Recent Submissions

  • POTATO: Automated pipeline for batch analysis of optical tweezers data.

    Buck, Stefan; Pekarek, Lukas; Caliskan, Neva; Helmholtz Institute for RNA-based Infection Research (HIRI), Würzburg, Germany ; Medical Faculty, Julius-Maximilians University Würzburg, Würzburg, Germany (Elsevier, Cell Press, 2022-06-30)
    Optical tweezers are a single-molecule technique that allows probing of intra- and intermolecular interactions that govern complex biological processes involving molecular motors, protein-nucleic acid interactions, and protein/RNA folding. Recent developments in instrumentation eased and accelerated optical tweezers data acquisition, but analysis of the data remains challenging. Here, to enable high-throughput data analysis, we developed an automated python-based analysis pipeline called POTATO (practical optical tweezers analysis tool). POTATO automatically processes the high-frequency raw data generated by force-ramp experiments and identifies (un)folding events using predefined parameters. After segmentation of the force-distance trajectories at the identified (un)folding events, sections of the curve can be fitted independently to a worm-like chain and freely jointed chain models, and the work applied on the molecule can be calculated by numerical integration. Furthermore, the tool allows plotting of constant force data and fitting of the Gaussian distance distribution over time. All these features are wrapped in a user-friendly graphical interface, which allows researchers without programming knowledge to perform sophisticated data analysis.
  • POTATO: Automated pipeline for batch analysis of optical tweezers data

    Buck, Stefan; Pekarek, Lukas; Caliskan, Neva; Helmholtz Institute for RNA-based Infection Research (HIRI), Würzburg, GermanyMedical Faculty, Julius-Maximilians University Würzburg, Würzburg, Germany; EC | T-FRAME (948636)
    Optical tweezers are a single-molecule technique that allows probing of intra- and intermolecular interactions that govern complex biological processes involving molecular motors, protein-nucleic acid interactions, and protein/RNA folding. Recent developments in instrumentation eased and accelerated optical tweezers data acquisition, but analysis of the data remains challenging. Here, to enable high-throughput data analysis, we developed an automated python-based analysis pipeline called POTATO (practical optical tweezers analysis tool). POTATO automatically processes the high-frequency raw data generated by force-ramp experiments and identifies (un)folding events using predefined parameters. After segmentation of the force-distance trajectories at the identified (un)folding events, sections of the curve can be fitted independently to a worm-like chain and freely jointed chain models, and the work applied on the molecule can be calculated by numerical integration. Furthermore, the tool allows plotting of constant force data and fitting of the Gaussian distance distribution over time. All these features are wrapped in a user-friendly graphical interface, which allows researchers without programming knowledge to perform sophisticated data analysis. -(c) 2022 Biophysical Society
  • An amphipathic peptide with antibiotic activity against multidrug-resistant Gram-negative bacteria.

    Elliott, Alysha G; Huang, Johnny X; Neve, Søren; Zuegg, Johannes; Edwards, Ingrid A; Cain, Amy K; Boinett, Christine J; Barquist, Lars; Lundberg, Carina Vingsbo; Steen, Jason; et al. (2020-06-23)
    Peptide antibiotics are an abundant and synthetically tractable source of molecular diversity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an amphipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina. The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC, a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3–4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the ‘no observable adverse effect level’ (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models
  • Dynamics of Cardiac Neutrophil Diversity in Murine Myocardial Infarction.

    Vafadarnejad, Ehsan; Rizzo, Giuseppe; Krampert, Laura; Arampatzi, Panagiota; Arias-Loza, Anahi-Paula; Nazzal, Yara; Rizakou, Anna; Knochenhauer, Tim; Bandi, Sourish Reddy; Nugroho, Vallery Audy; et al. (2020-08-19)
    We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G+ (lymphocyte antigen 6G) neutrophils could be delineated into 6 distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils (Cd177, Lcn2, Fpr1), and putative activity of transcriptional regulators involved in hypoxic response (Hif1a) and emergency granulopoiesis (Cebpb). At 3 and 5 days, 2 major subsets of Siglecfhi (enriched for eg, Icam1 and Tnf) and Siglecflow (Slpi, Ifitm1) neutrophils were found. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis in blood and heart revealed that while circulating neutrophils undergo a process of aging characterized by loss of surface CD62L and upregulation of Cxcr4, heart infiltrating neutrophils acquired a unique SiglecFhi signature. SiglecFhi neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecFhi signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecFhi neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecFhi neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation.
  • Merging bioresponsive release of insulin-like growth factor I with 3D printable thermogelling hydrogels

