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[This corrects the article DOI: 10.1371/journal.ppat.1005822.].
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Francisella tularensis is composed of a number of subspecies with varied geographic distribution, host ranges, and virulence. In view of these marked differences, comparative functional genomics may elucidate some of the molecular mechanism(s) behind these differences. In this study a shared probe microarray was designed that could be used to compare the transcriptomes of Francisella tularensis subsp. tularensis Schu S4 (Ftt), Francisella tularensis subsp. holarctica OR960246 (Fth), Francisella tularensis subsp. holarctica LVS (LVS), and Francisella novicida U112 (Fn). To gain insight into expression differences that may be related to the differences in virulence of these subspecies, transcriptomes were measured from each strain grown in vitro under identical conditions, utilizing a shared probe microarray. The human avirulent Fn strain exhibited high levels of transcription of genes involved in general metabolism, which are pseudogenes in the human virulent Ftt and Fth strains, consistent with the process of genome decay in the virulent strains. Genes encoding an efflux system (emrA2 cluster of genes), siderophore (fsl operon), acid phosphatase, LPS synthesis, polyamine synthesis, and citrulline ureidase were all highly expressed in Ftt when compared to Fn, suggesting that some of these may contribute to the relative high virulence of Ftt. Genes expressed at a higher level in Ftt when compared to the relatively less virulent Fth included genes encoding isochorismatases, cholylglycine hydrolase, polyamine synthesis, citrulline ureidase, Type IV pilus subunit, and the Francisella Pathogenicity Island protein PdpD. Fth and LVS had very few expression differences, consistent with the derivation of LVS from Fth. This study demonstrated that a shared probe microarray designed to detect transcripts in multiple species/subspecies of Francisella enabled comparative transcriptional analyses that may highlight critical differences that underlie the relative pathogenesis of these strains for humans. This strategy could be extended to other closely-related bacterial species for inter-strain and inter-species analyses.
Asunto(s)
Francisella tularensis/metabolismo , Francisella/metabolismo , Francisella/genética , Francisella/patogenicidad , Francisella tularensis/genética , Francisella tularensis/patogenicidad , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Tularemia/microbiologíaRESUMEN
Bacterial cell division predominantly occurs by a highly conserved process, termed binary fission, that requires the bacterial homologue of tubulin, FtsZ. Other mechanisms of bacterial cell division that are independent of FtsZ are rare. Although the obligate intracellular human pathogen Chlamydia trachomatis, the leading bacterial cause of sexually transmitted infections and trachoma, lacks FtsZ, it has been assumed to divide by binary fission. We show here that Chlamydia divides by a polarized cell division process similar to the budding process of a subset of the Planctomycetes that also lack FtsZ. Prior to cell division, the major outer-membrane protein of Chlamydia is restricted to one pole of the cell, and the nascent daughter cell emerges from this pole by an asymmetric expansion of the membrane. Components of the chlamydial cell division machinery accumulate at the site of polar growth prior to the initiation of asymmetric membrane expansion and inhibitors that disrupt the polarity of C. trachomatis prevent cell division. The polarized cell division of C. trachomatis is the result of the unipolar growth and FtsZ-independent fission of this coccoid organism. This mechanism of cell division has not been documented in other human bacterial pathogens suggesting the potential for developing Chlamydia-specific therapeutic treatments.
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División Celular/fisiología , Chlamydia trachomatis/fisiología , Chlamydia trachomatis/ultraestructura , Polaridad Celular , Células HeLa , Humanos , Immunoblotting , Microscopía Confocal , Microscopía Electrónica de TransmisiónRESUMEN
Phosphatidylcholine is a constituent of Chlamydia trachomatis membranes that must be acquired from its mammalian host to support bacterial proliferation. The CLA1 (SR-B1) receptor is a bi-directional phosphatidylcholine/cholesterol transporter that is recruited to the inclusion of Chlamydia-infected cells along with ABCA1. C. trachomatis growth was inhibited in a dose-dependent manner by BLT-1, a selective inhibitor of CLA1 function. Expression of a BLT-1-insensitive CLA1(C384S) mutant ameliorated the effect of the drug on chlamydial growth. CLA1 knockdown using shRNAs corroborated an important role for CLA1 in the growth of C. trachomatis. Trafficking of a fluorescent phosphatidylcholine analogue to Chlamydia was blocked by the inhibition of CLA1 or ABCA1 function, indicating a critical role for these transporters in phosphatidylcholine acquisition by this organism. Our analyses using a dual-labelled fluorescent phosphatidylcholine analogue and mass spectrometry showed that the phosphatidylcholine associated with isolated Chlamydia was unmodified host phosphatidylcholine. These results indicate that C. trachomatis co-opts host phospholipid transporters normally used to assemble lipoproteins to acquire host phosphatidylcholine essential for growth.
