Unknown,Transcriptomics,Genomics,Proteomics

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Pan-genomic analysis of bovine monocyte-derived macrophage gene expression in response to in vitro infection with Mycobacterium avium subspecies paratuberculosis

ABSTRACT: Background: Mycobacterium avium subspecies paratuberculosis (MPTb) is the causative agent of Johne’s disease, an intestinal disease of ruminants with major economic consequences. MPTb bacilli are phagocytosed by host macrophages upon exposure where they persist, resulting in lengthy subclinical phases of infection that can lead to immunopathology and disease dissemination. Consequently, analysis of the macrophage transcriptome in response to MPTb infection can provide valuable insights into the molecular mechanisms that underlie Johne’s disease. Here, we investigate pan-genomic gene expression in bovine monocyte-derived macrophages (MDM) purified from seven age-matched non-related females, in response to in vitro infection with MPTb (multiplicity of infection 2:1) at intervals of 2 hours, 6 hours and 24 hours post-infection. Results: Differentially expressed genes were identified by comparing the transcriptomes of the infected MDM to the control MDM at each time point (adjusted P-value threshold ≤ 0.10). 1,050 differentially expressed unique genes were identified 2 hours post-infection, with 974 and 84 differentially expressed unique genes detected 6 hours and 24 hours post-infection, respectively. Furthermore, in the infected MDM the number of upregulated genes exceeded the number of downregulated genes at each time point, with the fold-change in expression for the upregulated genes markedly higher than that for the downregulated genes. Conclusions: Inspection and systems analysis of the differentially expressed genes revealed changes in the expression profiles of genes involved in the inflammatory response, cell signalling pathways and apoptosis. The transcriptional changes associated with cellular signalling and the inflammatory response may reflect different immuno-modulatory mechanisms that underlie host-pathogen interactions during infection. Affymetrix GeneChip® Bovine Genome Arrays were used to examine gene expression of bovine monocyte-derived macrophages (MDM) after in vitro challenge with Mycobacterium avium subspecies paratuberculosis across a time series of 2 hr, 6 hr and 24 hr post-challenge. A 0 hr control treatment was also generated and seven different age-matched female Holstein-Friesian cattle were used for each time-point/treatment combination for a total of 49 microarrays, one of which was excluded from analysis since it did not pass quality control.

ORGANISM(S): Bos taurus

SUBMITTER: David MacHugh

PROVIDER: E-GEOD-35185 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major cause of mortality in global cattle populations. Macrophages are among the first cells types to encounter M. bovis following exposure and the response elicited by these cells is pivotal in determining the outcome of infection. Here, a functional genomics approach was undertaken to investigate global gene expression profiles in bovine monocyte-derived macrophages (MDM) purified from seven age-matched non-related females, in response to in vitro challenge with M. bovis (multiplicity of infection 2:1). Total cellular RNA was extracted from non-challenged control and M. bovis-challenged MDM for all animals at intervals of 2 hours, 6 hours and 24 hours post-challenge and prepared for global gene expression analysis using the Affymetrix® GeneChip® Bovine Genome Array. Results: Comparison of M. bovis-challenged MDM gene expression profiles with the non-challenged MDM controls at each time point identified 3,529 differentially expressed genes after 2 hours post-challenge, with 5,211 and 6,150 differentially expressed genes detected at the 6 hour and 24 hour time points, respectively (adjusted P-value threshold ≤ 0.05). Notably, the number of downregulated genes exceeded the number of upregulated genes in the M. bovis-challenged MDM across all time points; however, the fold-change in expression for the upregulated genes was markedly larger than that for the downregulated genes. Systems analysis revealed enrichment for genes involved in: (1) the inflammatory response; (2) cell signalling pathways, including Toll-like receptors and intracellular pathogen recognition receptors (PRRs)-receptors; and (3) apoptosis. Conclusions: The increased number of downregulated genes is consistent with previous studies showing that M. bovis infection is associated with the repression of host gene expression. The results also support important roles for MYD88-independent signalling and intracellular PRRs in mediating the host response to M. bovis, which to our knowledge have not been reported previously. Affymetrix GeneChip® Bovine Genome Arrays were used to examine gene expression of bovine monocyte-derived macrophages (MDM) after in vitro challenge with Mycobacterium bovis across a time series of 2 hr, 6 hr and 24 hr post-challenge. A 0 hr control treatment was also generated and seven different age-matched female Holstein-Friesian cattle were used for each time-point/treatment combination for a total of 49 microarrays.