    Beudert, Matthias; Hahn, Lukas; Horn, Anselm H.C.; Hauptstein, Niklas; Sticht, Heinrich; Meinel, Lorenz; Luxenhofer, Robert; Gutmann, Marcus; Lühmann, Tessa (2022-07-01)
    3D printing of biomaterials enables spatial control of drug incorporation during automated manufacturing. This study links bioresponsive release of the anabolic biologic, insulin-like growth factor-I (IGF-I) in response to matrix metalloproteinases (MMP) to 3D printing using the block copolymer of poly(2-methyl-2-oxazoline) and thermoresponsive poly(2-n-propyl-2-oxazine) (POx-b-POzi). For that, a chemo-enzymatic synthesis was deployed, ligating IGF-I enzymatically to a protease sensitive linker (PSL), which was conjugated to a POx-b-POzi copolymer. The product was blended with the plain thermogelling POx-b-POzi hydrogel. MMP exposure of the resulting hydrogel triggered bioactive IGF-I release. The bioresponsive IGF-I containing POx-b-POzi hydrogel system was further detailed for shape control and localized incorporation of IGF-I via extrusion 3D printing for future applications in biomedicine and biofabrication. © 2022 Elsevier B.V.
  • Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

    Pekarek, Lukas; Buck, Stefan; Caliskan, Neva; Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Zentrum für Infektionsforschung (Helmholtz Centre for Infection Research), Germany (JOVE, 2022-02-12)
    RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously. This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.
  • Complement activation induces excessive T cell cytotoxicity in severe COVID-19.

    Georg, Philipp; Astaburuaga-García, Rosario; Bonaguro, Lorenzo; Brumhard, Sophia; Michalick, Laura; Lippert, Lena J; Kostevc, Tomislav; Gäbel, Christiane; Schneider, Maria; Streitz, Mathias; et al. (Elsevier, 2021-12-28)
    Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. Proportions of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.
  • Global RNA interactome of Salmonella discovers a 5' UTR sponge for the MicF small RNA that connects membrane permeability to transport capacity.

    Matera, Gianluca; Altuvia, Yael; Gerovac, Milan; El Mouali, Youssef; Margalit, Hanah; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier (Cell Press), 2022-01-20)
    The envelope of Gram-negative bacteria is a vital barrier that must balance protection and nutrient uptake. Small RNAs are crucial regulators of the envelope composition and function. Here, using RIL-seq to capture the Hfq-mediated RNA-RNA interactome in Salmonella enterica, we discover envelope-related riboregulators, including OppX. We show that OppX acts as an RNA sponge of MicF sRNA, a prototypical porin repressor. OppX originates from the 5' UTR of oppABCDF, encoding the major inner-membrane oligopeptide transporter, and sequesters MicF's seed region to derepress the synthesis of the porin OmpF. Intriguingly, OppX operates as a true sponge, storing MicF in an inactive complex without affecting its levels or stability. Conservation of the opp-OppX-MicF-ompF axis in related bacteria suggests that it serves an important mechanism, adjusting envelope porosity to specific transport capacity. These data also highlight the resource value of this Salmonella RNA interactome, which will aid in unraveling RNA-centric regulation in enteric pathogens.
  • Comparative genomics provides structural and functional insights into Bacteroides RNA biology.

    Prezza, Gianluca; Ryan, Daniel; Mädler, Gohar; Reichardt, Sarah; Barquist, Lars; Westermann, Alexander J; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Wiley & Sons Ltd., 2021-08-28)
    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.
  • A Hyperthermoactive-Cas9 Editing Tool Reveals the Role of a Unique Arsenite Methyltransferase in the Arsenic Resistance System of Thermus thermophilus HB27.

