RESUMEN
Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.
Asunto(s)
Factor de Unión a CCCTC , Diferenciación Celular , Interferón gamma , Interleucina-22 , Interleucinas , Células TH1 , Animales , Factor de Unión a CCCTC/metabolismo , Factor de Unión a CCCTC/genética , Células TH1/inmunología , Ratones , Diferenciación Celular/inmunología , Interferón gamma/metabolismo , Sitios de Unión , Interleucinas/metabolismo , Interleucinas/genética , Elementos de Facilitación Genéticos/genética , Ratones Endogámicos C57BL , Cromatina/metabolismo , Toxoplasmosis/inmunología , Toxoplasmosis/parasitología , Toxoplasmosis/genética , Regulación de la Expresión Génica , Toxoplasma/inmunología , Citocinas/metabolismo , Linaje de la Célula , Células Th17/inmunologíaRESUMEN
Specification of the T helper 17 (Th17) cell lineage requires a well-defined set of transcription factors, but how these integrate with posttranscriptional and epigenetic programs to regulate gene expression is poorly understood. Here we found defective Th17 cell cytokine expression in miR-155-deficient CD4+ T cells in vitro and in vivo. Mir155 was bound by Th17 cell transcription factors and was highly expressed during Th17 cell differentiation. miR-155-deficient Th17 and T regulatory (Treg) cells expressed increased amounts of Jarid2, a DNA-binding protein that recruits the Polycomb Repressive Complex 2 (PRC2) to chromatin. PRC2 binding to chromatin and H3K27 histone methylation was increased in miR-155-deficient cells, coinciding with failure to express Il22, Il10, Il9, and Atf3. Defects in Th17 cell cytokine expression and Treg cell homeostasis in the absence of Mir155 could be partially suppressed by Jarid2 deletion. Thus, miR-155 contributes to Th17 cell function by suppressing the inhibitory effects of Jarid2.
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Citocinas/genética , Regulación de la Expresión Génica , MicroARNs/metabolismo , Complejo Represivo Polycomb 2/inmunología , Células Th17/inmunología , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Animales , Diferenciación Celular/inmunología , Células Cultivadas , Cromatina/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Unión Proteica , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Células TH1/inmunologíaRESUMEN
Th17 cells, which express the chemokine receptor CCR6, are implicated in many immune-mediated disorders, such as psoriasis and multiple sclerosis. We found that expression levels of CCR6 on human effector/memory CD4(+) T cells reflect a continuum of Th17 differentiation. By evaluating the transcriptome in cells with increasing CCR6, we detected progressive upregulation of ZBTB16, which encodes the broad complex, tramtrack, bric-à-brac-zinc finger transcription factor promyelocytic leukemia zinc finger protein (PLZF). Using chromatin immunoprecipitation for modified histones, p300, and PLZF, we identified enhancer-like sites at -9/-10 and -13/-14 kb from the upstream transcription start site of CCR6 that bind PLZF in CCR6(+) cells. For Th cells from adult blood, both in the CCR6(+) memory population and in naive cells activated ex vivo, knockdown of ZBTB16 downregulated CCR6 and other Th17-associated genes. ZBTB16 and RORC (which encodes the "master regulator" RORγt) cross-regulate each other, and PLZF binds at the RORC promoter in CCR6(+) cells. In naive Th cells from cord blood, ZBTB16 expression was confined to CD161(+) cells, which are Th17 cell precursors. ZBTB16 was not expressed in mouse Th17 cells, and Th17 cells could be made from luxoid mice, which harbor an inactivating mutation in Zbtb16. These studies demonstrate a role for PLZF as an activator of transcription important both for Th17 differentiation and the maintenance of the Th17 phenotype in human cells, expand the role of PLZF as a critical regulator in the human adaptive immune system, and identify a novel, essential element in a regulatory network that is of significant therapeutic interest.
