Project description:We are studying the host immune response to a bacterial toxin in the chicken spleen and bursa. We performed snRNAseq to analyze the various immune cell types that play a role in combating the infection and the cell-type transcriptional response.
Project description:In this study, we characterized the proteomic composition of extracellular vesicles released from human THP-1 monocytes challenged with the pneumococcal pore-forming toxin, pneumolysin. We found that the vesicles shed from pneumolysin challenged cells are selectively enriched in key inflammatory host proteins relative to vesicles from untreated cells.
Project description:Rickettsia spp. can cause mild to severe human disease. These intracellular bacteria are associated with arthropods, nematodes and trematodes, and usually, are efficiently transmitted transovarially to the progeny of the invertebrate host. We recently demonstrated foreign gene acquisition by lateral gene transfer in Rickettsia genomes. The unexpected presence of laterally transferred toxin-antitoxin (TA) genetic elements (including vapBC) in several Rickettsia genomes has not been connected with the pathogenic process or the host-bacteria relationship. We suspect that vapBC are selfish genetic elements that addict eukaryotic hosts to Rickettsia. We identified a statistical link between the transovarial transmission of Rickettsia in invertebrate hosts and the presence of TA operons, specifically vapBC, in the Rickettsia genome. These TA are neighboring to type IV secretion genes. Tunel assays and whole-genome expression of infected cells showed that antibiotic eradication of TA-containing Rickettsia from the host in cell culture initiates a proapoptotic program. Rickettsia VapC toxins inhibit the growth of transformed Escherichia coli and Saccharomyces cerevisiae. Rickettsia toxin presents in vitro RNase activity. Annexin-V staining and time-lapse video showed that intracytoplasmic injections of VapC toxins in cells cause apoptosis. These data demonstrate that host cells may develop a dependence on Rickettsia spp. expressing the vapBC operon. This would constitute a new evolutionary “mafia strategy” of intracellular bacteria based on host addiction.
Project description:Rickettsia spp. can cause mild to severe human disease. These intracellular bacteria are associated with arthropods, nematodes and trematodes, and usually, are efficiently transmitted transovarially to the progeny of the invertebrate host. We recently demonstrated foreign gene acquisition by lateral gene transfer in Rickettsia genomes. The unexpected presence of laterally transferred toxin-antitoxin (TA) genetic elements (including vapBC) in several Rickettsia genomes has not been connected with the pathogenic process or the host-bacteria relationship. We suspect that vapBC are selfish genetic elements that addict eukaryotic hosts to Rickettsia. We identified a statistical link between the transovarial transmission of Rickettsia in invertebrate hosts and the presence of TA operons, specifically vapBC, in the Rickettsia genome. These TA are neighboring to type IV secretion genes. Tunel assays and whole-genome expression of infected cells showed that antibiotic eradication of TA-containing Rickettsia from the host in cell culture initiates a proapoptotic program. Rickettsia VapC toxins inhibit the growth of transformed Escherichia coli and Saccharomyces cerevisiae. Rickettsia toxin presents in vitro RNase activity. Annexin-V staining and time-lapse video showed that intracytoplasmic injections of VapC toxins in cells cause apoptosis. These data demonstrate that host cells may develop a dependence on Rickettsia spp. expressing the vapBC operon. This would constitute a new evolutionary M-bM-^@M-^\mafia strategyM-bM-^@M-^] of intracellular bacteria based on host addiction. Fresh cells from the human microvascular endothelial cell line (HMEC-1) [26] were infected with R. felis California-2 strain in the presence and absence of antibiotics, at a rate of 5 bacteria per eukaryotic cell. Then, we added or not antibiotics (chloramphenicol 50 M-BM-5g/ml or doxycycline to 40 M-BM-5g/ml) in both experimental (R.felis-infected) and control, mock-infected cells for 6 hours. The cells were harvested and RNA was extracted using the RNeasy Mini Kit (Qiagen). DNA contamination was removed using the Turbo DNA-free Kit (Ambion). RNA were labeled using the Quick Amp Labeling Kit One-color (Agilent) and hybridized onto a Whole Human Genome Microarray, 4x44K (Agilent) as recommended by the manufacturer. Arrays were scanned with DNA Microarray Scanner (Agilent), and data were extracted using Feature Extractor (Agilent).
Project description:Here we applied a novel approach to isolate nuclei from complex plant tissues (https://doi.org/10.1371/journal.pone.0251149), to dissect the transcriptome profiling of the hybrid poplar (Populus tremula × alba) vegetative shoot apex at single-cell resolution.
Project description:This study examines the transcriptional profile at the single-nuclei resolution human gastrocnemius skeletal muscle from 20 patients with Peripheral artery disease (PAD) and 12 non-PAD controls. In all libraries, muscle specimens were pooled prior to nuclei isolations were performed.
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.