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Chlamydia trachomatis/crecimiento & desarrollo , Interacciones Huésped-Patógeno , Fosfatidilcolinas/metabolismo , Receptores Depuradores de Clase B/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Membrana Celular/metabolismo , Chlamydia trachomatis/efectos de los fármacos , Chlamydia trachomatis/patogenicidad , Ciclopentanos/farmacología , Células HeLa/efectos de los fármacos , Células HeLa/microbiología , Humanos , Receptores Depuradores de Clase B/genética , Esfingomielinas/metabolismo , Tiosemicarbazonas/farmacologíaRESUMEN
Chlamydia is amongst the rare bacteria that lack the critical cell division protein FtsZ. By annotation, Chlamydia also lacks several other essential cell division proteins including the FtsLBQ complex that links the early (e.g., FtsZ) and late (e.g., FtsI/Pbp3) components of the division machinery. Here, we report chlamydial FtsL and FtsQ homologs. Ct271 aligned well with Escherichia coli FtsL and shared sequence homology with it, including a predicted leucine-zipper like motif. Based on in silico modeling, we show that Ct764 has structural homology to FtsQ in spite of little sequence similarity. Importantly, ct271/ftsL and ct764/ftsQ are present within all sequenced chlamydial genomes and are expressed during the replicative phase of the chlamydial developmental cycle, two key characteristics for a chlamydial cell division gene. GFP-Ct764 localized to the division septum of dividing transformed chlamydiae, and, importantly, over-expression inhibited chlamydial development. Using a bacterial two-hybrid approach, we show that Ct764 interacted with other components of the chlamydial division apparatus. However, Ct764 was not capable of complementing an E. coli FtsQ depletion strain in spite of its ability to interact with many of the same division proteins as E. coli FtsQ, suggesting that chlamydial FtsQ may function differently. We previously proposed that Chlamydia uses MreB and other rod-shape determining proteins as an alternative system for organizing the division site and its apparatus. Chlamydial FtsL and FtsQ homologs expand the number of identified chlamydial cell division proteins and suggest that Chlamydia has likely kept the late components of the division machinery while substituting the Mre system for the early components.
RESUMEN
The antibiotic spectinomycin is a potent inhibitor of bacterial protein synthesis with a unique mechanism of action and an excellent safety index, but it lacks antibacterial activity against most clinically important pathogens. A series of N-benzyl-substituted 3'-(R)-3'-aminomethyl-3'-hydroxy spectinomycins was developed on the basis of a computational analysis of the aminomethyl spectinomycin binding site and structure-guided synthesis. These compounds had ribosomal inhibition values comparable to spectinomycin but showed increased potency against the common respiratory tract pathogens Streptococcus pneumoniae, Haemophilus influenzae, Legionella pneumophila, and Moraxella catarrhalis, as well as the sexually transmitted bacteria Neisseria gonorrhoeae and Chlamydia trachomatis. Non-ribosome-binding 3'-(S) isomers of the lead compounds demonstrated weak inhibitory activity in in vitro protein translation assays and poor antibacterial activity, indicating that the antibacterial activity of the series remains on target against the ribosome. Compounds also demonstrated no mammalian cytotoxicity, improved microsomal stability, and favorable pharmacokinetic properties in rats. The lead compound from the series exhibited excellent chemical stability superior to spectinomycin; no interaction with a panel of human receptors and drug metabolism enzymes, suggesting low potential for adverse reactions or drug-drug interactions in vivo; activity in vitro against a panel of penicillin-, macrolide-, and cephalosporin-resistant S. pneumoniae clinical isolates; and the ability to cure mice of fatal pneumococcal pneumonia and sepsis at a dose of 5 mg/kg. Together, these studies indicate that N-benzyl aminomethyl spectinomycins are suitable for further development to treat drug-resistant respiratory tract and sexually transmitted bacterial infections.