@article{HombergerHaywardBarquistetal.2023,
  author    = {Homberger, Christina and Hayward, Regan J. and Barquist, Lars and Vogel, J{\"o}rg},
  title     = {Improved bacterial single-cell RNA-seq through automated MATQ-seq and Cas9-based removal of rRNA reads},
  series = {mBio},
  volume    = {14},
  journal   = {mBio},
  number    = {2},
  doi       = {10.1128/mbio.03557-22},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350059},
  year      = {2023},
  abstract  = {Bulk RNA sequencing technologies have provided invaluable insights into host and bacterial gene expression and associated regulatory networks. Nevertheless, the majority of these approaches report average expression across cell populations, hiding the true underlying expression patterns that are often heterogeneous in nature. Due to technical advances, single-cell transcriptomics in bacteria has recently become reality, allowing exploration of these heterogeneous populations, which are often the result of environmental changes and stressors. In this work, we have improved our previously published bacterial single-cell RNA sequencing (scRNA-seq) protocol that is based on multiple annealing and deoxycytidine (dC) tailing-based quantitative scRNA-seq (MATQ-seq), achieving a higher throughput through the integration of automation. We also selected a more efficient reverse transcriptase, which led to reduced cell loss and higher workflow robustness. Moreover, we successfully implemented a Cas9-based rRNA depletion protocol into the MATQ-seq workflow. Applying our improved protocol on a large set of single Salmonella cells sampled over different growth conditions revealed improved gene coverage and a higher gene detection limit compared to our original protocol and allowed us to detect the expression of small regulatory RNAs, such as GcvB or CsrB at a single-cell level. In addition, we confirmed previously described phenotypic heterogeneity in Salmonella in regard to expression of pathogenicity-associated genes. Overall, the low percentage of cell loss and high gene detection limit makes the improved MATQ-seq protocol particularly well suited for studies with limited input material, such as analysis of small bacterial populations in host niches or intracellular bacteria. IMPORTANCE: Gene expression heterogeneity among isogenic bacteria is linked to clinically relevant scenarios, like biofilm formation and antibiotic tolerance. The recent development of bacterial single-cell RNA sequencing (scRNA-seq) enables the study of cell-to-cell variability in bacterial populations and the mechanisms underlying these phenomena. Here, we report a scRNA-seq workflow based on MATQ-seq with increased robustness, reduced cell loss, and improved transcript capture rate and gene coverage. Use of a more efficient reverse transcriptase and the integration of an rRNA depletion step, which can be adapted to other bacterial single-cell workflows, was instrumental for these improvements. Applying the protocol to the foodborne pathogen Salmonella, we confirmed transcriptional heterogeneity across and within different growth phases and demonstrated that our workflow captures small regulatory RNAs at a single-cell level. Due to low cell loss and high transcript capture rates, this protocol is uniquely suited for experimental settings in which the starting material is limited, such as infected tissues.},
  language  = {en}
}
@article{MichauxGerovacHansenetal.2023,
  author    = {Michaux, Charlotte and Gerovac, Milan and Hansen, Elisabeth E. and Barquist, Lars and Vogel, J{\"o}rg},
  title     = {Grad-seq analysis of Enterococcus faecalis and Enterococcus faecium provides a global view of RNA and protein complexes in these two opportunistic pathogens},
  series = {microLife},
  volume    = {4},
  journal   = {microLife},
  doi       = {10.1093/femsml/uqac027},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-313311},
  year      = {2023},
  abstract  = {Enterococcus faecalis and Enterococcus faecium are major nosocomial pathogens. Despite their relevance to public health and their role in the development of bacterial antibiotic resistance, relatively little is known about gene regulation in these species. RNA-protein complexes serve crucial functions in all cellular processes associated with gene expression, including post-transcriptional control mediated by small regulatory RNAs (sRNAs). Here, we present a new resource for the study of enterococcal RNA biology, employing the Grad-seq technique to comprehensively predict complexes formed by RNA and proteins in E. faecalis V583 and E. faecium AUS0004. Analysis of the generated global RNA and protein sedimentation profiles led to the identification of RNA-protein complexes and putative novel sRNAs. Validating our data sets, we observe well-established cellular RNA-protein complexes such as the 6S RNA-RNA polymerase complex, suggesting that 6S RNA-mediated global control of transcription is conserved in enterococci. Focusing on the largely uncharacterized RNA-binding protein KhpB, we use the RIP-seq technique to predict that KhpB interacts with sRNAs, tRNAs, and untranslated regions of mRNAs, and might be involved in the processing of specific tRNAs. Collectively, these datasets provide departure points for in-depth studies of the cellular interactome of enterococci that should facilitate functional discovery in these and related Gram-positive species. Our data are available to the community through a user-friendly Grad-seq browser that allows interactive searches of the sedimentation profiles (https://resources.helmholtz-hiri.de/gradseqef/).},
