@article{GomesWestermannSauerweinetal.2019, author = {Gomes, Sara F. Martins and Westermann, Alexander J. and Sauerwein, Till and Hertlein, Tobias and F{\"o}rstner, Konrad U. and Ohlsen, Knut and Metzger, Marco and Shusta, Eric V. and Kim, Brandon J. and Appelt-Menzel, Antje and Schubert-Unkmeir, Alexandra}, title = {Induced pluripotent stem cell-derived brain endothelial cells as a cellular model to study Neisseria meningitidis infection}, series = {Frontiers in Microbiology}, volume = {10}, journal = {Frontiers in Microbiology}, number = {1181}, doi = {10.3389/fmicb.2019.01181}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201562}, year = {2019}, abstract = {Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs.}, language = {en} } @phdthesis{Westermann2014, author = {Westermann, Alexander J.}, title = {Dual RNA-seq of pathogen and host}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-112462}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {The infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections. However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell's physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies. The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed "Dual RNA-seq". Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella. Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and implications for the host's immune response. These findings exemplify the scope of Dual RNA-seq for the identification and characterization of novel bacterial virulence factors during host infection.}, subject = {Transkriptomanalyse}, language = {en} } @article{HickeySridharWestermannetal.2012, author = {Hickey, Scott F. and Sridhar, Malathy and Westermann, Alexander J. and Qin, Qian and Vijayendra, Pooja and Liou, Geoffrey and Hammond, Ming C.}, title = {Transgene regulation in plants by alternative splicing of a suicide exon}, series = {Nucleic Acids Research}, volume = {40}, journal = {Nucleic Acids Research}, number = {10}, doi = {10.1093/nar/gks032}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134724}, pages = {4701-4710}, year = {2012}, abstract = {Compared to transcriptional activation, other mechanisms of gene regulation have not been widely exploited for the control of transgenes. One barrier to the general use and application of alternative splicing is that splicing-regulated transgenes have not been shown to be reliably and simply designed. Here, we demonstrate that a cassette bearing a suicide exon can be inserted into a variety of open reading frames (ORFs), generating transgenes whose expression is activated by exon skipping in response to a specific protein inducer. The surprisingly minimal sequence requirements for the maintenance of splicing fidelity and regulation indicate that this splicing cassette can be used to regulate any ORF containing one of the amino acids Glu, Gln or Lys. Furthermore, a single copy of the splicing cassette was optimized by rational design to confer robust gene activation with no background expression in plants. Thus, conditional splicing has the potential to be generally useful for transgene regulation.}, language = {en} } @article{SchulteWestermannVogel2013, author = {Schulte, Leon N. and Westermann, Alexander J. and Vogel, J{\"o}rg}, title = {Differential activation and functional specialization of miR-146 and miR-155 in innate immune sensing}, series = {Nucleic Acids Research}, volume = {41}, journal = {Nucleic Acids Research}, number = {1}, doi = {10.1093/nar/gks1030}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-129765}, pages = {542-553}, year = {2013}, abstract = {Many microRNAs (miRNAs) are co-regulated during the same physiological process but the underlying cellular logic is often little understood. The conserved, immunomodulatory miRNAs miR-146 and miR-155, for instance, are co-induced in many cell types in response to microbial lipopolysaccharide (LPS) to feedback-repress LPS signalling through Toll-like receptor TLR4. Here, we report that these seemingly co-induced regulatory RNAs dramatically differ in their induction behaviour under various stimuli strengths and act non-redundantly through functional specialization; although miR-146 expression saturates at sub-inflammatory doses of LPS that do not trigger the messengers of inflammation markers, miR-155 remains tightly associated with the pro-inflammatory transcriptional programmes. Consequently, we found that both miRNAs control distinct mRNA target profiles; although miR-146 targets the messengers of LPS signal transduction components and thus downregulates cellular LPS sensitivity, miR-155 targets the mRNAs of genes pervasively involved in pro-inflammatory transcriptional programmes. Thus, miR-155 acts as a broad limiter of pro-inflammatory gene expression once the miR-146 dependent barrier to LPS triggered inflammation has been breached. Importantly, we also report alternative miR-155 activation by the sensing of bacterial peptidoglycan through cytoplasmic NOD-like receptor, NOD2. We predict that dosedependent responses to environmental stimuli may involve functional specialization of seemingly coinduced miRNAs in other cellular circuitries as well.}, language = {en} } @article{GorskiVogelSalibaetal.2014, author = {Gorski, Stanislaw A. and Vogel, J{\"o}rg and Saliba, Antoine-Emmanuel and Westermann, Alexander J.