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Factores de Transcripción de Tipo Kruppel/metabolismo , Fenotipo , Receptores CCR6/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Elementos de Facilitación Genéticos , Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Memoria Inmunológica , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Transgénicos , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismo , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Unión Proteica , Receptores CCR6/genética , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células Th17/citologíaRESUMEN
Transcriptional profiles of 2 unrelated clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were analyzed following 10% (v/v) ethanol challenge (15 min), which arrested growth but did not reduce viability. Ethanol-induced stress (EIS) resulted in differential gene expression of 1091 genes, 600 common to both strains, of which 291 were upregulated. With the exception of the downregulation of genes involved with osmotic stress functions, EIS resulted in the upregulation of genes that contribute to stress response networks, notably those altered by oxidative stress, protein quality control in general, and heat shock in particular. In addition, genes involved with transcription, translation, and nucleotide biosynthesis were downregulated. relP, which encodes a small alarmone synthetase (RelP), was highly upregulated in both MRSA strains following ethanol challenge, and relP inactivation experiments indicated that this gene contributed to EIS growth arrest. A number of persistence-associated genes were also upregulated during EIS, including those that encode toxin-antitoxin systems. Overall, transcriptional profiling indicated that the MRSA investigated responded to EIS by entering a state of dormancy and by altering the expression of elements from cross protective stress response systems in an effort to protect preexisting proteins.
Asunto(s)
Etanol/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Respuesta al Choque Térmico , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Infecciones Estafilocócicas/microbiología , Estrés FisiológicoRESUMEN
BACKGROUND: Preeclampsia is a significant cause of maternal and fetal mortality and morbidity worldwide. We previously reported associations between trichothiodystrophy (TTD) nucleotide excision repair (NER) and transcription gene mutations in the fetus and the risk of gestational complications including preeclampsia. TTD NER/transcription genes, XPD, XPB and TTD-A, code for subunits of Transcription Factor (TF)IIH. Interpreting XPD mutations in the context of available biochemical data led us to propose adverse effects on CDK-activating kinase (CAK) subunit of TFIIH and TFIIH-mediated functions as a relevant mechanism in preeclampsia. In order to gain deeper insight into the underlying biologic mechanisms involving TFIIH-mediated functions in placenta, we analyzed NER/transcription and global gene expression profiles of normal and preeclamptic placentas and studied gene regulatory networks. RESULTS: We found high expression of TTD NER/transcription genes in normal human placenta, above the mean of their expression in all organs. XPD and XPB were consistently expressed from 14 to 40 weeks gestation while expression of TTD-A was strongly negatively correlated (r=-0.7, P<0.0001) with gestational age. Analysis of gene expression patterns of placentas from a case-control study of preeclampsia using Algorithm for Reconstruction of Accurate Cellular Networks (ARACNE) revealed GTF2E1, a component of TFIIE which modulates TFIIH, among major regulators of differentially-expressed genes in preeclampsia. The basal transcription pathway was among the largest dysregulated protein-protein interaction networks in this preeclampsia dataset. Within the basal transcription pathway, significantly down-regulated genes besides GTF2E1 included those coding for the CAK complex of TFIIH, namely CDK7, CCNH, and MNAT1. Analysis of other relevant gene expression and gene regulatory network data also underscored the involvement of transcription pathways and identified JUNB and JUND (components of transcription factor AP-1) as transcription regulators of the network involving the TTD genes, GTF2E1, and selected gene regulators implicated in preeclampsia. CONCLUSIONS: Our results indicate that TTD NER/transcription genes are expressed in placenta during gestational periods critical to preeclampsia development. Our overall findings suggest that impairment of TFIIH-mediated function in transcription in placenta is a likely mechanism leading to preeclampsia and provide etiologic clues which may be translated into therapeutic and preventive measures.
