Lam.) is an ornamental plant that also produces secondary metabolites with potential value to the fragrance and pharmaceutical industries. In addition to providing base notes for the fragrance industry, iris tissues and extracts possess antioxidant, anti-inflammatory and immunomodulatory effects. However, study of these secondary metabolites has been hampered by a lack of genomic information, requiring difficult extraction and analysis techniques. Here, we report the genome sequence of Iris pallida Lam., generated with Pacific Bioscience long-read sequencing, resulting in a 10.04-Gbp assembly with a scaffold N50 of 14.34 Mbp and 91.8% complete BUSCOs. This reference genome will allow researchers to study the biosynthesis of these secondary metabolites in much greater detail, opening new avenues of investigation for drug discovery and fragrance formulations.
RESUMO
The majority of mammalian promoters are CpG islands; regions of high CG density that require protection from DNA methylation to be functional. Importantly, how sequence architecture mediates this unmethylated state remains unclear. To address this question in a comprehensive manner, we developed a method to interrogate methylation states of hundreds of sequence variants inserted at the same genomic site in mouse embryonic stem cells. Using this assay, we were able to quantify the contribution of various sequence motifs towards the resulting DNA methylation state. Modeling of this comprehensive dataset revealed that CG density alone is a minor determinant of their unmethylated state. Instead, these data argue for a principal role for transcription factor binding sites, a prediction confirmed by testing synthetic mutant libraries. Taken together, these findings establish the hierarchy between the two cis-encoded mechanisms that define the DNA methylation state and thus the transcriptional competence of CpG islands.
Assuntos
Ilhas de CpG/genética , Metilação de DNA/genética , Engenharia Genética/métodos , Genoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Sequência de Bases , Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Biblioteca Gênica , Humanos , Mamíferos/genética , Camundongos , Modelos Genéticos , Ligação Proteica , Recombinases/metabolismo , Fatores de Transcrição/metabolismoRESUMO
For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs), recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%-40%) of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS). Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R(2)) of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.
Assuntos
Ilhas de CpG/genética , Metilação de DNA/genética , Genoma , Animais , Linhagem Celular , DNA/genética , Mamíferos/genética , Análise de Sequência de DNARESUMO
BACKGROUND: Airway wall remodelling is a key pathology of asthma. It includes thickening of the airway wall, hypertrophy and hyperplasia of bronchial smooth muscle cells (BSMC), as well as an increased vascularity of the sub-epithelial cell layer. BSMC are known to be the effector cells of bronchoconstriction, but they are increasingly recognized as an important source of inflammatory mediators and angiogenic factors. OBJECTIVE: To compare the angiogenic potential of BSMC of asthmatic and non-asthmatic patients and to identify asthma-specific angiogenic factors. METHODS: Primary BSMC were isolated from human airway tissue of asthmatic and non-asthmatic patients. Conditioned medium (CM) collected from BSMC isolates was tested for angiogenic capacity using the endothelial cell (EC)-spheroid in vitro angiogenesis assay. Angiogenic factors in CM were quantified using a human angiogenesis antibody array and enzyme linked immunosorbent assay. RESULTS: Induction of sprout outgrowth from EC-spheroids by CM of BSMC obtained from asthma patients was increased compared with CM of control BSMC (twofold, p < 0.001). Levels of ENA-78, GRO-α and IL-8 were significantly elevated in CM of BSMC from asthma patients (p < 0.05 vs. non-asthmatic patients). SB 265610, a competitive antagonist of chemokine (CXC-motif) receptor 2 (CXCR2), attenuated the increased sprout outgrowth induced by CM of asthma patient-derived BSMC. CONCLUSIONS: BSMC isolated from asthma patients exhibit increased angiogenic potential. This effect is mediated through the CXCR2 ligands (ENA78, GRO-α and IL-8) produced by BSMC. IMPLICATIONS: CXCR2 ligands may play a decisive role in directing the neovascularization in the sub-epithelial cell layers of the lungs of asthma patients. Counteracting the CXCR2-mediated neovascularization by pharmaceutical compounds may represent a novel strategy to reduce airway remodelling in asthma.