  language  = {en}
}
@article{McFlederMakhotkinaGrohetal.2023,
  author    = {McFleder, Rhonda L. and Makhotkina, Anastasiia and Groh, Janos and Keber, Ursula and Imdahl, Fabian and Pe{\~n}a Mosca, Josefina and Peteranderl, Alina and Wu, Jingjing and Tabuchi, Sawako and Hoffmann, Jan and Karl, Ann-Kathrin and Pagenstecher, Axel and Vogel, J{\"o}rg and Beilhack, Andreas and Koprich, James B. and Brotchie, Jonathan M. and Saliba, Antoine-Emmanuel and Volkmann, Jens and Ip, Chi Wang},
  title     = {Brain-to-gut trafficking of alpha-synuclein by CD11c\(^+\) cells in a mouse model of Parkinson's disease},
  series = {Nature Communications},
  volume    = {14},
  journal   = {Nature Communications},
  doi       = {10.1038/s41467-023-43224-z},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-357696},
  year      = {2023},
  abstract  = {Inflammation in the brain and gut is a critical component of several neurological diseases, such as Parkinson's disease (PD). One trigger of the immune system in PD is aggregation of the pre-synaptic protein, α-synuclein (αSyn). Understanding the mechanism of propagation of αSyn aggregates is essential to developing disease-modifying therapeutics. Using a brain-first mouse model of PD, we demonstrate αSyn trafficking from the brain to the ileum of male mice. Immunohistochemistry revealed that the ileal αSyn aggregations are contained within CD11c+ cells. Using single-cell RNA sequencing, we demonstrate that ileal CD11c\(^+\) cells are microglia-like and the same subtype of cells is activated in the brain and ileum of PD mice. Moreover, by utilizing mice expressing the photo-convertible protein, Dendra2, we show that CD11c\(^+\) cells traffic from the brain to the ileum. Together these data provide a mechanism of αSyn trafficking between the brain and gut.},
  language  = {en}
}
@article{OkudaLenzSeitzetal.2023,
  author    = {Okuda, Takumi and Lenz, Ann-Kathrin and Seitz, Florian and Vogel, J{\"o}rg and H{\"o}bartner, Claudia},
  title     = {A SAM analogue-utilizing ribozyme for site-specific RNA alkylation in living cells},
  series = {Nature Chemistry},
  journal   = {Nature Chemistry},
  doi       = {10.1038/s41557-023-01320-z},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-328762},
  year      = {2023},
  abstract  = {Post-transcriptional RNA modification methods are in high demand for site-specific RNA labelling and analysis of RNA functions. In vitro-selected ribozymes are attractive tools for RNA research and have the potential to overcome some of the limitations of chemoenzymatic approaches with repurposed methyltransferases. Here we report an alkyltransferase ribozyme that uses a synthetic, stabilized S-adenosylmethionine (SAM) analogue and catalyses the transfer of a propargyl group to a specific adenosine in the target RNA. Almost quantitative conversion was achieved within 1 h under a wide range of reaction conditions in vitro, including physiological magnesium ion concentrations. A genetically encoded version of the SAM analogue-utilizing ribozyme (SAMURI) was expressed in HEK293T cells, and intracellular propargylation of the target adenosine was confirmed by specific fluorescent labelling. SAMURI is a general tool for the site-specific installation of the smallest tag for azide-alkyne click chemistry, which can be further functionalized with fluorophores, affinity tags or other functional probes.},
  language  = {en}
}
@article{DaeullaryImdahlDietrichetal.2023,
  author    = {D{\"a}ullary, Thomas and Imdahl, Fabian and Dietrich, Oliver and Hepp, Laura and Krammer, Tobias and Fey, Christina and Neuhaus, Winfried and Metzger, Marco and Vogel, J{\"o}rg and Westermann, Alexander J. and Saliba, Antoine-Emmanuel and Zdzieblo, Daniela},
  title     = {A primary cell-based in vitro model of the human small intestine reveals host olfactomedin 4 induction in response to Salmonella Typhimurium infection},
  series = {Gut Microbes},
  volume    = {15},
  journal   = {Gut Microbes},
  number    = {1},
  doi       = {10.1080/19490976.2023.2186109},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350451},
  year      = {2023},
  abstract  = {Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.},
  language  = {en}
}