Asunto(s)
Reparación del ADN , Feto/metabolismo , Placenta/metabolismo , Preeclampsia/patología , Factor de Transcripción TFIIH/metabolismo , Estudios de Casos y Controles , Regulación hacia Abajo , Femenino , Edad Gestacional , Células Endoteliales de la Vena Umbilical Humana , Humanos , Preeclampsia/genética , Embarazo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Interferente Pequeño/metabolismo , Factor de Transcripción TFIIH/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Transcriptoma , Síndromes de Tricotiodistrofia/genética , Síndromes de Tricotiodistrofia/patología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo D/metabolismoRESUMEN
Proinflammatory T-lymphocytes recruited into the brain and spinal cord mediate multiple sclerosis (MS) and currently there is no cure for MS. IFN-γ-producing Th1 cells induce ascending paralysis in the spinal cord while IL-17-producing Th17 cells mediate cerebellar ataxia. STAT1 and STAT3 are required for Th1 and Th17 development, respectively, and the simultaneous targeting of STAT1 and STAT3 pathways is therefore a potential therapeutic strategy for suppressing disease in the spinal cord and brain. However, the pharmacological targeting of STAT1 and STAT3 presents significant challenges because of their intracellular localization. We have developed a STAT-specific single-domain nanobody (SBT-100) derived from camelids that targets conserved residues in Src homolog 2 (SH2) domains of STAT1 and STAT3. This study investigated whether SBT-100 could suppress experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We show that SBT-100 ameliorates encephalomyelitis through suppressing the expansion of Th17 and Th1 cells in the brain and spinal cord. Adoptive transfer experiments revealed that lymphocytes from SBT-100-treated EAE mice have reduced capacity to induce EAE, indicating that the immunosuppressive effects derived from the direct suppression of encephalitogenic T-cells. The small size of SBT-100 makes this STAT-specific nanobody a promising immunotherapy for CNS autoimmune diseases, including multiple sclerosis.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Anticuerpos de Dominio Único , Células Th17 , Animales , Femenino , Ratones , Camélidos del Nuevo Mundo , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Ratones Endogámicos C57BL , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/uso terapéutico , Médula Espinal/patología , Médula Espinal/efectos de los fármacos , Médula Espinal/inmunología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Células TH1/inmunología , Células TH1/efectos de los fármacos , Células Th17/inmunología , Células Th17/efectos de los fármacosRESUMEN
The ocular surface is a mucosal barrier tissue colonized by commensal microbes, which tune local immunity by eliciting IL-17 from conjunctival γδ T cells to prevent pathogenic infection. The commensal Corynebacterium mastitidis (C. mast) elicits protective IL-17 responses from conjunctival Vγ4 T cells through a combination of γδ TCR ligation and IL-1 signaling. Here, we identify Vγ6 T cells as a major C. mast-responsive subset in the conjunctiva and uncover its unique activation requirements. We demonstrate that Vγ6 cells require not only extrinsic (via dendritic cells) but also intrinsic TLR2 stimulation for optimal IL-17A response. Mechanistically, intrinsic TLR2 signaling was associated with epigenetic changes and enhanced expression of genes responsible for metabolic shift to fatty acid oxidation to support Il17a transcription. We identify one key transcription factor, IκBζ, which is upregulated by TLR2 stimulation and is essential for this program. Our study highlights the importance of intrinsic TLR2 signaling in driving metabolic reprogramming and production of IL-17A in microbiome-specific mucosal γδ T cells.
RESUMEN
Tea tree oil (TTO) is a steam distillate of Melaleuca alternifolia that demonstrates broad-spectrum antibacterial activity. This study was designed to document how TTO challenge influences the Staphylococcus aureus transcriptome. Overall, bioinformatic analyses (S. aureus microarray meta-database) revealed that both ethanol and TTO induce related transcriptional alterations. TTO challenge led to the down-regulation of genes involved with energy-intensive transcription and translation, and altered the regulation of genes involved with heat shock (e.g. clpC, clpL, ctsR, dnaK, groES, groEL, grpE and hrcA) and cell wall metabolism (e.g. cwrA, isaA, sle1, vraSR and vraX). Inactivation of the heat shock gene dnaK or vraSR which encodes a two-component regulatory system that responds to peptidoglycan biosynthesis inhibition led to an increase in TTO susceptibility which demonstrates a protective role for these genes in the S. aureus TTO response. A gene (mmpL) encoding a putative resistance, nodulation and cell division efflux pump was also highly induced by TTO. The principal antimicrobial TTO terpene, terpinen-4-ol, altered ten genes in a transcriptional direction analogous to TTO. Collectively, this study provides additional insight into the response of a bacterial pathogen to the antimicrobial terpene mixture TTO.