Assuntos
Asma/patologia , Asma/fisiopatologia , Brônquios/patologia , Quimiocinas CXC/metabolismo , Miócitos de Músculo Liso/metabolismo , Neovascularização Patológica , Adulto , Asma/metabolismo , Quimiocina CXCL1/metabolismo , Quimiocina CXCL5/metabolismo , Feminino , Humanos , Interleucina-8/metabolismo , Ligantes , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/metabolismo , Triazóis/farmacologia , Adulto JovemRESUMO
At the end of mammalian spermatogenesis, chromatin in differentiating germ cells is extensively remodeled, with the majority of nucleosomes being removed and ultimately exchanged by highly basic proteins named protamines. Residual nucleosomes are, to various degrees, retained at regulatory sequences in human and mouse sperm. Moreover, certain histone variants and modifications remain present in regulatory sequences of subsets of genes in spermatozoa, providing opportunities for paternal inheritance of chromatin states and epigenetic control of gene expression in the subsequent generation. Here we describe in detail a method that enables the generation of soluble chromatin samples from mouse and human spermatozoa within 1 d. These samples are amendable to chromatin immunoprecipitation and high-throughput sequencing of nucleosome-associated genomic DNA, which require several additional days. We also provide computational scripts that allow straightforward analysis of large genome-wide data sets by biologists with limited computational experience. This protocol will facilitate studies of mechanisms of chromatin remodeling and epigenetic reprogramming during spermatogenesis and of paternal epigenetic inheritance. Similarly, it will help in the study of the causes of human male infertility.
Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/química , Espermatozoides/ultraestrutura , Animais , Montagem e Desmontagem da Cromatina , DNA/química , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/química , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nucleossomos/metabolismo , Reprodutibilidade dos TestesRESUMO
The oestrogen receptor (ER) α-ß+ HEC-1B and the ERα+ß+ Ishikawa (IK) cell lines were investigated to dissect the effects of oestrogen exposure on several parameters of Chlamydia trachomatis infection. Antibody blockage of ERα or ERß alone or simultaneously significantly decreased C. trachomatis infectivity (45-68%). Addition of the ERß antagonist, tamoxifen, to IK or HEC-1B prior to or after chlamydial infection caused a 30-90% decrease in infectivity, the latter due to disrupted eukaryotic organelles. In vivo, endometrial glandular epithelial cells are stimulated by hormonally influenced stromal signals. Accordingly, chlamydial infectivity was significantly increased by 27% and 21% in IK and HEC-1B cells co-cultured with SHT-290 stromal cells exposed to oestrogen. Endometrial stromal cell/epithelial cell co-culture revealed indirect effects of oestrogen on phosphorylation of extracellular signal-regulated kinase and calcium-dependant phospholipase A2 and significantly increased production of interleukin (IL)-8 and IL-6 in both uninfected and chlamydiae-infected epithelial cells. These results indicate that oestrogen and its receptors play multiple roles in chlamydial infection: (i) membrane oestrogen receptors (mERs) aid in chlamydial entry into host cells, and (ii) mER signalling may contribute to inclusion development during infection. Additionally, enhancement of chlamydial infection is affected by hormonally influenced stromal signals in conjunction with direct oestrogen stimulation of the human epithelia.
Assuntos
Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydia trachomatis/patogenicidade , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Estrogênios/metabolismo , Linhagem Celular , Técnicas de Cocultura , Perfilação da Expressão Gênica , Humanos , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Receptores de Estrogênio/metabolismoRESUMO
Regions along the Mediterranean and in southern Asia have lower prostate cancer incidence compared to the rest of the world. It has been hypothesized that one of the potential contributing factors for this low incidence includes a higher intake of tocotrienols. Here we examine the potential of γ-tocotrienol (GT3) to reduce prostate cancer proliferation and focus on elucidating pathways by which GT3 could exert a growth-inhibitory effect on prostate cancer cells. We find that the γ and δ isoforms of tocotrienol are more effective at inhibiting the growth of prostate cancer cell lines (PC-3 and LNCaP) compared with the γ and δ forms of tocopherol. Knockout of PPAR-γ and GT3 treatment show inhibition of prostate cancer cell growth, through a partially PPAR-γ-dependent mechanism. GT3 treatment increases the levels of the 15-lipoxygenase-2 enzyme, which is responsible for the conversion of arachidonic acid to the PPAR-γ-activating ligand 15-S-hydroxyeicosatrienoic acid. In addition, the latent precursor and the mature forms of TGFß2 are down-regulated after treatment with GT3, with concomitant disruptions in TGFß receptor I, SMAD-2, p38, and NF-κB signaling.