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Descubrimiento de Drogas , Farmacorresistencia Bacteriana , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Enfermedades Bacterianas de Transmisión Sexual/tratamiento farmacológico , Espectinomicina/farmacología , Animales , Antibacterianos/efectos adversos , Antibacterianos/síntesis química , Antibacterianos/farmacocinética , Bacterias/metabolismo , Bacterias/patogenicidad , Proteínas Bacterianas/biosíntesis , Chlorocebus aethiops , Simulación por Computador , Diseño Asistido por Computadora , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Estabilidad de Medicamentos , Humanos , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/microbiología , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Enfermedades Bacterianas de Transmisión Sexual/diagnóstico , Enfermedades Bacterianas de Transmisión Sexual/microbiología , Espectinomicina/efectos adversos , Espectinomicina/análogos & derivados , Espectinomicina/síntesis química , Espectinomicina/farmacocinética , Relación Estructura-Actividad , Células VeroAsunto(s)
Aterosclerosis/etiología , Aterosclerosis/microbiología , Infecciones por Chlamydophila/complicaciones , Infecciones por Chlamydophila/microbiología , Chlamydophila pneumoniae/aislamiento & purificación , Enfermedad Coronaria/etiología , Enfermedad Coronaria/microbiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC50 of 0.9 µM at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis.
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Chlamydia trachomatis/metabolismo , Ácidos Grasos/biosíntesis , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Benzofuranos/farmacología , Proliferación Celular/efectos de los fármacos , Chlamydia trachomatis/genética , Chlamydia trachomatis/patogenicidad , Cristalografía por Rayos X , Enoil-ACP Reductasa (NADH)/antagonistas & inhibidores , Enoil-ACP Reductasa (NADH)/genética , Enoil-ACP Reductasa (NADH)/metabolismo , Inhibidores Enzimáticos/farmacología , Acido Graso Sintasa Tipo II/antagonistas & inhibidores , Acido Graso Sintasa Tipo II/genética , Acido Graso Sintasa Tipo II/metabolismo , Genes Bacterianos , Células HeLa , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Pironas/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
In vitro models of Chlamydia trachomatis growth have long been studied to predict growth in vivo. Alternative or persistent growth modes in vitro have been shown to occur under the influence of numerous stressors but have not been studied in vivo. Here, we report the development of methods for sampling human infections from the endocervix in a manner that permits a multifaceted analysis of the bacteria, host and the endocervical environment. Our approach permits evaluating total bacterial load, transcriptional patterns, morphology by immunofluorescence and electron microscopy, and levels of cytokines and nutrients in the infection microenvironment. By applying this approach to two pilot patients with disparate infections, we have determined that their contrasting growth patterns correlate with strikingly distinct transcriptional biomarkers, and are associated with differences in local levels of IFNγ. Our multifaceted approach will be useful to dissect infections in the human host and be useful in identifying patients at risk for chronic disease. Importantly, the molecular and morphological analyses described here indicate that persistent growth forms can be isolated from the human endocervix when the infection microenvironment resembles the in vitro model of IFNγ-induced persistence.