Asunto(s)
Antibacterianos/farmacología , Staphylococcus aureus/efectos de los fármacos , Aceite de Árbol de Té/farmacología , Transcriptoma/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Etanol/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Staphylococcus aureus/genética , Terpenos/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Purpose: Using the model of experimental autoimmune uveitis (EAU) induced by immunization with a retinal antigen, two studies have reported contradictory results on disease development following oral antibiotic treatment (ABX). We showed that long-term ABX did not affect EAU, but another study showed that short-term ABX was protective. We therefore studied the effects of ABX on EAU, gut microbiota, and host immune responses as a function of treatment duration. Methods: EAU-susceptible mice were treated orally with broad-spectrum antibiotics starting at least 10 weeks (long-term) or 1 week (short-term) before immunization until termination of the experiment. Gut microbiota were characterized by 16S amplicon sequencing, and host gut immune elements were studied phenotypically and functionally. Results: Long-term ABX had no effect, whereas short-term ABX delayed EAU, as previously reported by us and others, respectively. Microbial sequencing revealed progressive reduction of gut microbiota that showed some differences in the two ABX groups. Interestingly, duration of ABX was associated with a gradual disappearance of the CD4+ and CD4+CD8+ subset of gut intraepithelial lymphocytes (IELs). This IEL subset is microbiota dependent and is absent in germ-free mice. Relative abundance of Lactobacillus reuteri correlated with the frequencies of CD4+CD8+ IELs. IELs suppressed antigen-specific activation of autoreactive T cells in culture. Conclusions: Gut microbiota may play dual roles in uveitis development: They promote EAU development but also help maintain IEL populations that have regulatory function against autoreactive T cells. We propose that the progressive loss of this population during long-term ABX reverses the EAU-ameliorating effects of microbiota depletion.
Asunto(s)
Microbioma Gastrointestinal , Microbiota , Uveítis , Animales , Ratones , Inmunización , Administración Oral , Antibacterianos , Uveítis/prevención & controlRESUMEN
Alzheimer's disease (AD) is the leading cause of dementia worldwide, but there are limited therapeutic options and no current cure. While the involvement of microglia in AD has been highly appreciated, the role of other innate and adaptive immune cells remains largely unknown, partly due to their scarcity and heterogeneity. This study aimed to study non-microglial immune cells in wild type and AD-transgenic mouse brains across different ages. Our results uncovered the presence of a unique CD8+ T cell population that were selectively increased in aging AD mouse brains, here referred to as "disease-associated T cells (DATs)". These DATs were found to express an elevated tissue-resident memory and Type I interferon-responsive gene signature. Further analysis of aged AD mouse brains showed that these CD8+ T cells were not present in peripheral or meningeal tissues. Preventing CD8+ T cell development in AD-transgenic mice via genetic deletion of beta-2 microglobulin ( B2m ) led to a reduction of amyloid-ß plaque formation in aged mice, and improved memory in AD-transgenic mice as early as four months of age. The integration of transcriptomic and epigenomic profiles at the single-cell level revealed potential transcription factors that reshape the regulomes of CD8+ T cells. These findings highlight a critical role for DATs in the progression of AD and provide a new avenue for treatment.
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Eukaryotic cells utilize complex signaling systems to detect their environments, responding and adapting as new conditions arise during evolution. The basidiomycete fungus Cryptococcus neoformans is a leading cause of AIDS-related death worldwide and utilizes the calcineurin and protein kinase C-1 (Pkc1) signaling pathways for host adaptation and expression of virulence. In the present studies, a C-terminal zinc finger transcription factor, homologous both to the calcineurin-responsive zinc fingers (Crz1) of ascomycetes and to the Pkc1-dependent specificity protein-1 (Sp1) transcription factors of metazoans, was identified and named SP1 because of its greater similarity to the metazoan factors. Structurally, the Cryptococcus neoformans Sp1 (Cn Sp1) protein was found to have acquired an additional zinc finger motif from that of Crz1 and showed Pkc1-dependent phosphorylation, nuclear localization, and whole genome epistatic associations under starvation conditions. Transcriptional targets of Cn Sp1 shared functional similarities with Crz1 factors, such as cell wall synthesis, but gained the regulation of processes involved in carbohydrate metabolism, including trehalose metabolism, and lost others, such as the induction of autophagy. In addition, overexpression of Cn Sp1 in a pkc1Δ mutant showed restoration of altered phenotypes involved in virulence, including cell wall stability, nitrosative stress, and extracellular capsule production. Cn Sp1 was also found to be important for virulence of the fungus using a mouse model. In summary, these data suggest an evolutionary shift in C-terminal zinc finger proteins during fungal evolution, transforming them from calcineurin-dependent to PKC1-dependent transcription factors, helping to shape the role of fungal pathogenesis of C. neoformans.
