RESUMO
Brucellosis is a worldwide zoonotic infectious disease that has a significant economic impact on animal production and human public health. We characterized the gene expression profile of B. abortus-infected monocyte-derived macrophages (MDMs) from naïve cattle naturally resistant (R) or susceptible (S) to brucellosis using a cDNA microarray technology. Our data indicate that (1) B. abortus induced a slightly increased genome activation in R MDMs and a down-regulated transcriptome in S MDMs, during the onset of infection, (2) R MDMs had the ability to mount a type 1 immune response against B. abortus infection which was impaired in S cells, and (3) the host cell activity was not altered after 12 h post-B. abortus infection in R MDMs while the cell cycle was largely arrested in infected S MDMs at 12 h p.i. These results contribute to an improved understanding of how host responses may be manipulated to prevent infection by brucellae.
Assuntos
Brucella abortus/imunologia , Brucelose Bovina/genética , Suscetibilidade a Doenças/veterinária , Imunidade Inata , Macrófagos , Animais , Brucelose Bovina/imunologia , Bovinos , Suscetibilidade a Doenças/imunologia , Regulação para Baixo/imunologia , Perfilação da Expressão Gênica/veterinária , Análise de Sequência com Séries de Oligonucleotídeos/veterináriaRESUMO
We sequenced the 5' UTR of the estrogen-related receptor gamma gene (ERR-γ) in ~500 patient and volunteer samples and found that longer alleles of the (AAAG)(n) microsatellite were statistically and significantly more likely to exist in the germlines of breast cancer patients when compared to healthy volunteers. This microsatellite region contains multiple binding sites for a number of transcription factors, and we hypothesized that the polymorphic AAAG-containing sequence in the 5' UTR region of ERR-γ might modulate expression of ERR-γ. We found that the 369 bp PCR product containing the AAAG repeat drove expression of a reporter gene in estrogen receptor positive breast cancer cells. Our results support a role for the 5' UTR region in ERR-γ expression, which is potentially mediated via binding to the variable tandem AAAG repeat, the length of which correlates with breast cancer pre-disposition. Our study indicates that the AAAG tetranucleotide repeat polymorphism in ERR-γ gene 5' UTR region may be a new biomarker for genetic susceptibility to breast cancer.
Assuntos
Regiões 5' não Traduzidas , Alelos , Neoplasias da Mama/genética , Predisposição Genética para Doença , Repetições de Microssatélites , Regiões Promotoras Genéticas , Receptores de Estrogênio/genética , Animais , Sequência de Bases , Biomarcadores Tumorais/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Genes Reporter , Genótipo , Humanos , Dados de Sequência Molecular , Polimorfismo Genético , Receptores de Estrogênio/metabolismoRESUMO
Microsatellites are highly mutable, repetitive sequences commonly used as genetic markers, but they have never been studied en masse. Using a custom microarray to measure hybridization intensities of every possible repetitive nucleotide motif from 1-mers to 6-mers, we examined 25 genomes. Here, we show that global microsatellite content varies predictably by species, as measured by array hybridization signal intensities, correlating with established taxonomic relationships, and particular motifs are characteristic of one species versus another. For instance, hominid-specific microsatellite motifs were identified despite alignment of the human reference, Celera, and Venter genomic sequences indicating substantial variation (30-50%) among individuals. Differential microsatellite motifs were mainly associated with genes involved in developmental processes, whereas those found in intergenic regions exhibited no discernible pattern. This is the first description of a method for evaluating microsatellite content to classify individual genomes.
Assuntos
Composição de Bases/genética , Repetições de Microssatélites/genética , Plantas/genética , Primatas/genética , Animais , Loci Gênicos/genética , Genoma/genética , Humanos , Hibridização de Ácido Nucleico/genética , Análise de Sequência com Séries de Oligonucleotídeos , Pan troglodytes/genética , Especificidade da EspécieRESUMO
Host immune mechanisms were proposed to decline under microgravity conditions during spaceflights, which might result in severe infections in astronauts. Therefore, it was important to investigate the effects of microgravity on infecting organisms and their interaction with host cells. Data showed that simulated microgravity (SMG) conditions markedly increased production of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin, which induced fluid secretory responses in a mouse model. SMG also enhanced production of tumor necrosis factor-alpha in murine macrophages infected with enteropathogenic E. coli (EPEC). In a similar fashion, simulated microgravity conditions augmented the invasive potential of Salmonella enterica serovar typhimurium and enhanced production of tumor necrosis-factor alpha in S. typhimurium-infected epithelial cells. Furthermore, coculturing of macrophages and S. typhimurium in a simulated microgravity environment resulted in activation of stress-associated mitogen-activated protein kinase kinase 4. Using the antiorthostatic tail suspension mouse model, which simulates some aspects of microgravity, oral inoculation of S. typhimurium markedly reduced the 50% lethal dose compared to mice infected under normal gravitational conditions. Microarray analysis revealed simulated microgravity-induced alterations in the expression of 22 genes in S. typhimurium, and protein expression profiles were altered in both EPEC and S. typhimurium, based on two-dimensional gel electrophoresis. These studies indicated alterations in the virulence potential of bacteria and in host responses to these pathogens under simulated microgravity conditions, which may represent an important environmental signal. Such studies are essential for better understanding bacterial-host cell interactions, particularly in the context of spaceflights and space habitations of long duration.