Assuntos
Antineoplásicos/farmacologia , Cromanos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Vitamina E/análogos & derivados , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Neoplasias da Próstata/patologia , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Vitamina E/farmacologiaRESUMO
Chronic inflammation and dietary fat consumption correlates with an increase in prostate cancer. Our previous studies in the colon have demonstrated that gamma-tocopherol treatment could upregulate the expression of peroxisome proliferator-activated preceptors (PPAR) gamma, a nuclear receptor involved in fatty acid metabolism as well modulation of cell proliferation and differentiation. In this study, we explored the possibility that gamma-tocopherol could induce growth arrest in PC-3 prostate cancer cells through the regulation of fatty acid metabolism. Growth arrest (40%) and PPAR gamma mRNA and protein upregulation was achieved with gamma-tocopherol within 6 h. gamma-Tocopherol-mediated growth arrest was demonstrated to be PPAR gamma dependent using the agonist GW9662 and a PPAR gamma dominant negative vector. gamma-tocopherol was shown not to be a direct PPAR gamma ligand, but rather 15-S-HETE (an endogenous PPAR gamma ligand) was upregulated by gamma-tocopherol treatment. 15-Lipoxygenase-2, a tumor suppressor and the enzyme that converts arachidonic acid to 15-S-HETE, was upregulated at 3 h following gamma-tocopherol treatment. Expression of proteins downstream of the PPAR gamma pathway were examined. Cyclin D1, cyclin D3, bcl-2, and NFkappa B proteins were found to be downregulated following gamma-tocopherol treatment. These data demonstrate that the growth arrest mediated by gamma-tocopherol follows a PPAR-gamma-dependent mechanism.
Assuntos
Proliferação de Células/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Ácidos Hidroxieicosatetraenoicos/metabolismo , PPAR gama/metabolismo , Neoplasias da Próstata/patologia , gama-Tocoferol/farmacologia , Adenocarcinoma/patologia , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais , Técnicas de Inativação de Genes , Humanos , Ácidos Hidroxieicosatetraenoicos/química , Ligantes , Masculino , PPAR gama/agonistas , PPAR gama/genética , Próstata/citologia , Ligação Proteica , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , gama-Tocoferol/metabolismoRESUMO
Laser capture microdissection (LCM) is a versatile computer-assisted dissection method that permits collection of tissue samples with a remarkable level of anatomical resolution. LCM's application to the study of human brain pathology is growing, although it is still relatively underutilized, compared with other areas of research. The present study examined factors that affect the utility of LCM, as performed with an Arcturus Veritas, in the study of gene expression in the human brain using frozen tissue sections. LCM performance was ascertained by determining cell capture efficiency and the quality of RNA extracted from human brain tissue under varying conditions. Among these, the relative humidity of the laboratory where tissue sections are stained, handled, and submitted to LCM had a profound effect on the performance of the instrument and on the quality of RNA extracted from tissue sections. Low relative humidity in the laboratory, i.e., 6-23%, was conducive to little or no degradation of RNA extracted from tissue following staining and fixation and to high capture efficiency by the LCM instrument. LCM settings were optimized as described herein to permit the selective capture of astrocytes, oligodendrocytes, and noradrenergic neurons from tissue sections containing the human locus coeruleus, as determined by the gene expression of cell-specific markers. With due regard for specific limitations, LCM can be used to evaluate the molecular pathology of individual cell types in post-mortem human brain.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Umidade/efeitos adversos , Microdissecção/métodos , Neurônios/metabolismo , Oligodendroglia/metabolismo , Adolescente , Adulto , Contagem de Células , Perfilação da Expressão Gênica/métodos , Humanos , Lasers , Locus Cerúleo/metabolismo , Pessoa de Meia-Idade , Norepinefrina/metabolismo , RNA/isolamento & purificação , RNA/metabolismo , Adulto JovemRESUMO
The obligate intracellular bacterium Chlamydia trachomatis serovar E is the most prevalent cause of bacterial sexually transmitted disease. With an established requirement for iron, the developmental cycle arrests at the intracellular reticulate body stage during iron restriction, resulting in a phenomenon termed persistence. Persistence has implications in natural infections for altered expression of virulence factors and antigens, in addition to a potential role in producing chronic infection. In this study, chlamydial proteins in iron-restricted, infected HEC-1B cells were radiolabelled during mid-developmental cycle growth, harvested, and separated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Of approximately 250 radiolabelled protein species