Asunto(s)
Pared Celular/metabolismo , Criptococosis/metabolismo , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidad , Proteínas Fúngicas/metabolismo , Proteína Quinasa C/metabolismo , Factores de Transcripción/metabolismo , Factores de Virulencia/metabolismo , Animales , Pared Celular/genética , Criptococosis/genética , Criptococosis/microbiología , Cryptococcus neoformans/genética , Modelos Animales de Enfermedad , Proteínas Fúngicas/genética , Humanos , Ratones , Proteína Quinasa C/genética , Factores de Transcripción/genética , Factores de Virulencia/genética , Dedos de ZincRESUMEN
Here, we report the genome sequence of a protective commensal, Corynebacterium mastitidis RC, isolated from mouse conjunctiva. The C. mastitidis RC genome sequence is 2,153,054 bp in size and 96.95% complete, and we believe that it can contribute to the understanding of the functional immune attributes of the ocular commensal microbiome.
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Staphylococcus aureus possesses 16 two-component systems (TCSs), two of which (GraRS and NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the firmicutes. NsaRS has recently been documented as being important for nisin resistance in S. aureus. In this study, we present a characterization of NsaRS and reveal that, as with other IM-HK TCSs, it responds to disruptions in the cell envelope. Analysis using a lacZ reporter-gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-envelope-damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, carbonyl cyanide m-chlorophenylhydrazone and penicillin G. Additionally, we reveal that NsaRS regulates a downstream transporter NsaAB during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall-targeting antibiotic bacitracin. Microarray analysis reveals that the transcription of 245 genes is altered in an nsaS mutant, with the vast majority being downregulated. Included within this list are genes involved in transport, drug resistance, cell envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using inductively coupled plasma-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in cell envelope structure. Finally, a variety of virulence-related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus, NsaRS is important in sensing cell damage in S. aureus and functions to reprogram gene expression to modify cell envelope architecture, facilitating adaptation and survival.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/fisiología , Regulación Bacteriana de la Expresión Génica , Staphylococcus aureus/fisiología , Estrés Fisiológico , Antibacterianos/toxicidad , Fusión Artificial Génica , Proteínas Bacterianas/genética , Membrana Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Genes Reporteros , Histidina Quinasa , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Transducción de Señal , Staphylococcus aureus/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) which has been shown to increase the susceptibility of various bacteria to antimicrobials and demonstrated to have broad antimicrobial activity. This study describes transcriptome alterations in S. aureus strain COL grown with diclofenac and characterizes the effects of this NSAID on antibiotic susceptibility in laboratory, clinical and diclofenac reduced-susceptibility (DcRS) S. aureus strains. METHODS: Transcriptional alterations in response to growth with diclofenac were measured using S. aureus gene expression microarrays and quantitative real-time PCR. Antimicrobial susceptibility was determined by agar diffusion MICs and gradient plate analysis. Ciprofloxacin accumulation was measured by fluorescence spectrophotometry. RESULTS: Growth of S. aureus strain COL with 80 µg/ml (0.2 × MIC) of diclofenac resulted in the significant alteration by ≥2-fold of 458 genes. These represented genes encoding proteins for transport and binding, protein and DNA synthesis, and the cell envelope. Notable alterations included the strong down-regulation of antimicrobial efflux pumps including mepRAB and a putative emrAB/qacA-family pump. Diclofenac up-regulated sigB (σB), encoding an alternative sigma factor which has been shown to be important for antimicrobial resistance. Staphylococcus aureus microarray metadatabase (SAMMD) analysis further revealed that 46% of genes differentially-expressed with diclofenac are also σB-regulated. Diclofenac altered S. aureus susceptibility to multiple antibiotics in a strain-dependent manner. Susceptibility increased for ciprofloxacin, ofloxacin and norfloxacin, decreased for oxacillin and vancomycin, and did not change for tetracycline or chloramphenicol. Mutation to DcRS did not affect susceptibility to the above antibiotics. Reduced ciprofloxacin MICs with diclofenac in strain BB255, were not associated with increased drug accumulation. CONCLUSIONS: The results of this study suggest that diclofenac influences antibiotic susceptibility in S. aureus, in part, by altering the expression of regulatory and structural genes associated with cell wall biosynthesis/turnover and transport.