}, title = {Single-cell RNA-seq: advances and future challenges}, doi = {10.1093/nar/gku555}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110993}, year = {2014}, abstract = {Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Singlecell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNAseq protocols have been introduced and are reviewed here - each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field.}, subject = {RNS}, language = {en} } @article{SchulteWestermannVogel2012, author = {Schulte, Leon N. and Westermann, Alexander J. and Vogel, J{\"o}rg}, title = {Differential activation and functional specialization of miR-146 and miR-155 in innate immune sensing}, edition = {Advance Access}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76365}, year = {2012}, abstract = {Many microRNAs (miRNAs) are co-regulated during the same physiological process but the underlying cellular logic is often little understood. The conserved, immunomodulatory miRNAs miR-146 and miR-155, for instance, are co-induced in many cell types in response to microbial lipopolysaccharide (LPS) to feedback-repress LPS signalling through Toll-like receptor TLR4. Here, we report that these seemingly co-induced regulatory RNAs dramatically differ in their induction behaviour under various stimuli strengths and act non-redundantly through functional specialization; although miR-146 expression saturates at sub-inflammatory doses of LPS that do not trigger the messengers of inflammation markers, miR-155 remains tightly associated with the pro-inflammatory transcriptional programmes. Consequently, we found that both miRNAs control distinct mRNA target profiles; although miR-146 targets the messengers of LPS signal transduction components and thus downregulates cellular LPS sensitivity, miR-155 targets the mRNAs of genes pervasively involved in pro-inflammatory transcriptional programmes. Thus, miR-155 acts as a broad limiter of pro-inflammatory gene expression once the miR-146 dependent barrier to LPS triggered inflammation has been breached. Importantly, we also report alternative miR-155 activation by the sensing of bacterial peptidoglycan through cytoplasmic NOD-like receptor, NOD2. We predict that dosedependent responses to environmental stimuli may involve functional specialization of seemingly coinduced miRNAs in other cellular circuitries as well.}, subject = {Medizin}, language = {en} } @article{PrezzaRyanMaedleretal.2022, author = {Prezza, Gianluca and Ryan, Daniel and M{\"a}dler, Gohar and Reichardt, Sarah and Barquist, Lars and Westermann, Alexander J.}, title = {Comparative genomics provides structural and functional insights into Bacteroides RNA biology}, series = {Molecular Microbiology}, volume = {117}, journal = {Molecular Microbiology}, number = {1}, doi = {10.1111/mmi.14793}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259594}, pages = {67-85}, year = {2022}, abstract = {Bacteria employ noncoding RNA molecules for a wide range of biological processes, including scaffolding large molecular complexes, catalyzing chemical reactions, defending against phages, and controlling gene expression. Secondary structures, binding partners, and molecular mechanisms have been determined for numerous small noncoding RNAs (sRNAs) in model aerobic bacteria. However, technical hurdles have largely prevented analogous analyses in the anaerobic gut microbiota. While experimental techniques are being developed to investigate the sRNAs of gut commensals, computational tools and comparative genomics can provide immediate functional insight. Here, using Bacteroides thetaiotaomicron as a representative microbiota member, we illustrate how comparative genomics improves our understanding of RNA biology in an understudied gut bacterium. We investigate putative RNA-binding proteins and predict a Bacteroides cold-shock protein homolog to have an RNA-related function. We apply an in silico protocol incorporating both sequence and structural analysis to determine the consensus structures and conservation of nine Bacteroides noncoding RNA families. Using structure probing, we validate and refine these predictions and deposit them in the Rfam database. Through synteny analyses, we illustrate how genomic coconservation can serve as a predictor of sRNA function. Altogether, this work showcases the power of RNA informatics for investigating the RNA biology of anaerobic microbiota members.}, 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{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{WestermannBarquistVogel2017, author = {Westermann, Alexander J. and Barquist, Lars and Vogel, J{\"o}rg}, title = {Resolving host-pathogen interactions by dual RNA-seq}, series = {PLoS Pathogens}, volume = {13}, journal = {PLoS Pathogens}, number = {2}, doi = {10.1371/journal.ppat.1006033}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171921}, year = {2017}, abstract = {The transcriptome is a powerful proxy for the physiological state of a cell, healthy or diseased. As a result, transcriptome analysis has become a key tool in understanding the molecular changes that accompany bacterial infections of eukaryotic cells. Until recently, such transcriptomic studies have been technically limited to analyzing mRNA expression changes in either the bacterial pathogen or the infected eukaryotic host cell. However, the increasing sensitivity of high-throughput RNA sequencing now enables "dual RNA-seq" studies, simultaneously capturing all classes of coding and noncoding transcripts in both the pathogen and the host. In the five years since the concept of dual RNA-seq was introduced, the technique has been applied to a range of infection models. This has not only led to a better understanding of the physiological changes in pathogen and host during the course of an infection but has also revealed hidden molecular phenotypes of virulence-associated small noncoding RNAs that were not visible in standard infection assays. Here, we use the knowledge gained from these recent studies to suggest experimental and computational guidelines for the design of future dual RNA-seq studies. We conclude this review by discussing prospective applications of the technique.