    Gallo, Giovanni; Mougiakos, Ioannis; Bianco, Mauricio; Carbonaro, Miriam; Carpentieri, Andrea; Illiano, Anna; Pucci, Pietro; Bartolucci, Simonetta; van der Oost, John; Fiorentino, Gabriella; et al. (ASM, 2021-12-07)
    Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to humans. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pulldown assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosyl-l-methionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyze SAM-dependent arsenite methylation with formation of monomethylarsenites (MMAs) and dimethylarsenites (DMAs). In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene encoding a stabilized yellow fluorescent protein (sYFP) to create a sensitive genome-based bioreporter system for the detection of arsenic ions. IMPORTANCE We here describe the discovery of an unknown protein by using a proteomics approach with a transcriptional regulator as bait. Remarkably, we successfully obtained a novel type of enzyme through the interaction with a transcriptional regulator controlling the expression of this enzyme. Employing this strategy, we isolated TtArsM, the first thermophilic prokaryotic arsenite methyltransferase, as a new enzyme of the arsenic resistance mechanism in T. thermophilus HB27. The atypical arsenite binding site of TtArsM categorizes the enzyme as the first member of a new arsenite methyltransferase type, exclusively present in the Thermus genus. The enzyme methylates arsenite-producing MMAs and DMAs. Furthermore, we developed an hyperthermophilic Cas9-based genome-editing tool, active up to 65°C. The tool allowed us to perform highly efficient, marker-free modifications (either gene deletion or insertion) in the T. thermophilus genome. With these modifications, we confirmed the critical role of TtArsM in the arsenite detoxification system and developed a sensitive whole-cell bioreporter for arsenic ions. We anticipate that the developed tool can be easily adapted for editing the genomes of other thermophilic bacteria, significantly boosting fundamental and metabolic engineering in hyperthermophilic microorganisms.
  • Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch.

    Hill, Chris H; Pekarek, Lukas; Napthine, Sawsan; Kibe, Anuja; Firth, Andrew E; Graham, Stephen C; Caliskan, Neva; Brierley, Ian; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Nature Publishing Group (NPG), 2021-12-09)
    rogrammed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection. © 2021. The Au
  • Dysregulated Immunometabolism Is Associated with the Generation of Myeloid-Derived Suppressor Cells in Staphylococcus aureus Chronic Infection.

    Dietrich, Oliver; Heinz, Alexander; Goldmann, Oliver; Geffers, Robert; Beineke, Andreas; Hiller, Karsten; Saliba, Antoine-Emmanuel; Medina, Eva; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Karger, 2021-11-11)
    Myeloid-derived suppressor cells (MDSCs) are a compendium of immature myeloid cells that exhibit potent T-cell suppressive capacity and expand during pathological conditions such as cancer and chronic infections. Although well-characterized in cancer, the physiology of MDSCs in the infection setting remains enigmatic. Here, we integrated single-cell RNA sequencing (scRNA-seq) and functional metabolic profiling to gain deeper insights into the factors governing the generation and maintenance of MDSCs in chronic Staphylococcus aureus infection. We found that MDSCs originate not only in the bone marrow but also at extramedullary sites in S. aureus-infected mice. scRNA-seq showed that infection-driven MDSCs encompass a spectrum of myeloid precursors in different stages of differentiation, ranging from promyelocytes to mature neutrophils. Furthermore, the scRNA-seq analysis has also uncovered valuable phenotypic markers to distinguish mature myeloid cells from immature MDSCs. Metabolic profiling indicates that MDSCs exhibit high glycolytic activity and high glucose consumption rates, which are required for undergoing terminal maturation. However, rapid glucose consumption by MDSCs added to infection-induced perturbations in the glucose supplies in infected mice hinders the terminal maturation of MDSCs and promotes their accumulation in an immature stage. In a proof-of-concept in vivo experiment, we demonstrate the beneficial effect of increasing glucose availability in promoting MDSC terminal differentiation in infected mice. Our results provide valuable information of how metabolic alterations induced by infection influence reprogramming and differentiation of MDSCs.
  • The ambivalent role of Bacteroides in enteric infections.

    Bornet, Elise; Westermann, Alexander J; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier (Cell Press), 2021-12-07)
    Bacteroides spp. are increasingly used as model gut commensals in cocolonization studies with enteropathogens. The collective findings imply common themes of colonization resistance but also pathogen crossfeeding. We discuss how cutting-edge transcriptomics may help to disentangle the molecular basis of the divergent roles of Bacteroides in either protecting against or promoting infection.
  • Concentration and composition dependent aggregation of Pluronic- and Poly-(2-oxazolin)-Efavirenz formulations in biorelevant media.