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Cuello del Útero/microbiología , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/citología , Chlamydia trachomatis/genética , Infecciones del Sistema Genital/microbiología , Adolescente , Adulto , Carga Bacteriana , Chlamydia trachomatis/aislamiento & purificación , Citocinas/análisis , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Técnicas Microbiológicas/métodos , Microscopía Electrónica , Patología/métodos , Adulto JovenRESUMEN
The natural history of genital Chlamydia trachomatis infections can vary widely; infections can spontaneously resolve but can also last from months to years, potentially progressing to cause significant pathology. The host and bacterial factors underlying this wide variation are not completely understood, but emphasize the bacterium's capacity to evade/adapt to the genital immune response, and/or exploit local environmental conditions to survive this immune response. IFNγ is considered to be a primary host protective cytokine against endocervical C. trachomatis infections. IFNγ acts by inducing the host enzyme indoleamine 2,3-dioxgenase, which catabolizes tryptophan, thereby depriving the bacterium of this essential amino acid. In vitro studies have revealed that tryptophan deprivation causes Chlamydia to enter a viable but non-infectious growth pattern that is termed a persistent growth form, characterized by a unique morphology and gene expression pattern. Provision of tryptophan can reactivate the bacterium to the normal developmental cycle. There is a significant difference in the capacity of ocular and genital C. trachomatis serovars to counter tryptophan deprivation. The latter uniquely encode a functional tryptophan synthase to synthesize tryptophan via indole salvage, should indole be available in the infection microenvironment. In vitro studies have confirmed the capacity of indole to mitigate the effects of IFNγ; it has been suggested that a perturbed vaginal microbiome may provide a source of indole in vivo. Consistent with this hypothesis, the microbiome associated with bacterial vaginosis includes species that encode a tryptophanase to produce indole. In this review, we discuss the natural history of genital chlamydial infections, morphological and molecular changes imposed by IFNγ on Chlamydia, and finally, the microenvironmental conditions associated with vaginal co-infections that can ameliorate the effects of IFNγ on C. trachomatis.
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Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/inmunología , Indoles/metabolismo , Interferón gamma/metabolismo , Infecciones del Sistema Genital/inmunología , Triptófano/metabolismo , Vagina/microbiología , Chlamydia trachomatis/crecimiento & desarrollo , Chlamydia trachomatis/metabolismo , Femenino , HumanosRESUMEN
Chlamydia trachomatis is an obligate intracellular bacterial pathogen that is the most common cause of sexually transmitted bacterial infections and is the etiological agent of trachoma, the leading cause of preventable blindness. The organism infects epithelial cells of the genital tract and eyelid resulting in a damaging inflammatory response. Chlamydia trachomatis grows within a vacuole termed the inclusion, and its growth depends on numerous host factors, including lipids. Although a variety of mechanisms are involved in the acquisition of host cell cholesterol and glycosphingolipids by C. trachomatis, none of the previously documented pathways for lipid acquisition are absolutely required for growth. Here we demonstrate that multiple components of the host high-density lipoprotein (HDL) biogenesis machinery including the lipid effluxers, ABCA1 and CLA 1, and their extracellular lipid acceptor, apoA-1, are recruited to the inclusion of C. trachomatis-infected cells. Furthermore, the apoA-1 that accumulates within the inclusion colocalizes with pools of phosphatidylcholine. Knockdown of ABCA1, which mediates the cellular efflux of cholesterol and phospholipids to initiate the formation of HDL in the serum, prevents the growth of C. trachomatis in infected HeLa cells. In addition, drugs that inhibit the lipid transport activities of ABCA1 and CLA 1 also inhibit the recruitment of phospholipids to the inclusion and prevent chlamydial growth.These results strongly suggest that C. trachomatis co-opts the host cell lipid transport system involved in the formation of HDL to acquire lipids, such as phosphatidylcholine, that are necessary for growth.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Chlamydia trachomatis/crecimiento & desarrollo , Interacciones Huésped-Patógeno , Cuerpos de Inclusión/enzimología , Cuerpos de Inclusión/microbiología , Lipoproteínas HDL/metabolismo , Receptores Depuradores de Clase B/metabolismo , Transportador 1 de Casete de Unión a ATP , Apolipoproteína A-I/metabolismo , Células HeLa , Humanos , Fosfolípidos/metabolismo , Vacuolas/enzimología , Vacuolas/microbiologíaRESUMEN
Loss of the conserved "cryptic" plasmid from C. trachomatis and C. muridarum is pleiotropic, resulting in reduced innate inflammatory activation via TLR2, glycogen accumulation and infectivity. The more genetically distant C. caviae GPIC is a natural pathogen of guinea pigs and induces upper genital tract pathology when inoculated intravaginally, modeling human disease. To examine the contribution of pCpGP1 to C. caviae pathogenesis, a cured derivative of GPIC, strain CC13, was derived and evaluated in vitro and in vivo. Transcriptional profiling of CC13 revealed only partial conservation of previously identified plasmid-responsive chromosomal loci (PRCL) in C. caviae. However, 2-deoxyglucose (2DG) treatment of GPIC and CC13 resulted in reduced transcription of all identified PRCL, including glgA, indicating the presence of a plasmid-independent glucose response in this species. In contrast to plasmid-cured C. muridarum and C. trachomatis, plasmid-cured C. caviae strain CC13 signaled via TLR2 in vitro and elicited cytokine production in vivo similar to wild-type C. caviae. Furthermore, inflammatory pathology induced by infection of guinea pigs with CC13 was similar to that induced by GPIC, although we observed more rapid resolution of CC13 infection in estrogen-treated guinea pigs. These data indicate that either the plasmid is not involved in expression or regulation of virulence in C. caviae or that redundant effectors prevent these phenotypic changes from being observed in C. caviae plasmid-cured strains.
