Project description:In order to uncover transcriptional changes occurring in Brucella- infected mouse brains that might lead to neurologic complications, we infected susceptible mice intranasally with Brucella and harvested their brains.
Project description:Using Solexa/Illumina's digital gene expression (DGE) system, a tag-based transcriptome sequencing method, we investigated the kinetic transcriptional profile of gene expression in macrophages infected with Brucella melitensis strain 16M. A key aspect of Brucella virulence is their ability to proliferate within professional and nonprofessional phagocytic host cells, thereby successfully bypassing the bactericidal effects of phagocytes. Their virulence and chronic infections are thought to be due to their ability to avoid the killing mechanisms within host cells. Defining the interaction between a host cell and Brucella is crucial to understanding the infectious process. Most researchers have studied the pathogens, but the host plays a very important role during infections. To date, relatively few host factors have been shown important in Brucella infections. However, little is known about the host networks that mediate infection. The objective of the study is to analyze the genes and cellular components related to the innate immunity response to determine the mechanisms through which Brucella avoids the host innate immunity. A total of 3576 and 3962 genes that are differentially expressed between 0 and 4 h and between 0 and 24 h were identified. The identified genes are related to immune processes, signal transduction, inflammation, apoptosis, cell membrane, transcriptional regulation, and intracellular trafficking. Our data have added to the current understanding of different host gene expressions during different infection phases by Brucella spp.
Project description:Gene expression analysis of wild-type and STING knock-out mouse bone marrow-derived macrophages (mBMDM) infected with Brucella abortus or transfected with Brucella abortus DNA. Genes whose expression are affected by Brucella abortus in a STING-dependent manner will be identified and signaling pathways regulated by STING will be elucidated.
Project description:In a time course study, we characterized global gene expression profile of B. melitensis-infected epithelium-like cells at the onset of infection. B. melitensis-infected HeLa cells exhibited a down-regulated expression profile at 4 h (48 up- and 109 down-regulated genes) that transitioned to an activated transcriptional profile at 12 h post-infection (733 up- and 224 down-regulated genes). The analysis of the results indicates that infected cells undergo an adaptation period during the first 4 h p.i. that is overcome by 12 h p.i., permitting Brucella to replicate intracellularly while minimally affecting host physiological processes. Specific genes and biological processes identified in this study will further help elucidate how both host and Brucella interact during the early infectious process to the eventual benefit of the pathogen and to the detriment of the naïve host. Keywords: Time course study of gene expression profile of Brucella melitensis-infected HeLa cells
Project description:Using Solexa/Illumina's digital gene expression (DGE) system, a tag-based transcriptome sequencing method, we investigated the kinetic transcriptional profile of gene expression in macrophages infected with Brucella melitensis strain 16M. A key aspect of Brucella virulence is their ability to proliferate within professional and nonprofessional phagocytic host cells, thereby successfully bypassing the bactericidal effects of phagocytes. Their virulence and chronic infections are thought to be due to their ability to avoid the killing mechanisms within host cells. Defining the interaction between a host cell and Brucella is crucial to understanding the infectious process. Most researchers have studied the pathogens, but the host plays a very important role during infections. To date, relatively few host factors have been shown important in Brucella infections. However, little is known about the host networks that mediate infection. The objective of the study is to analyze the genes and cellular components related to the innate immunity response to determine the mechanisms through which Brucella avoids the host innate immunity. A total of 3576 and 3962 genes that are differentially expressed between 0 and 4 h and between 0 and 24 h were identified. The identified genes are related to immune processes, signal transduction, inflammation, apoptosis, cell membrane, transcriptional regulation, and intracellular trafficking. Our data have added to the current understanding of different host gene expressions during different infection phases by Brucella spp. The RAW264.7 cells were seeded into 24-well plates at 5-105 cells/well, incubated in 5% CO2 at 37 M-BM-0C for 24 h, and then infected with Brucella at a multiplicity of infection of 200. To synchronize the infection, the infected plates were centrifuged at 200 ug for 5 min at room temperature and then incubated at 37 M-BM-0C for 20 minutes. The infected cell monolayers were washed three times with PBS, overlaid with 0.5 ml of DMEM containing 100 mg/ml of ampicillin and 50 mg/ml of kanamycin at 37 M-BM-0C for 0, 4, and 24 h. At the end of the incubation period, the culture medium was removed and centrifuged at 1500 rpm for 15 min. The cell pellet and adherent cells were resuspended in TRIzol. Total RNA were extracted from the cells at 0, 4 and 24 h post infection.
Project description:In a time course study, we characterized global gene expression profile of B. melitensis-infected epithelium-like cells at the onset of infection. B. melitensis-infected HeLa cells exhibited a down-regulated expression profile at 4 h (48 up- and 109 down-regulated genes) that transitioned to an activated transcriptional profile at 12 h post-infection (733 up- and 224 down-regulated genes). The analysis of the results indicates that infected cells undergo an adaptation period during the first 4 h p.i. that is overcome by 12 h p.i., permitting Brucella to replicate intracellularly while minimally affecting host physiological processes. Specific genes and biological processes identified in this study will further help elucidate how both host and Brucella interact during the early infectious process to the eventual benefit of the pathogen and to the detriment of the naM-CM-/ve host. Keywords: Time course study of gene expression profile of Brucella melitensis-infected HeLa cells We generated 4 different samples: A) RNA isolated from Brucella melitensis-infected HeLa cells at 4 h p.i.; B) RNA isolated from non-infected HeLa cells at 4 h p.i. of the A samples; C) RNA isolated from Brucella melitensis-infected HeLa cells at 12 h p.i., and D) RNA isolated from non-infected HeLa cells at 12 h p.i. of the D samples. The B. melitensis-infected HeLa cells gene expression was compared with gene expression from non-infected HeLa cells treated similarly. There are four biological replicates of each sample and time point (n=16). ORFs were single spotted on each microarray. Each replicate was normalized against labeled universal human reference RNA.
Project description:The macrophage-Brucella interaction is critical for the establishment of a chronic Brucella infection. Smooth virulent B. suis strain 1330 (S1330) prevents macrophage cell death. However, rough attenuated B. suis strain VTRS1 induces strong macrophage cell death. To further investigate the mechanism of VTRS1-induced macrophage cell death, microarrays were used to analyze temporal transcriptional responses of murine macrophage-like J774. A1 cells infected with S1330 or VTRS1.
Project description:Transcriptional profile compairing infected human macrophages at 2 and 4 h p.i. with virulent strain Brucella melitensis BM16M vs wildtype BM133 , BM16M vs mutant LVM31, BM133 vs 16M.
Project description:Brucella suis infects macrophages and dendritic cells. Wild boars act as reservoirs and carriers of Brucella suis biovar 2, and there is evidence that wild boar can be the main source of infection for domestic pigs through the venereal route. Transmission through this route could be an important path for disesease dissemination. The result from this study will contribute to the overall understanding of the molecular pathogenic mechanisms involved during Brucella suis infection in European wild boar. Experiment Overall Design: In this study we preliminarily characterized differential gene expression in European wild boar naturally infected with Brucella suis biovar 2 using Microarray hybridization and Real Time RT-PCR analysis. Since Brucella suis acts by infecting macrophages, we used spleen cells to analyze the gene expression response to Brucella suis infection.
Project description:We describe the isolation of sufficient Brucella abortus RNA from primary host cell environment using modified reported methods for RNA-seq analysis, and simultaneously characterize the transcriptional profiles of intracellular B. abortus and bone marrow-derived macrophages (BMM) from BALB/c mice at 24 h (replicative phase) post-infection.