Probe-based spatial host-pathogen genes expression to study bacterial pathogenesis and the regulation of bacterial virulence factors in tissue.
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ABSTRACT: Co-localization of host and pathogen spatial transcriptome information can improve our understanding on how inflammatory environments modulate pathogen virulence in tissues and vice versa. Here, we demonstrate for the first time that bacterial probes can be used to simultaneously identify host-pathogen mutual interactions in formalin-fixed, paraffin-embedded (FFPE) tissues using spatial transcriptomics technology. With properly designed probes targeting pathogens of interest, our approach could provide an improved definition of infection processes by detecting pathogen and host genes expression simultaneously, spatially distributed, and unbiased.
Project description:To test specificity of human microarray probes against mouse mRNA serially mixed human MDA-MB231 cancer cells' mRNAs with mouse Astrocytes were hybridized against human microarray and vice versa by changing the ratios of human and mouse samples from 100% to 0%.
Project description:To advance our understanding of cellular host-pathogen interactions, technologies that facilitate the co-capture of both host and pathogen spatial transcriptome information are needed. Here, we present an approach to simultaneously capture host and pathogen spatial gene expression information from the same formalin-fixed paraffin embedded (FFPE) tissue section using the spatial transcriptomics technology. We applied the method to COVID-19 patient lung samples and enabled the dual detection of human and SARS-CoV-2 transcriptomes at 55 µm resolution. We validated our spatial detection of SARS-CoV-2 and identified an average specificity of 94.92% in comparison to RNAScope and 82.20% in comparison to in situ sequencing (ISS). COVID-19 tissues showed an upregulation of host immune response, such as increased expression of inflammatory cytokines, lymphocyte and fibroblast markers. Our colocalization analysis revealed that SARS-CoV-2+ spots presented shifts in host RNA metabolism, autophagy, NFκB, and interferon response pathways. Future applications of our approach will enable new insights into host response to pathogen infection through the simultaneous, unbiased detection of two transcriptomes.
Project description:AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro. Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology we here introduce a novel UMP pronucleotide probe and utilize it in the profiling of uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases which can largely differ compared to their in vitro activity.
Project description:To holistically unravel the complexity of pathogen-host interactions within infected tissues we leverage a dual spatial transcriptomic approach that, for the first time, simultaneously captures the expression of Pseudomonas aeruginosa genes alongside the entire host transcriptome in a model of ocular infection. This innovative method reveals differential pathogen and host-specific gene expression patterns across specific anatomical regions generating a unified transcriptional map of infection. By integrating these data, we developed a predictive ridge regression model trained on images from infected tissues.
Project description:Clonal bacterial populations rely on transcriptional variation across individual cells to commit to specialized states that increase the population’s fitness. Such heterogeneous gene expression is implicated in fundamental microbial processes including sporulation, cell communication, detoxification, substrate utilization, competence, biofilm formation, and motility1. To identify specialized cell states and determine the processes by which they develop, isogenic bacterial populations need to be studied at the single cell level2,3. Here, we developed ProBac-seq a method that uses libraries of DNA probes and leverages an existing commercial microfluidic platform to conduct bacterial single cell RNA sequencing. We sequenced the transcriptome of thousands of individual bacterial cells per experiment, detecting several hundred transcripts per cell on average. When applying this method to the model organisms Bacillus subtilis and Escherichia coli, we correctly identify known cell states and uncover previously unreported transcriptional heterogeneity. In the context of bacterial pathogenesis, single cell RNA-seq of the pathogen Clostridium perfringens reveals that toxin is differentially expressed by a subpopulation of cells with a distinct transcriptional profile. We further show that the size of the toxin producing subpopulation and the secreted toxin levels can be downregulated by providing acetate, a short chain fatty acid highly prevalent in the gut. Overall, we demonstrate that our high throughput, highly resolved single cell transcriptomic platform can be broadly used to uncover heterogeneity in isogenic microbial populations and identify perturbations that can impact pathogenicity.
Project description:Probe-based spatial host-pathogen genes expression to study bacterial pathogenesis and the regulation of bacterial virulence factors in tissue.
Project description:To test specificity of human microarray probes against mouse mRNA serially mixed human MDA-MB231 cancer cells' mRNAs with mouse Astrocytes were hybridized against human microarray and vice versa by changing the ratios of human and mouse samples from 100% to 0%. Mixed human: 1 MDA-MB231, 1 MDA231_Astro(91), 2 MDA231_Astro(73), 2 MDA231_Astro(55),2 MDA231_Astro(37),1 MDA231_Astro(19), 1 Astrocytes Mixed mouse: 1 MDA-MB231, 1 MDA231_Astro(91), 2 MDA231_Astro(73), 2 MDA231_Astro(55),2 MDA231_Astro(37),1 MDA231_Astro(19), 1 Astrocytes
Project description:In this study cDNA microarrays were used to identify genes with sex-biased expression in mouse whole hearts of two age classes (2 months and 8 months of age). Experiment Overall Design: A total of 24 male and female C57BL/6 mice were sacrificed at two and eight months of age, respectively (n=6 per group). Isolated total RNAs were pooled according to sex and age and hybridized on Agilent cDNA microarrays (age-matched, male vs. female or vice versa). Four technical replicates were carried out with exchanged dye-labelled RNA probes (dye-swap) on two of them.