visualized, densitometric analysis revealed 25 proteins that increased in expression under iron restriction compared to iron-sufficient control samples; ten protein species identified by mass spectrometry are involved in the oxidative damage response (alkyl hydroperoxide reductase, 6-phosphogluconolactonase and acyl carrier protein synthase), transcription (RNA polymerase subunit alpha and transcription anti-termination factors NusA and NusG), protein modification (peptide deformylase and trigger factor), and virulence (Chlamydia protein associating with death domains, CADD). Transcript-level expression patterns of ahpC, devB, cadd, fabF and ct538 were measured by quantitative RT-PCR throughout the developmental cycle, and each gene examined demonstrated a significant but small mid-cycle increase in transcript level in iron-restricted cultures compared to iron-replete controls. Taken together, these data suggest that the primary response of chlamydiae to reduced iron availability is to increase expression of proteins involved in protection against oxidative damage via iron-catalysed generation of reactive oxygen species and adaptation to stress by increasing expression of transcriptional machinery and other stress-responsive proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/crescimento & desenvolvimento , Endométrio/microbiologia , Células Epiteliais/microbiologia , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Chlamydia trachomatis/metabolismo , Endométrio/citologia , Feminino , Humanos , Espectrometria de Massas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
While much is known about the attachment of the chlamydiae to the host cell and intracellular events during the developmental cycle, little is known about the mechanism(s) by which elementary bodies exit the cell. In this report, we use the guinea-pig conjunctival model of Chlamydia caviae infection to present in vivo ultrastructural evidence supporting two mechanisms for release of chlamydiae from the mucosal epithelia. Four days after infection, histopathologic observation shows an intense infiltration of polymorphonuclear leukocytes (PMN) in the conjunctival epithelium. Using transmission electron microscopy, a gradient-directed PMN response to chlamydiae-infected epithelial cells was observed. As PMN infiltration intensifies, epithelial hemidesmosome/integrin/focal adhesion adherence with the basal lamina is disconnected and PMNs literally lift off and release infected superficial epithelia from the mucosa. Many of these infected cells appear to be healthy with intact microvilli, nuclei, and mitochondria. While lysis of some infected cells occurs with release of chlamydiae into the extracellular surface milieu, the majority of infected cells are pushed off the epithelium. We propose that PMNs play an active role in detaching infected cells from the epithelium and that these infected cells eventually die releasing organisms but, in the process, move to new tissue sites via fluid dynamics.
Assuntos
Chlamydia/patogenicidade , Túnica Conjuntiva/imunologia , Conjuntivite de Inclusão/transmissão , Células Epiteliais/microbiologia , Neutrófilos/imunologia , Animais , Adesão Celular , Quimiotaxia de Leucócito , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/microbiologia , Infecções por Chlamydia/transmissão , Túnica Conjuntiva/citologia , Túnica Conjuntiva/microbiologia , Túnica Conjuntiva/ultraestrutura , Conjuntivite de Inclusão/imunologia , Conjuntivite de Inclusão/microbiologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/ultraestrutura , Feminino , Cobaias , Humanos , Microscopia Eletrônica de Transmissão , Especificidade de ÓrgãosRESUMO
A common model for studying Chlamydia trachomatis and growing chlamydial stocks uses Lymphogranuloma venereum serovar L2 and non-polarized HeLa cells. However, recent publications indicate that the growth rate and progeny yields can vary considerably for a particular strain depending on the cell line/type used, and seem to be partially related to cell tropism. In the present study, the growth of invasive serovar L2 was compared in endometrial HEC-1B and endocervical HeLa cells polarized on collagen-coated microcarrier beads, as well as in HeLa cells grown in tissue culture flasks. Microscopy analysis revealed no difference in chlamydial attachment/entry patterns or in inclusion development throughout the developmental cycle between cell lines. Very comparable growth curves in both cell lines were also found using real-time PCR analysis, with increases in chlamydial DNA content of 400-500-fold between 2 and 36 h post-inoculation. Similar progeny yields with comparable infectivity were recovered from HEC-1B and HeLa cell bead cultures, and no difference in chlamydial growth was found in polarized vs. non-polarized HeLa cells. In conclusion, unlike other C. trachomatis strains such as urogenital serovar E, invasive serovar L2 grows equally well in physiologically different endometrial and endocervical environments, regardless of the host cell polarization state.