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Infecciones por Chlamydia/microbiología , Chlamydia/genética , Chlamydia/fisiología , Chlamydia/patogenicidad , Plásmidos/genética , Infecciones del Sistema Genital/microbiología , Receptor Toll-Like 2/fisiología , Virulencia/genética , Animales , Células Cultivadas , Chlamydia/inmunología , Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/patología , Modelos Animales de Enfermedad , Evolución Molecular , Femenino , Eliminación de Gen , Cobayas , Células HEK293 , Humanos , Activación de Linfocitos/genética , Plásmidos/fisiología , Infecciones del Sistema Genital/inmunología , Infecciones del Sistema Genital/patología , Transducción de Señal/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismoRESUMEN
Gamma interferon (IFN-γ) induces expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO1) in human epithelial cells, the permissive cells for the obligate intracellular bacterium Chlamydia trachomatis. IDO1 depletes tryptophan by catabolizing it to kynurenine with consequences for C. trachomatis, which is a tryptophan auxotroph. In vitro studies reveal that tryptophan depletion can result in the formation of persistent (viable but noncultivable) chlamydial forms. Here, we tested the effects of the IDO1 inhibitor, levo-1-methyl-tryptophan (L-1MT), on IFN-γ-induced C. trachomatis persistence. We found that addition of 0.2 mM L-1MT to IFN-γ-exposed infected HeLa cell cultures restricted IDO1 activity at the mid-stage (20 h postinfection [hpi]) of the chlamydial developmental cycle. This delayed tryptophan depletion until the late stage (38 hpi) of the cycle. Parallel morphological and gene expression studies indicated a consequence of the delay was a block in the induction of C. trachomatis persistence by IFN-γ. Furthermore, L-1MT addition allowed C. trachomatis to undergo secondary differentiation, albeit with limited productive multiplication of the bacterium. IFN-γ-induced persistent infections in epithelial cells have been previously reported to be more resistant to doxycycline than normal productive infections in vitro. Pertinent to this observation, we found that L-1MT significantly improved the efficacy of doxycycline in clearing persistent C. trachomatis forms. It has been postulated that persistent forms of C. trachomatis may contribute to chronic chlamydial disease. Our findings suggest that IDO1 inhibitors such as L-1MT might provide a novel means to investigate, and potentially target, persistent chlamydial forms, particularly in conjunction with conventional therapeutics.
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Chlamydia trachomatis/efectos de los fármacos , Células Epiteliales/microbiología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Interferón gamma/farmacología , Triptófano/análogos & derivados , Antibacterianos/farmacología , Chlamydia trachomatis/fisiología , Relación Dosis-Respuesta a Droga , Doxiciclina/farmacología , Células HeLa , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Quinurenina/análisis , Factores de Tiempo , Triptófano/análisis , Triptófano/farmacologíaRESUMEN
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.
