@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} } @article{CorreiaSantosBischlerWestermannetal.2021, author = {Correia Santos, Sara and Bischler, Thorsten and Westermann, Alexander J. and Vogel, J{\"o}rg}, title = {MAPS integrates regulation of actin-targeting effector SteC into the virulence control network of Salmonella small RNA PinT}, series = {Cell Reports}, volume = {34}, journal = {Cell Reports}, number = {5}, doi = {10.1016/j.celrep.2021.108722}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259134}, pages = {108722}, year = {2021}, abstract = {A full understanding of the contribution of small RNAs (sRNAs) to bacterial virulence demands knowledge of their target suites under infection-relevant conditions. Here, we take an integrative approach to capturing targets of the Hfq-associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica. Using MS2 affinity purification and RNA sequencing (MAPS), we identify PinT ligands in bacteria under in vitro conditions mimicking specific stages of the infection cycle and in bacteria growing inside macrophages. This reveals PinT-mediated translational inhibition of the secreted effector kinase SteC, which had gone unnoticed in previous target searches. Using genetic, biochemical, and microscopic assays, we provide evidence for PinT-mediated repression of steC mRNA, eventually delaying actin rearrangements in infected host cells. Our findings support the role of PinT as a central post-transcriptional regulator in Salmonella virulence and illustrate the need for complementary methods to reveal the full target suites of sRNAs.}, language = {en} } @article{GerovaWickeChiharaetal.2021, author = {Gerova, Milan and Wicke, Laura and Chihara, Kotaro and Schneider, Cornelius and Lavigne, Rob and Vogel, J{\"o}rg}, title = {A grad-seq view of RNA and protein complexes in Pseudomonas aeruginosa under standard and bacteriophage predation conditions}, series = {mbio}, volume = {12}, journal = {mbio}, number = {1}, doi = {10.1128/mBio.03454-20}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259054}, pages = {e03454-20}, year = {2021}, abstract = {The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73\% of all transcripts and ∼40\% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell. IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.}, language = {en} } @article{GarciaBetancurGoniMorenoHorgeretal.2017, author = {Garc{\´i}a-Betancur, Juan-Carlos and Go{\~n}i-Moreno, Angel and Horger, Thomas and Schott, Melanie and Sharan, Malvika and Eikmeier, Julian and Wohlmuth, Barbara and Zernecke, Alma and Ohlsen, Knut and Kuttler, Christina and Lopez, Daniel}, title = {Cell differentiation defines acute and chronic infection cell types in Staphylococcus aureus}, series = {eLife}, volume = {6}, journal = {eLife}, number = {e28023}, doi = {10.7554/eLife.28023}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170346}, year = {2017}, abstract = {A central question to biology is how pathogenic bacteria initiate acute or chronic infections. Here we describe a genetic program for cell-fate decision in the opportunistic human pathogen Staphylococcus aureus, which generates the phenotypic bifurcation of the cells into two genetically identical but different cell types during the course of an infection. Whereas one cell type promotes the formation of biofilms that contribute to chronic infections, the second type is planktonic and produces the toxins that contribute to acute bacteremia. We identified a bimodal switch in the agr quorum sensing system that antagonistically regulates the differentiation of these two physiologically distinct cell types. We found that extracellular signals affect the behavior of the agr bimodal switch and modify the size of the specialized subpopulations in specific colonization niches. For instance, magnesium-enriched colonization niches causes magnesium binding to S. aureusteichoic acids and increases bacterial cell wall rigidity. This signal triggers a genetic program that ultimately downregulates the agr bimodal switch. Colonization niches with different magnesium concentrations influence the bimodal system activity, which defines a distinct ratio between these subpopulations; this in turn leads to distinct infection outcomes in vitro and in an in vivo murine infection model. Cell differentiation generates physiological heterogeneity in clonal bacterial infections and helps to determine the distinct infection types.}, language = {en} } @article{DembekBarquistBoinettetal.2015, author = {Dembek, Marcin and Barquist, Lars and Boinett, Christine J. and Cain, Amy K. and Mayho, Matthew and Lawley, Trevor D. and Fairweather, Neil F. and Fagan, Robert P.}, title = {High-throughput analysis of gene essentiality and sporulation in Clostridium difficile}, series = {mBio}, volume = {6}, journal = {mBio}, number = {2}, doi = {10.1128/mBio.02383-14}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143745}, pages = {e02383-14}, year = {2015}, abstract = {Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen.}, language = {en} } @article{OkoroBarquistConnoretal.2015, author = {Okoro, Chinyere K. and Barquist, Lars and Connor, Thomas R. and Harris, Simon R. and Clare, Simon and Stevens, Mark P. and Arends, Mark J. and Hale, Christine and Kane, Leanne and Pickard, Derek J. and Hill, Jennifer and Harcourt, Katherine and Parkhill, Julian and Dougan, Gordon and Kingsley, Robert A.