}, language = {en} } @article{HennessenMiethkeZaburannyietal.2020, author = {Hennessen, Fabienne and Miethke, Marcus and Zaburannyi, Nestor and Loose, Maria and Lukežič, Tadeja and Bernecker, Steffen and H{\"u}ttel, Stephan and Jansen, Rolf and Schmiedel, Judith and Fritzenwanker, Moritz and Imirzalioglu, Can and Vogel, J{\"o}rg and Westermann, Alexander J. and Hesterkamp, Thomas and Stadler, Marc and Wagenlehner, Florian and Petković, Hrvoje and Herrmann, Jennifer and M{\"u}ller, Rolf}, title = {Amidochelocardin overcomes resistance mechanisms exerted on tetracyclines and natural chelocardin}, series = {Antibiotics}, volume = {9}, journal = {Antibiotics}, number = {9}, issn = {2079-6382}, doi = {10.3390/antibiotics9090619}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213149}, year = {2020}, abstract = {The reassessment of known but neglected natural compounds is a vital strategy for providing novel lead structures urgently needed to overcome antimicrobial resistance. Scaffolds with resistance-breaking properties represent the most promising candidates for a successful translation into future therapeutics. Our study focuses on chelocardin, a member of the atypical tetracyclines, and its bioengineered derivative amidochelocardin, both showing broad-spectrum antibacterial activity within the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) panel. Further lead development of chelocardins requires extensive biological and chemical profiling to achieve favorable pharmaceutical properties and efficacy. This study shows that both molecules possess resistance-breaking properties enabling the escape from most common tetracycline resistance mechanisms. Further, we show that these compounds are potent candidates for treatment of urinary tract infections due to their in vitro activity against a large panel of multidrug-resistant uropathogenic clinical isolates. In addition, the mechanism of resistance to natural chelocardin was identified as relying on efflux processes, both in the chelocardin producer Amycolatopsis sulphurea and in the pathogen Klebsiella pneumoniae. Resistance development in Klebsiella led primarily to mutations in ramR, causing increased expression of the acrAB-tolC efflux pump. Most importantly, amidochelocardin overcomes this resistance mechanism, revealing not only the improved activity profile but also superior resistance-breaking properties of this novel antibacterial compound.}, language = {en} } @article{SchulteSchweinlinWestermannetal.2020, author = {Schulte, Leon N. and Schweinlin, Matthias and Westermann, Alexander J. and Janga, Harshavardhan and Santos, Sara C. and Appenzeller, Silke and Walles, Heike and Vogel, J{\"o}rg and Metzger, Marco}, title = {An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Salmonella Infection}, series = {mBio}, volume = {11, 2020}, journal = {mBio}, number = {1}, doi = {10.1128/mBio.03348-19}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229428}, year = {2020}, abstract = {A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens. IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.}, language = {en} } @article{MuellerDolowschiakSellinetal.2016, author = {M{\"u}ller, Anna A. and Dolowschiak, Tamas and Sellin, Mikael E. and Felmy, Boas and Verbree, Carolin and Gadient, Sandra and Westermann, Alexander J. and Vogel, J{\"o}rg and LeibundGut-Landmann, Salome and Hardt, Wolf-Dietrich}, title = {An NK Cell Perforin Response Elicited via IL-18 Controls Mucosal Inflammation Kinetics during Salmonella Gut Infection}, series = {PLoS Pathogens}, volume = {12}, journal = {PLoS Pathogens}, number = {6}, doi = {10.1371/journal.ppat.1005723}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167429}, pages = {e1005723}, year = {2016}, abstract = {Salmonella Typhimurium (S.Tm) is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S.Tm infections in knockout mice, cytokine inhibition and -injection experiments revealed that caspase-1 (not -11) dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf\(^{-/-}\) ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S.Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens.}, language = {en} } @article{WestermannVenturiniSellinetal.2019, author = {Westermann, Alexander J. and Venturini, Elisa and Sellin, Mikael E. and F{\"o}rstner, Konrad U. and Hardt, Wolf-Dietrich and Vogel, J{\"o}rg}, title = {The major RNA-binding protein ProQ impacts virulence gene expression in Salmonella enterica serovar Typhimurium}, series = {mBio}, volume = {10}, journal = {mBio}, number = {1}, doi = {10.1128/mBio.02504-18}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-177722}, pages = {e02504-18}, year = {2019}, abstract = {FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella, Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3′UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs. IMPORTANCE The protein ProQ has recently been discovered as the centerpiece of a previously overlooked "third domain" of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria.}, 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} }