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Antibacterianos/farmacología , Antiinflamatorios no Esteroideos/farmacología , Diclofenaco/farmacología , Perfilación de la Expresión Génica , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/metabolismoRESUMEN
OBJECTIVES: Fusidic acid interferes with the release of elongation factor G (EF-G) after the translocation step of protein synthesis. The objective of this study was to characterize the fusidic acid stimulon of a fusidic acid-susceptible strain of Staphylococcus aureus (SH1000). METHODS: S. aureus microarrays and real-time PCR determined transcriptome alterations occurring in SH1000 grown with fusidic acid. The Staphylococcus aureus microarray meta-database (SAMMD) compared and contrasted the SH1000 fusidic stimulon with 89 other S. aureus transcriptional datasets. Fusidic acid gradient analyses with mutant-parent strain pairs were used to identify genes required for intrinsic fusidic acid susceptibility identified during transcriptional analysis. RESULTS: Many genes altered by fusidic acid challenge are associated with protein synthesis. SAMMD analysis determined that the fusidic acid stimulon has the greatest overlap with the S. aureus cold shock and stringent responses. Six out of nine peptidoglycan hydrolase genes making up the two component YycFG regulon were also up-regulated by fusidic acid, as were a carboxylesterase gene (est) and two putative drug efflux pump genes (emr-qac1 and macA). Genes down-regulated by fusidic acid induction encoded a putative secreted acid phosphatase and a number of protease genes. Roles for the agr operon, the peptidoglycan hydrolase gene isaA and two proteases (htrA1 and htrA2) in the expression of fusidic acid susceptibility were revealed. CONCLUSIONS: The SH1000 fusidic acid stimulon includes genes involved with two stress responses, YycFG-regulated cell wall metabolism, drug efflux, and protein synthesis and turnover.
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Antibacterianos/farmacología , Ácido Fusídico/farmacología , Regulación Bacteriana de la Expresión Génica , Staphylococcus aureus/efectos de los fármacos , Perfilación de la Expresión Génica , Genes Bacterianos , Análisis de Secuencia por Matrices de Oligonucleótidos , Biosíntesis de Proteínas/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Staphylococcus aureus/genéticaRESUMEN
BACKGROUND: Staphylococcus aureus is an important pathogen that forms biofilms. The global regulator sarA is essential for biofilm formation. Since the modulator of sarA (msa) is required for full expression of sarA and regulates several virulence factors, we examined the capacity of the msa mutant to form biofilm. RESULTS: We found that mutation of msa results in reduced expression of sarA in biofilm and that the msa mutant formed a weak and unstable biofilm. The msa mutant is able to adhere to surfaces and begins to form biofilm but fails to mature indicating that the defect of the msa mutant biofilm is in the accumulation stage but not in primary adhesion. CONCLUSION: The msa gene plays an important role in biofilm development which is likely due to its role in modulating the expression of sarA. This finding is significant because it identifies a new gene that plays a role in the development of biofilm.
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Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Mutación/genética , Staphylococcus aureus/metabolismo , Transactivadores/metabolismoRESUMEN
BACKGROUND: Staphylococcus aureus is a human pathogen that causes a wide variety of life-threatening infections using a large number of virulence factors. One of the major global regulators used by S. aureus is the staphylococcal accessory regulator (sarA). We have identified and characterized a new gene (modulator of sarA: msa) that modulates the expression of sarA. Genetic and functional analysis shows that msa has a global effect on gene expression in S. aureus. However, the mechanism of Msa function is still unknown. Function predictions of Msa are complicated by the fact that it does not have a homologous partner in any other organism. This work aims at predicting the structure and function of the Msa protein. RESULTS: Preliminary sequence analysis showed that Msa is a putative membrane protein. It would therefore be very difficult to purify and crystallize Msa in order to acquire structure information about this protein. We have used several computational tools to predict the physico-chemical properties, secondary structural features, topology, 3D tertiary structure, binding sites, motifs/patterns/domains and cellular location. We have built a consensus that is derived from analysis using different algorithms to predict several structural features. We confirm that Msa is a putative membrane protein with three transmembrane regions. We also predict that Msa has phosphorylation sites and binding sites suggesting functions in signal transduction. CONCLUSION: Based on our predictions we hypothesise that Msa is a novel signal transducer that might be involved in the interaction of the S. aureus with its environment.