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Infecciones por Chlamydia/genética , Chlamydia muridarum/genética , Chlamydia trachomatis/genética , Regulación Bacteriana de la Expresión Génica/genética , Plásmidos/genética , Receptor Toll-Like 2/metabolismo , Animales , Línea Celular , Infecciones por Chlamydia/metabolismo , Chlamydia muridarum/metabolismo , Chlamydia muridarum/patogenicidad , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/patogenicidad , Cromosomas Bacterianos/genética , Expresión Génica , Sitios Genéticos , Glucosa/metabolismo , Glucógeno/metabolismo , Glucógeno Sintasa/biosíntesis , Glucógeno Sintasa/genética , Humanos , Cuerpos de Inclusión/metabolismo , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virulencia/genéticaRESUMEN
Chlamydia trachomatis is an obligate intracellular bacterium that exhibits a unique biphasic developmental cycle that can be disrupted by growth in the presence of IFN-γ and ß-lactams, giving rise to an abnormal growth state termed persistence. Here we have examined the expression of a family of non-coding RNAs (ncRNAs) that are differentially expressed during the developmental cycle and the induction of persistence and reactivation. ncRNAs were initially identified using an intergenic tiling microarray and were confirmed by northern blotting. ncRNAs were mapped, characterized and compared with the previously described chlamydial ncRNAs. The 5'- and 3'-ends of the ncRNAs were determined using an RNA circularization procedure. Promoter predictions indicated that all ncRNAs were expressed from σ(66) promoters and eight ncRNAs contained non-templated 3'-poly-A or poly-AG additions. Expression of ncRNAs was studied by northern blotting during (i) the normal developmental cycle, (ii) IFN-γ-induced persistence and (iii) carbenicillin-induced persistence. Differential temporal expression during the developmental cycle was seen for all ncRNAs and distinct differences in expression were seen during IFN-γ and carbenicillin-induced persistence and reactivation. A heterologous co-expression system was used to demonstrate that one of the identified ncRNAs regulated the expression of FtsI by inducing degradation of ftsI mRNA.
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Chlamydia trachomatis/genética , ARN no Traducido/metabolismo , Carbenicilina/farmacología , Chlamydia trachomatis/crecimiento & desarrollo , Chlamydia trachomatis/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Interferón gamma/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano Glicosiltransferasa/genética , Peptidoglicano Glicosiltransferasa/metabolismo , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN no Traducido/química , ARN no Traducido/genéticaRESUMEN
The protein complement of whole cell extract of the bacterium Francisella tularensis tularensis was analyzed using two-dimensional electrophoresis with preparative isoelectric focusing in the first dimension. The format allows the quantification of relative protein abundance by linear densitometry and extends the potential dynamic range of protein detection by as much as an order of magnitude. The relative abundance and rank order of 136 unique proteins identified in F. tularensis tularensis were established. It is estimated that 16% of the moderately to highly expressed proteins and 8% of all predicted non-pseudogenes were identified by comparing this proteome information with the relative abundance of mRNA as measured by microarray. This rank-ordered proteome list provides an important resource for understanding the pathogenesis of F. tularensis and is a tool for the selection and design of synthetic vaccines. This method represents a useful additional technique to improve whole proteome analyses of simple organisms.
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Electroforesis en Gel Bidimensional/métodos , Francisella tularensis/metabolismo , Focalización Isoeléctrica/métodos , Análisis por Matrices de Proteínas/métodos , Proteómica/métodos , Proteínas Bacterianas/química , Biología Computacional/métodos , Regulación Bacteriana de la Expresión Génica , Focalización Isoeléctrica/instrumentación , Proteoma , ARN Mensajero/metabolismo , Tinción con Nitrato de PlataRESUMEN
C57BL/6J mice were 10(5)-fold more resistant to Chlamydia psittaci infection than DBA/2J mice by LD(100) determinations. Linkage analysis using BXD recombinant inbred strains revealed a single effector locus at a 1.5-Mbp region on chromosome 11 encoding a cluster of three p47 GTPases (Irgb10, Igtp, and Iigp2). Western blots of infected tissue showed that Irgb10 was elevated in resistant mice and one of the two possible Iigp2 protein isoforms was preferentially expressed in susceptible mice. The BXD39 strain, susceptible at Irgb10 and resistant at Iigp2, had an intermediate phenotype implicating the nonredundant role of these p47 GTPases. C57BL/6J and DBA/2J exhibited a difference in IFN-gamma-dependent chlamydial control, which was reversible by Iigp2 small interfering RNA knockdown. Microarrays of infected peritoneal lavage revealed >10-fold up-regulation of neutrophil-recruiting chemokines in susceptible mice and >100-fold increase in macrophage differentiation genes in resistant mice, indicating that the susceptibility pattern involves the stimulation of different inflammatory cell-recruiting pathways. Massive neutrophil recruitment was seen in susceptible mice by histology and flow cytometry, and neutrophil chemokine receptor (CXCR2) knockout mice on a susceptible background survived a lethal challenge, confirming that neutrophil recruitment was required for susceptibility. Congenic Igtp knockout mice also susceptible at Irgb10 and Iigp2 on a resistant background recruited neutrophils and succumbed to infection. We conclude that Irgb10 and Iigp2 act together to confer differential susceptibility against murine chlamydial infection. Data indicate that these p47 GTPases have cell-autonomous effects that result in vastly different inflammatory stimulations, leading to either recovery or death.