Assuntos
Chlamydia trachomatis/crescimento & desenvolvimento , Endométrio/microbiologia , Chlamydia trachomatis/isolamento & purificação , DNA Bacteriano/análise , Endométrio/ultraestrutura , Feminino , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Microesferas , Reação em Cadeia da PolimeraseRESUMO
In vitro studies of obligate intracellular chlamydia biology and pathogenesis are highly dependent on the use of experimental models and growth conditions that mimic the mucosal architecture and environment these pathogens encounter during natural infections. In this study, the growth of Chlamydia trachomatis genital serovar E was monitored in mouse fibroblast McCoy cells and compared to more relevant host human epithelial endometrium-derived HEC-1B and cervix-derived HeLa cells, seeded and polarized on collagen-coated microcarrier beads, using a three-dimensional culture system. Microscopy analysis of these cell lines prior to infection revealed morphological differences reminiscent of their in vivo architecture. Upon infection, early chlamydial inclusion distribution was uniform in McCoy cells but patchy in both epithelial cell lines. Although no difference in chlamydial attachment to or entry into the two genital epithelial cell lines was noted, active bacterial genome replication and transcription, as well as initial transformation of elementary bodies to reticulate bodies, were detected earlier in HEC-1B than in HeLa cells, suggesting a faster growth, which led to higher progeny counts and titers in HEC-1B cells upon completion of the developmental cycle. Chlamydial development in the less relevant McCoy cells was very similar to that in HeLa cells, although higher progeny counts were obtained. In conclusion, this three-dimensional bead culture system represents an improved model for harvesting large quantities of infectious chlamydia progeny from their more natural polarized epithelial host cells.
Assuntos
Técnicas de Cultura de Células/métodos , Chlamydia trachomatis/crescimento & desenvolvimento , Células Epiteliais/microbiologia , Animais , Linhagem Celular , Polaridade Celular , Citoplasma/microbiologia , Replicação do DNA , DNA Bacteriano/análise , Endométrio/citologia , Células Epiteliais/citologia , Feminino , Fibroblastos/microbiologia , Células HeLa , Humanos , Corpos de Inclusão , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Microesferas , RNA Bacteriano/análise , Transcrição GênicaRESUMO
Epidemiological and clinical studies have shown that double infection with herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occurs in vivo. We hypothesized that co-infection would alter replication of these agents. To test this hypothesis, HeLa cells were infected with C. trachomatis serovar E, followed 24 h later by HSV-2 strain 333. Transmission electron microscopic (TEM) analyses indicated that, by 10 h after HSV addition, reticulate bodies (RBs) in co-infected cells were swollen, aberrantly shaped and electron-lucent. In infectious titre assays, HSV-2 co-infection abrogated production of infectious chlamydial progeny. Western blot analyses indicated that accumulation of chlamydial major outer membrane protein (MOMP) was decreased by HSV co-infection while accumulation of chlamydial heat-shock protein 60-1 (HSP60-1) was increased. Polymerase chain reaction (PCR) experiments indicated that chlamydial genome copy number was unaltered by HSV-2 superinfection. Semi-quantitative, reverse transcription PCR (RT-PCR) experiments demonstrated that levels of chlamydial groEL, ftsK, ftsW, dnaA and unprocessed 16S rRNA transcripts were not changed by HSV-2 super-infection. These data indicate that HSV-2 superinfection drives chlamydia into a viable but non-cultivable state, which is the hallmark of persistence. Because chlamydial HSP60-1 has been associated with immunopathology in vivo, these results also suggest that disease severity might be increased in co-infected individuals.
Assuntos
Chlamydia trachomatis/fisiologia , Herpesvirus Humano 2/fisiologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Chaperonina 60/metabolismo , Infecções por Chlamydia/complicações , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , Chlamydia trachomatis/ultraestrutura , Dosagem de Genes , Células HeLa , Herpes Genital/complicações , Herpes Genital/virologia , Herpesvirus Humano 2/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/metabolismo , Superinfecção/complicações , Superinfecção/virologiaRESUMO
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.