Asunto(s)
Modelos Químicos , Modelos Moleculares , Alineación de Secuencia/métodos , Análisis de Secuencia/métodos , Staphylococcus aureus/metabolismo , Factores de Transcripción/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Simulación por Computador , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Staphylococcus aureus is an important human pathogen, causing a wide variety of diseases ranging from superficial skin infections to severe life threatening infections. S. aureus is one of the leading causes of nosocomial infections. Its ability to resist multiple antibiotics poses a growing public health problem. In order to understand the mechanism of pathogenesis of S. aureus, several global expression profiles have been developed. These transcriptional profiles included regulatory mutants of S. aureus and growth of wild type under different growth conditions. The abundance of these profiles has generated a large amount of data without a uniform annotation system to comprehensively examine them. We report the development of the Staphylococcus aureus Microarray meta-database (SAMMD) which includes data from all the published transcriptional profiles. SAMMD is a web-accessible database that helps users to perform a variety of analysis against and within the existing transcriptional profiles. DESCRIPTION: SAMMD is a relational database that uses MySQL as the back end and PHP/JavaScript/DHTML as the front end. The database is normalized and consists of five tables, which holds information about gene annotations, regulated gene lists, experimental details, references, and other details. SAMMD data is collected from the peer-reviewed published articles. Data extraction and conversion was done using perl scripts while data entry was done through phpMyAdmin tool. The database is accessible via a web interface that contains several features such as a simple search by ORF ID, gene name, gene product name, advanced search using gene lists, comparing among datasets, browsing, downloading, statistics, and help. The database is licensed under General Public License (GPL). CONCLUSION: SAMMD is hosted and available at http://www.bioinformatics.org/sammd/. Currently there are over 9500 entries for regulated genes, from 67 microarray experiments. SAMMD will help staphylococcal scientists to analyze their expression data and understand it at global level. It will also allow scientists to compare and contrast their transcriptome to that of the other published transcriptomes.
Asunto(s)
Bases de Datos Genéticas , Perfilación de la Expresión Génica , Análisis por Micromatrices , Staphylococcus aureus/genética , Biología Computacional , Interfaz Usuario-ComputadorRESUMEN
Infection of mice with strains of Plasmodium yoelii parasites can result in different pathology, but molecular mechanisms to explain this variation are unclear. Here we show that a P. yoelii gene encoding a HECT-like E3 ubiquitin ligase (Pyheul) influences parasitemia and host mortality. We genetically cross two lethal parasites with distinct disease phenotypes, and identify 43 genetically diverse progeny by typing with microsatellites and 9230 single-nucleotide polymorphisms. A genome-wide quantitative trait loci scan links parasite growth and host mortality to two major loci on chromosomes 1 and 7 with LOD (logarithm of the odds) scores = 6.1 and 8.1, respectively. Allelic exchange of partial sequences of Pyheul in the chromosome 7 locus and modification of the gene expression alter parasite growth and host mortality. This study identifies a gene that may have a function in parasite growth, virulence, and host-parasite interaction, and therefore could be a target for drug or vaccine development.Many strains of Plasmodium differ in virulence, but factors that control these distinctions are not known. Here the authors comparatively map virulence loci using the offspring from a P. yoelii YM and N67 genetic cross, and identify a putative HECT E3 ubiquitin ligase that may explain the variance.
Asunto(s)
Interacciones Huésped-Parásitos/genética , Malaria/parasitología , Plasmodium yoelii/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Cruzamientos Genéticos , Femenino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Parasitemia/genética , Parasitemia/parasitología , Plasmodium yoelii/crecimiento & desarrollo , Plasmodium yoelii/patogenicidad , Virulencia/genéticaRESUMEN
The symptoms of malaria are brought about by blood-stage parasites, which are established when merozoites invade human erythrocytes. Our understanding of the molecular events that underpin erythrocyte invasion remains hampered by the short-period of time that merozoites are invasive. To address this challenge, a Plasmodium falciparum gamma-irradiated long-lived merozoite (LLM) line was developed and investigated. Purified LLMs invaded erythrocytes by an increase of 10-300 fold compared to wild-type (WT) merozoites. Using an integrated omics approach, we investigated the basis for the phenotypic difference. Only a few single nucleotide polymorphisms within the P. falciparum genome were identified and only marginal differences were observed in the merozoite transcriptomes. By contrast, using label-free quantitative mass-spectrometry, a significant change in protein abundance was noted, of which 200 were proteins of unknown function. We determined the relative molar abundance of over 1100 proteins in LLMs and further characterized the major merozoite surface protein complex. A unique processed MSP1 intermediate was identified in LLM but not observed in WT suggesting that delayed processing may be important for the observed phenotype. This integrated approach has demonstrated the significant role of the merozoite proteome during erythrocyte invasion, while identifying numerous unknown proteins likely to be involved in invasion.