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Chlamydophila psittaci/inmunología , GTP Fosfohidrolasas/fisiología , Inmunidad Innata , Psitacosis/inmunología , Psitacosis/patología , Animales , Chlamydophila psittaci/crecimiento & desarrollo , Femenino , GTP Fosfohidrolasas/deficiencia , GTP Fosfohidrolasas/genética , Predisposición Genética a la Enfermedad , Inflamación/enzimología , Inflamación/genética , Inflamación/inmunología , Inflamación/microbiología , Interferón gamma/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Fenotipo , Proteoma/genética , Psitacosis/enzimología , Psitacosis/prevención & controlRESUMEN
The developmentally regulated intracellular pathogen Chlamydia pneumoniae is a natural tryptophan auxotroph. These organisms survive tryptophan starvation induced by host cell activation with IFNgamma by blocking maturation to the infectious form. In most bacteria, the stringent response is induced during amino acid starvation to promote survival. However, the response of obligate intracellular pathogens, which are predicted to lack stringent responses to amino acid starvation, is poorly characterized. Chlamydial transcription and translation were analysed during IFNgamma-mediated tryptophan starvation using genomic normalization methods, and the data revealed the novel findings that: (i) global chlamydial transcription was upregulated; and (ii) protein synthesis was dramatically reduced. These results indicate a dysregulation of developmental gene expression and an uncoupling of transcription from translation. These observations represent an alternative survival strategy for host-adapted obligate intracellular bacterial pathogens that have lost the genes for stringent control during reductive evolution.
Asunto(s)
Chlamydophila pneumoniae/fisiología , Interferón gamma/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Triptófano/deficiencia , Línea Celular , Infecciones por Chlamydia/genética , Infecciones por Chlamydia/metabolismo , Chlamydophila pneumoniae/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Regulación hacia ArribaRESUMEN
BACKGROUND: Despite small genomic differences, Chlamydia trachomatis biovars exhibit diverse disease manifestations and different growth rates in vivo and in cell culture models. METHODS: Chlamydial inclusion-forming units were enumerated over time in HeLa cells, to evaluate the length of the developmental cycle for C. trachomatis strains A, B, C, and E/Bour (ocular strains) as well as D, E/UW5/Cx, F, and L2 (genital strains). Prototype strains A, D, and L2 were selected for detailed analysis of reticulate body growth, division, and genomic replication. The impact that changing host cells and that coinfection with different strains has on growth was also assessed. RESULTS: The genital strains completed the developmental cycle in 36-44 h, whereas the ocular strains lagged behind considerably. Differences were the result of a longer lag phase (entry plus differentiation) and generation time for the ocular strains. A prototype ocular strain grew faster in conjunctival cells than in cervical cells. Coinfection with genital (D or L2) and ocular strains expedited recovery of the ocular strain. CONCLUSIONS: Precise temporal evaluation of the chlamydial developmental cycle for selected genital and ocular C. trachomatis biovars provides a means for investigating genomic differences that define chlamydial pathotype.
Asunto(s)
Chlamydia trachomatis/crecimiento & desarrollo , Chlamydia trachomatis/efectos de los fármacos , Chlamydia trachomatis/genética , Chlamydia trachomatis/patogenicidad , Conjuntiva/citología , Conjuntiva/microbiología , Citocinesis/genética , Citocinesis/fisiología , Replicación del ADN , Células Epiteliales/microbiología , Femenino , Células HeLa , Humanos , Penicilinas/farmacología , Tracoma/microbiología , Cervicitis Uterina/microbiologíaRESUMEN
Two chlamydial homologues of the Yersinia lcrH chaperone for type III secretion system structural components are present within separate gene clusters. Quantitative transcriptional analyses demonstrated that each cluster is differentially regulated and expressed as an operon using major sigma factor elements, suggesting the presence of more elaborate developmental regulation mechanisms in chlamydiae.