B. pseudomallei genes differentially expressed during host cell infection
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PROVIDER: PRJNA659553 | ENA |
REPOSITORIES: ENA
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Project description:Prokaryotic cell transcriptomics have been limited to mixed or sub-population dynamics and individuality of cells within heterogeneous populations. This significantly hampers further knowledge into spatiotemporal and stage-specific processes of prokaryotic cells within complex environments. Herein, we developed a ‘TRANSITomic’ approach to profile transcriptomes of single Burkholderia pseudomallei (Bp) cells as they transit through host cell infection at defined stages, yielding pathophysiological insights. Bp transits through host cells during infection in three observable stages: i) vacuole entry; ii) cytoplasmic escape and replication; and iii) membrane protrusion, promoting cell-to-cell spread. The Bp ‘TRANSITome’ revealed dynamic gene-expression flux during transit in host cells. Some genes undergoing changes are required for pathogenesis. We discovered several hypothetical proteins and assigned them to virulence mechanisms such as attachment, cytoskeletal modulation, and autophagy evasion. The Bp ‘TRANSITome’ has provided high-resolution understanding of host-pathogen interactions and opens the door for future single-cell transcriptomic analysis of other prokaryotic processes.
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Project description:Burkholderia pseudomallei is a Gram-negative pathogen responsible for melioidosis, a life-threatening disease endemic to Southeast Asia. LysR-type transcriptional regulators (LTTRs) are known key regulators of bacterial pathogenesis and metabolism, yet many remain uncharacterized. This study investigates the function of UKMD286_5923, a novel LTTR, in B. pseudomallei UKMD286. We constructed a deletion mutant and performed transcriptomic analysis via RNA sequencing. This analysis identified 67 differentially expressed genes, with 45 genes upregulated and 22 genes downregulated in the mutant compared to the wild-type. Functional enrichment analysis of these genes highlighted significant roles in metabolism, transport and secretion system. To further characterize the phenotypic impact of the gene deletion, we conducted biofilm formation and plaque assays. Biofilm formation assays showed increased biofilm production in the mutant, suggesting a regulatory role in bacterial adhesion. Plaque assays revealed reduced plaque formation in the mutant, indicating impaired host cell invasion. These findings collectively suggest that UKMD286_5923 influences genes essential for bacterial survival and host-pathogen interaction, including components of the Type III secretion system. Understanding the function of this regulator enhances our knowledge of B. pseudomallei pathogenesis and may contribute to future diagnostic and treatment strategies for melioidosis.
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Project description:Burkholderia pseudomallei can adapt to and thrive in a variety of environments, including soil and water, and also can infect different hosts, including humans, leading to the tropical disease melioidosis. Modulation of gene and protein expression is one of this pathogen's adaptive survival mechanisms, which could lead to changes in the bacteria's cell membrane, metabolism, and virulence. To better understand bacterial adaptation and host-pathogen interactions, this study compared the expression profiles of B. pseudomallei from infected mice to B. pseudomallei cultivated in soil extract media. B. pseudomallei in vivo was created by infecting mice through the intraperitoneal route and harvesting the spleens on day 5 post infection. Total RNA was isolated and sequenced from the harvested spleen. Sequence reads were mapped to the B. pseudomallei UKMD286 strain genome sequence.
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Project description:Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a disease endemic to South-East Asia and Northern Australia. Clinical presentation is highly variable, ranging from asymptomatic to fatal septicaemia, and thus the outcome of infection can depend on the host immune responses. The aim of this study was to characterise the macrophage immune response to B. pseudomallei in the presence of novel inhibitors targeting the virulence factor, Macrophage Infectivity Potentiator (Mip) protein. To do this. murine macrophage J774A.1 cells were infected with B. pseudomallei K96243 in the presence and absence of two small-molecule inhibitors designed to target the Mip protein. Global transcriptional profiling of macrophages infected with B. pseudomallei was analysed by RNA-Seq four hours post-infection. In the presence of Mip inhibitors, we found a significant reduction in the expression of pro-inflammatory cytokines highlighting the potential to utilize Mip inhibitors to dampen potentially harmful pro-inflammatory responses resulting from B. pseudomallei infection in macrophages. We then performed gene expression profiling analysis using data obtained from RNA-seq of J774A.1 macrophages infected with Burkholderia pseudomallei in the presence of two Mip inhibitors or vehicle control 4 hours post-infection
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Project description:The potential for epigenetic changes in host cells following microbial infection has been widely suggested, but few examples have been reported. We assessed genome-wide patterns of DNA methylation in human macrophage-like U937 cells following infection with Burkholderia pseudomallei, an intracellular bacterial pathogen and the causative agent of human melioidosis. Our analyses revealed significant changes in host cell DNA methylation, at multiple CpG sites in the host cell genome, following infection. Infection induced differentially methylated probes (iDMPs) showing the greatest changes in DNA methylation were found to be in the vicinity of genes involved in inflammatory responses, intracellular signalling, apoptosis and pathogen-induced signalling. A comparison of our data with reported methylome changes in cells infected with M. tuberculosis revealed commonality of differentially methylated genes, including genes involved in T cell responses (BCL11B, FOXO1, KIF13B, PAWR, SOX4, SYK), actin cytoskeleton organisation (ACTR3, CDC42BPA, DTNBP1, FERMT2, PRKCZ, RAC1), and cytokine production (FOXP1, IRF8, MR1). Overall our findings show that pathogenic-specific and pathogen-common changes in the methylome occur following infection.
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