Assuntos
Chlamydia trachomatis/crescimento & desenvolvimento , Células Epiteliais/microbiologia , Estrogênios/fisiologia , Western Blotting , Linhagem Celular , Membrana Celular/química , Citoplasma/microbiologia , Citoplasma/ultraestrutura , Células Epiteliais/ultraestrutura , Estrogênios/análise , Citometria de Fluxo , Expressão Gênica , Humanos , Corpos de Inclusão/microbiologia , Corpos de Inclusão/ultraestrutura , Microscopia Confocal , RNA Mensageiro/análise , Receptores de Estrogênio/análise , Receptores de Estrogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Staphylococcus aureus undergoes a density-dependent conversion in phenotype from tissue-adhering to tissue-damaging and phagocyte-evading that is mediated in part by the quorum-sensing operon, agr, and its effector, RNAIII. Contributions of host factors to this mechanism for regulating virulence have not been studied. We hypothesized that fibrinogen, as a component of the inflammatory response, could create spatially constrained microenvironments around bacteria that increase density independently of bacterial numbers and thus potentiate quorum-sensing-dependent virulence gene expression. Here we show that transient fibrinogen depletion significantly reduces the bacterial burden and the consequential morbidity and mortality during experimental infection with wild-type S. aureus, but not with bacteria that lack expression of the quorum-sensing operon, agr. In addition, it inhibits in vivo activation of the promoter for the agr effector, RNAIII, and downstream targets of RNAIII, including alpha hemolysin and capsule production. Moreover, both in vitro and in vivo, the mechanism for promoting this phenotypic switch in virulence involves clumping of the bacteria, demonstrating that S. aureus responds to fibrinogen-mediated bacterial clumping by enhancing density-dependent virulence gene expression. These data demonstrate that down-modulation of specific inflammatory components of the host that augment bacterial quorum sensing can be a strategy for enhancing host defense against infection.
Assuntos
Regulação para Baixo/genética , Fibrinogênio/metabolismo , Regulação Bacteriana da Expressão Gênica , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Regulação para Cima/genética , Afibrinogenemia/induzido quimicamente , Afibrinogenemia/microbiologia , Ancrod/administração & dosagem , Animais , Aderência Bacteriana/genética , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/fisiologia , Feminino , Fibrinogênio/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Morbidade , Fenótipo , Infecções Estafilocócicas/mortalidade , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/crescimento & desenvolvimento , Transativadores/antagonistas & inibidores , Transativadores/fisiologia , Virulência/genéticaRESUMO
The inflammatory response associated with Chlamydia trachomatis genital infections is thought to be initiated by the release of proinflammatory cytokines from infected epithelial cells. This study focuses on the interactions between C. trachomatis-infected HeLa cells and immune cells involved in the early stages of infection, i.e., neutrophils and macrophages. First, we showed that the expression of interleukin-11 (IL-11), an anti-inflammatory cytokine mainly active on macrophages, was upregulated at the mRNA level in the genital tracts of infected mice. Second, incubation of differentiated THP-1 (dTHP-1) cells or monocyte-derived macrophages (MdM) with basal culture supernatants from C. trachomatis serovar E- or serovar L2-infected HeLa cells resulted in macrophage activation with a differential release of tumor necrosis factor alpha (TNF-alpha) and upregulation of indoleamine 2,3-deoxygenase (IDO) but not of Toll-like receptor 2 and 4 mRNA expression. Third, coculture of infected HeLa cells with dTHP-1 cells resulted in a reduction in chlamydial growth, which was more dramatic for serovar E than for L2 and which was partially reversed by the addition of anti-TNF-alpha antibodies for serovar E or exogenous tryptophan for both serovars but was not reversed by the addition of superoxide dismutase or anti-IL-8 or anti-IL-1beta antibodies. A gamma interferon-independent IDO mRNA upregulation was also detected in dTHP-1 cells from infected cocultures. Lastly, with a two-stage coculture system, we found that (i) supernatants from neutrophils added to the apical side of infected HeLa cell cultures were chlamydicidal and induced MdM to express antichlamydial activity and (ii) although polymorphonuclear leukocytes released more proinflammatory cytokines in response to serovar E- than in response to L2-infected cells, MdM were strongly activated by serovar L2 infection, indicating that the early inflammatory response generated with a nondisseminating or a disseminating strain is different.