Project description:Background: MycobacteriumM-BM- bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M.M-BM- bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density AffymetrixM-BM-. GeneChipM-BM-. Bovine Genome Array platform from the same MDM-extracted RNA. Results: A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single BosM-BM- taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value M-bM-^IM-$ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology.M-BM- Conclusions: This study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA. Description of the transcriptomic host response after infection of bovine monocyte-derived macrophages with Mycobacterium bovis

Project description:Bbr_0838 from Bifidobacterium breve UCC2003 encodes a 683 residue membrane protein, that contains a permease domain displaying similarity to transporters belonging to the major facilitator superfamily, as well as a CBS (cystathionine beta synthase) domain. The high level of similarity to bile-efflux pumps from other bifidobacteria, suggests a significant role for Bbr_0838 in bile tolerance of B. breve UCC2003. Bbr_0838 transcription was shown to be monocistronic and strongly induced upon exposure to bile. Further analysis delineated the transcriptional start site and the minimal region required for promoter activity and bile regulation. Insertional inactivation of Bbr_0838 in B. breve UCC2003 resulted in a strain that exhibited reduced survival upon cholate exposure as compared to the parent strain, a phenotype that was reversed when a functional Bbr_0838 gene was introduced into UCC2003::838800. Transcriptome analysis of UCC2003::838800 grown in the presence or absence of bile demonstrated that transcription of Bbr_0832, which is predicted to encode a macrolide-efflux transporter gene, was significantly increased in the presence of bile, representing a likely compensatory mechanism for bile removal in the absence of Bbr_0838. This study represents the first in depth analysis of a bile-inducible locus in bifidobacteria, identifying a key gene relevant for bifidobacterial bile tolerance. In order to investigate differences in global gene expression upon growth or exposure of B. breve UCC2003-delta0838 to cholic acid compared to normal growing cells, DNA microarray experiments were performed. Total RNA was isolated from B. breve UCC2003-delta0838 cultures under normal conditions and cultures grown on or exposed to cholic acid. All experiments were performed in duplo and targets where confirmed with QRT-PCR. In addition transcriptome analyse was performed of B. breve UCC2003 compared to that of B. breve UCC2003-delta0838 both exposed to 0.1 % cholic acid. This was performed as a single experiment and targets were confirmed by QRT-PCR

Project description:In this study, we describe the functional characterisation of the B. breve UCC2003 gal locus, which is dedicated to the utilisation of galactan, a plant-derived polysaccharide. Using a combination of molecular approaches we conclude that the galA gene of B. breve UCC2003 encodes a beta-1,4-endogalactanase producing galacto-oligosaccharides, which are specifically internalised by an ABC transport system, encoded by galBCDE, and which are then hydrolysed to galactose moieties by a dedicated intracellular beta-galactosidase, specified by galG. The generated galactose molecules are presumed to be fed into the fructose-6-phosphate phosphoketolase pathway via the Leloir pathway, thereby allowing B. breve UCC2003 to use galactan as its sole carbon and energy source. In addition to these findings we demonstrate that GalR is a LacI-type DNA-binding protein, which not only appears to control transcription of the galCDEGR operon, but also that of the galA gene. DNA-microarrays containing oligonucleotide primers representing each of the 1864 annotated genes on the genome of B. breve UCC2003 were designed by and obtained from Agilent Technologies (Palo Alto, Ca., USA). Methods for cell disruption, RNA isolation, RNA quality control, complementary DNA synthesis and labeling were performed as described previously (Pokusaeva et al., 2009). Labeled cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis v4.0 manual (G4140-90050). Following hybridization, microarrays were washed in accordance with Agilent’s standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A). Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.5). DNA-microarray data were processed as previously described (Garcia De La Nava et al., 2003). Differential expression tests were performed with the Cyber-T implementation of a variant of the t-test (Long et al., 2001). A gene was considered differentially expressed when p < 0.001 and an expression ratio of >3 or <0.33 relative to the control.

Project description:In the present study we show that a functional luxS gene is present in all members of the genus Bifidobacterium and that AI-2 production by Bifidobacterium breve UCC2003 is necessary to confer protection against Salmonella infection in Caenorhabditis elegans. Mutants carrying a disrupted luxS gene conferred less protection than the WT, whereas administration of a complemented strain, a LuxS-overexpression strain and the WT resulted in similar survival. Transcriptome data obtained for both the WT and the luxS-insertion mutant grown in vitro suggest that LuxS has a metabolic role that impacts on iron metabolism. In line with this, we demonstrated that the luxS mutant is significantly more susceptible to the ferrous ion chelator 2,2-dipyridyl than the WT strain. Furthermore, qRT-PCR shows that the iron regulated genes are upregulated under in vivo conditions, while we also show that LuxS is required for colonization of BALB/c mice. Altogether, our findings provide detailed insights into the molecular mechanism underlying gut pathogen protection conferred by bifidobacteria. DNA-microarrays containing oligonucleotide primers representing each of the 1864 annotated genes on the genome of B. breve UCC2003 (O'Connell Motherway et al., 2011) were designed by and obtained from Agilent Technologies (Palo Alto, Ca., USA). Methods for cell disruption, RNA isolation, RNA quality control, complementary DNA synthesis and labeling were performed as described previously (Pokusaeva et al., 2009). Labeled cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis v4.0 manual (G4140-90050). Following hybridization, microarrays were washed in accordance with Agilentâs standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A). Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.5). DNA-microarray data were processed as previously described (Garcia De La Nava et al., 2003). Differential expression tests were performed with the Cyber-T implementation of a variant of the t-test (Long et al., 2001). A gene was considered differentially expressed when p < 0.001 and an expression ratio of >3 or <0.33 relative to the control.

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Pan-genomic analysis of bovine monocyte-derived macrophage gene expression in response to in vitro infection with Mycobacterium avium subspecies paratuberculosis

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