Assuntos
Escherichia coli/patogenicidade , Salmonelose Animal/fisiopatologia , Salmonella typhimurium/patogenicidade , Simulação de Ausência de Peso/efeitos adversos , Animais , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Dinoprostona/análise , Enterobacteriaceae/patogenicidade , Proteínas de Escherichia coli/biossíntese , Genes Bacterianos/fisiologia , Humanos , MAP Quinase Quinase 4/imunologia , Macrófagos/microbiologia , Camundongos , Salmonelose Animal/genética , Salmonelose Animal/imunologia , Fator de Necrose Tumoral alfa/análise , Virulência/genética , Virulência/fisiologiaRESUMO
Braun/murein lipoprotein (Lpp) is one of the major outer membrane components of gram-negative enteric bacteria involved in inflammatory responses and septic shock. In previous studies, we reported that two copies of the lipoprotein (lpp) gene (designated as lppA and lppB) existed on the chromosome of Salmonella enterica serovar Typhimurium. Deletion of both lppA and lppB genes rendered Salmonella defective in invasion, motility, induction of cytotoxicity, and production of inflammatory cytokines/chemokines. The lppAB double-knockout (DKO) mutant was attenuated in mice, and animals immunized with this mutant were protected against subsequent challenge with lethal doses of wild-type (wt) S. Typhimurium. To better understand how deletion of the lpp gene might affect Salmonella virulence, we performed global transcriptional profiling of the genes in the wt and the lppAB DKO mutant of S. Typhimurium using microarrays. Our data revealed alterations in the expression of flagellar genes, invasion-associated type III secretion system genes, and transcriptional virulence gene regulators in the lppAB DKO mutant compared to wt S. Typhimurium. These data correlated with the lppAB DKO mutant phenotype and provided possible mechanism(s) of Lpp-associated attenuation in S. Typhimurium. Although these studies were performed in in vitro grown bacteria, our future research will be targeted at global transcriptional profiling of the genes in in vivo grown wt S. Typhimurium and its Lpp mutant.
Assuntos
Expressão Gênica/fisiologia , Lipoproteínas/genética , Lipoproteínas/metabolismo , Análise em Microsséries , Salmonella typhimurium/metabolismo , Flagelos/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Mutação , Salmonella typhimurium/classificação , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/ultraestrutura , Sorologia , Virulência/genética , Virulência/fisiologiaRESUMO
We investigated the potential of the cytotoxic enterotoxin (Act) of Aeromonas hydrophila to bind to 1869 human and 4319 yeast proteins, using protein microarray technology. Act was capable of binding nine different human proteins, including the SNARE complex scaffolding protein synaptosomal-associated protein 23 (SNAP23), galectin-3, and guanylate kinase 1 (GUK-1). Act was also able to bind to four of the yeast proteins examined, which included the vesicle tethering protein Vsp52. We verified interaction of Act with murine and human SNAP23, galectin-3, and GUK-1 by sandwich Western blot analysis. In order to determine the physiological relevance of Act binding to these three proteins, we performed small interfering RNA (siRNA) gene knockdown experiments in RAW 264.7 cells, a murine macrophage cell line in which Act-induced signaling and cell death is well characterized. Based on real-time reverse transcriptase-polymerase chain reaction, siRNA transfection of RAW 264.7 cells with specific oligonucleotides reduced the expression of genes encoding SNAP23, galectin-3, and GUK-1 by 62, 63, and 99%, respectively. Knockdown of galectin-3 and SNAP23, but not GUK-1, significantly reduced Act-induced apoptosis of host cells, as determined by TUNEL (TdT-mediated dUTP nick end labeling) assay, lactate dehydrogenase release, Giemsa staining, and reduction in activation of caspase 3, compared to toxin-treated macrophages that were transfected with a random sequence control siRNA. We also performed these assays using a human intestinal epithelial cell line (HT-29) and observed a similar trend of galectin-3 and SNAP23 association with Act-induced apoptosis. This is the first report of putative protein binding partners for this toxin and potential mediators/regulators of Act-induced apoptosis.