}, title = {Signatures of adaptation in human invasive Salmonella Typhimurium ST313 populations from sub-Saharan Africa}, series = {PLoS Neglected Tropical Diseases}, volume = {9}, journal = {PLoS Neglected Tropical Diseases}, number = {3}, doi = {10.1371/journal.pntd.0003611}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143779}, pages = {e0003611}, year = {2015}, abstract = {Two lineages of Salmonella enterica serovar Typhimurium (S. Typhimurium) of multi-locus sequence type ST313 have been linked with the emergence of invasive Salmonella disease across sub-Saharan Africa. The expansion of these lineages has a temporal association with the HIV pandemic and antibiotic usage. We analysed the whole genome sequence of 129 ST313 isolates representative of the two lineages and found evidence of lineage-specific genome degradation, with some similarities to that observed in S. Typhi. Individual ST313 S. Typhimurium isolates exhibit a distinct metabolic signature and modified enteropathogenesis in both a murine and cattle model of colitis, compared to S. Typhimurium outside of the ST313 lineages. These data define phenotypes that distinguish ST313 isolates from other S. Typhimurium and may represent adaptation to a distinct pathogenesis and lifestyle linked to an-immuno-compromised human population.}, language = {en} } @article{AfonsoGrunzHoffmeierMuelleretal.2015, author = {Afonso-Grunz, Fabian and Hoffmeier, Klaus and M{\"u}ller, S{\"o}ren and Westermann, Alexander J. and Rotter, Bj{\"o}rn and Vogel, J{\"o}rg and Winter, Peter and Kahl, G{\"u}nter}, title = {Dual 3'Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells}, series = {BMC Genomics}, volume = {16}, journal = {BMC Genomics}, number = {323}, doi = {10.1186/s12864-015-1489-1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143230}, year = {2015}, abstract = {Background: The interaction of eukaryotic host and prokaryotic pathogen cells is linked to specific changes in the cellular proteome, and consequently to infection-related gene expression patterns of the involved cells. To simultaneously assess the transcriptomes of both organisms during their interaction we developed dual 3'Seq, a tag-based sequencing protocol that allows for exact quantification of differentially expressed transcripts in interacting pro-and eukaryotic cells without prior fixation or physical disruption of the interaction. Results: Human epithelial cells were infected with Salmonella enterica Typhimurium as a model system for invasion of the intestinal epithelium, and the transcriptional response of the infected host cells together with the differential expression of invading and intracellular pathogen cells was determined by dual 3'Seq coupled with the next-generation sequencing-based transcriptome profiling technique deepSuperSAGE (deep Serial Analysis of Gene Expression). Annotation to reference transcriptomes comprising the operon structure of the employed S. enterica Typhimurium strain allowed for in silico separation of the interacting cells including quantification of polycistronic RNAs. Eighty-nine percent of the known loci are found to be transcribed in prokaryotic cells prior or subsequent to infection of the host, while 75\% of all protein-coding loci are represented in the polyadenylated transcriptomes of human host cells. Conclusions: Dual 3'Seq was alternatively coupled to MACE (Massive Analysis of cDNA ends) to assess the advantages and drawbacks of a library preparation procedure that allows for sequencing of longer fragments. Additionally, the identified expression patterns of both organisms were validated by qRT-PCR using three independent biological replicates, which confirmed that RELB along with NFKB1 and NFKB2 are involved in the initial immune response of epithelial cells after infection with S. enterica Typhimurium.}, language = {en} } @article{MakgotlhoMarincolaSchaeferetal.2013, author = {Makgotlho, Phuti E. and Marincola, Gabriella and Sch{\"a}fer, Daniel and Liu, Quian and Bae, Taeok and Geiger, Tobias and Wasserman, Elizabeth and Wolz, Christine and Ziebuhr, Wilma and Sinha, Bhanu}, title = {SDS Interferes with SaeS Signaling of Staphylococcus aureus Independently of SaePQ}, series = {PLOS ONE}, volume = {8}, journal = {PLOS ONE}, number = {8}, issn = {1932-6203}, doi = {10.1371/journal.pone.0071644}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-128469}, pages = {e71644}, year = {2013}, abstract = {The Staphylococcus aureus regulatory saePQRS system controls the expression of numerous virulence factors, including extracellular adherence protein (Eap), which amongst others facilitates invasion of host cells. The saePQRS operon codes for 4 proteins: the histidine kinase SaeS, the response regulator SaeR, the lipoprotein SaeP and the transmembrane protein SaeQ. S. aureus strain Newman has a single amino acid substitution in the transmembrane domain of SaeS (L18P) which results in constitutive kinase activity. SDS was shown to be one of the signals interfering with SaeS activity leading to inhibition of the sae target gene eap in strains with SaeS(L) but causing activation in strains containing SaeS(P). Here, we analyzed the possible involvement of the SaeP protein and saePQ region in SDS-mediated sae/eap expression. We found that SaePQ is not needed for SDS-mediated SaeS signaling. Furthermore, we could show that SaeS activity is closely linked to the expression of Eap and the capacity to invade host cells in a number of clinical isolates. This suggests that SaeS activity might be directly modulated by structurally non-complex environmental signals, as SDS, which possibly altering its kinase/phosphatase activity.}, language = {en} }