Assuntos
Aeromonas hydrophila/metabolismo , Apoptose/fisiologia , Proteínas de Bactérias/metabolismo , Enterotoxinas/metabolismo , Galectina 3/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Aeromonas hydrophila/química , Animais , Proteínas de Bactérias/isolamento & purificação , Western Blotting/métodos , Caspase 3 , Caspases/análise , Linhagem Celular , Enterotoxinas/isolamento & purificação , Guanilato Quinases/metabolismo , Células HT29 , Humanos , Imuno-Histoquímica/métodos , L-Lactato Desidrogenase/análise , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Análise Serial de Proteínas/métodos , Ligação Proteica , RNA Interferente Pequeno/metabolismoRESUMO
We constructed Salmonella enterica serovar Typhimurium double-knockout mutants in which either the lipoprotein A (lppA) or the lipoprotein B (lppB) gene was deleted from an msbB-negative background strain by marker exchange mutagenesis. These mutants were highly attenuated when tested with in vitro and in vivo models of Salmonella pathogenesis.
Assuntos
Proteínas de Bactérias/genética , Lipoproteínas/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Aciltransferases/genética , Animais , Proteínas de Bactérias/imunologia , Deleção de Genes , Soros Imunes/imunologia , Lipopolissacarídeos/imunologia , Lipoproteína(a)/genética , Lipoproteína(a)/imunologia , Lipoproteínas/imunologia , Camundongos , Mutação , Infecções por Salmonella/imunologia , Salmonella typhimurium/genética , Salmonella typhimurium/imunologiaRESUMO
We performed microarray analyses on RNA from human intestinal epithelial (HT-29) cells treated with the cytotoxic enterotoxin (Act) of Aeromonas hydrophila to examine global cellular transcriptional responses. Based on three independent experiments, Act upregulated the expression of 34 genes involved in cell growth, adhesion, signaling, immune responses (including interleukin-8 [IL-8] production), and apoptosis. We verified the upregulation of 14 genes by real-time reverse transcriptase-PCR and confirmed Act-induced production of IL-8 by enzyme-linked immunosorbent assay on supernatants from nonpolarized and polarized HT-29 cells. Maximal production of IL-8 in response to Act required the presence of intracellular calcium, since chelation of calcium with BAPTA-AM significantly reduced Act-induced IL-8 production in HT-29 cells. We also examined activation of mitogen-activated protein kinases and, as demonstrated by Western blot analysis of apical side-treated polarized HT-29 cells, Act induced phosphorylation of p38, c-Jun NH(2)-terminal kinase, and extracellular signal-regulated kinase 1/2. In addition, KinetWorks proteomics screening of whole-cell lysates revealed Act-induced phosphorylation of cyclic AMP-response element binding protein (CREB), c-Jun, adducin, protein kinase C, and signal transducer and activator of transcription 3 (STAT3) and decreased phosphorylation of protein kinase Balpha, v-raf-1 murine leukemia viral oncogene homolog 1 (i.e., Raf1), and STAT1. We verified activation of CREB and activator protein 1 in polarized cells by gel shift assay. This is the first description of human intestinal epithelial cell transcriptional alterations, phosphorylation or activation of signaling molecules, cytokine production, and calcium mobilization in response to this toxin.
Assuntos
Proteínas de Bactérias/farmacologia , Enterotoxinas/farmacologia , Células Epiteliais/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas/metabolismo , Proteômica , Aeromonas hydrophila/metabolismo , Aeromonas hydrophila/patogenicidade , Proteínas de Bactérias/metabolismo , Linhagem Celular , Enterotoxinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Intestinos/citologia , Proteínas/genéticaRESUMO
We performed microarray analyses of murine peritoneal macrophages to examine cellular transcriptional responses to a cytotoxic enterotoxin of Aeromonas hydrophila. While 66% of altered genes were common to both primary macrophages and the murine macrophage cell line RAW 264.7, Act caused expression changes of 28 genes specifically in murine peritoneal macrophages.
Assuntos
Aeromonas hydrophila/metabolismo , Proteínas de Bactérias/farmacologia , Enterotoxinas/farmacologia , Regulação da Expressão Gênica , Macrófagos Peritoneais/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Camundongos , Proteínas/genéticaRESUMO
Aeromonas hydrophila is an emerging human pathogen that leads to gastroenteritis and other invasive diseases. By using a murine peritoneal culture (MPC) model, we identified via restriction fragment differential display PCR (RFDDPCR) five genes of A. hydrophila that were differentially expressed under in vivo versus in vitro growth conditions. The gene encoding enolase was among those five genes that were differentially up regulated. Enolase is a glycolytic enzyme and its surface expression was recently shown to be important in the pathogenesis of a gram-positive bacterium Streptococcus pyogenes. By Western blot analysis and Immunogold staining, we demonstrated secretion and surface expression of enolase in A. hydrophila. We also showed that the whole cells of A. hydrophila had strong enolase activity. Using an enzyme-linked immunosorbant assay and sandwich Western blot analysis, we demonstrated binding of enolase to human plasminogen, which is involved in the fibrinolytic system of the host. We cloned the A. hydrophila enolase gene, which exhibited 62% homology at the DNA level and 57% homology at the amino acid level when compared to S. pyogenes enolase. This is a first report describing the increased expression of enolase gene in vivo that could potentially contribute to the pathogenesis of A. hydrophila infections.