    Endres, Sebastian; Karaev, Emil; Hanio, Simon; Schlauersbach, Jonas; Kraft, Christian; Rasmussen, Tim; Luxenhofer, Robert; Böttcher, Bettina; Meinel, Lorenz; Pöppler, Ann-Christin; et al. (Elsevier, 2021-08-10)
    Many drugs and drug candidates are poorly water-soluble. Intestinal fluids play an important role in their solubilization. However, the interactions of intestinal fluids with polymer excipients, drugs and their formulations are not fully understood. Here, diffusion ordered spectroscopy (DOSY) and nuclear Overhauser effect spectroscopy (NOESY), complemented by cryo-TEM were employed to address this. Efavirenz (EFV) as model drug, the triblock copolymers Pluronic® F-127 (PF127) and poly(2-oxazoline) based pMeOx-b-pPrOzi-b-pMeOx (pOx/pOzi) and their respective formulations were studied in simulated fed-state intestinal fluid (FeSSIF). For the individual polymers, the bile interfering nature of PF127 was confirmed and pure pOx/pOzi was newly classified as non-interfering. A different and more complex behaviour was however observed if EFV was involved. PF127/EFV formulations in FeSSIF showed concentration dependent aggregation with separate colloids at low formulation concentrations, a merging of individual particles at the solubility limit of EFV in FeSSIF and joint aggregates above this concentration. In the case of pOx/pOzi/EFV formulations, coincident diffusion coefficients for pOx/pOzi, lipids and EFV indicate joint aggregates across the studied concentration range. This demonstrates that separate evaluation of polymers and drugs in biorelevant media is not sufficient and their mixtures need to be studied to learn about concentration and composition dependent behaviour.
  • Complete Genome Sequencing Leptospira interrogans Isolates from Malaysia Reveals Massive Genome Rearrangement but High Conservation of Virulence-Associated Genes

    Ramli, Siti Roszilawati; Bunk, Boyke; Spröer, Cathrin; Geffers, Robert; Jarek, Michael; Bhuju, Sabin; Goris, Marga; Mustakim, Sahlawati; Pessler, Frank; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (PLOS, 2021-09-15)
    The ability of Leptospirae to persist in environments and animal hosts but to cause clinically highly variable disease in humans has made leptospirosis the most common zoonotic disease. Considering the paucity of data on variation in complete genomes of human pathogenic Leptospirae, we have used a combination of Single Molecule Real-Time (SMRT) and Illumina sequencing to obtain complete genome sequences of six human clinical L. interrogans isolates from Malaysia. All six contained the larger (4.28-4.56 Mb) and smaller (0.34-0.395 Mb) chromosome typical of human pathogenic Leptospirae and 0-7 plasmids. Only 24% of the plasmid sequences could be matched to databases. We identified a chromosomal core genome of 3318 coding sequences and strain-specific accessory genomes of 49-179 coding sequences. These sequences enabled detailed genomic strain typing (Genome BLAST Distance Phylogeny, DNA-DNA hybridization, and multi locus sequence typing) and phylogenetic classification (whole-genome SNP genotyping). Even though there was some shared synteny and collinearity across the six genomes, there was evidence of major genome rearrangement, likely driven by horizontal gene transfer and homologous recombination. Mobile genetic elements were identified in all strains in highly varying numbers, including in the rfb locus, which defines serogroups and contributes to immune escape and pathogenesis. On the other hand, there was high conservation of virulence-associated genes including those relating to sialic acid, alginate, and lipid A biosynthesis. These findings suggest (i) that the antigenic variation, adaption to various host environments, and broad spectrum of virulence of L. interrogans are in part due to a high degree of genomic plasticity and (ii) that human pathogenic strains maintain a core set of genes required for virulence.
  • A Review of the Multipronged Attack of Herpes Simplex Virus 1 on the Host Transcriptional Machinery.

    Hennig, Thomas; Djakovic, Lara; Dölken, Lars; Whisnant, Adam W; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (MDPI, 2021-09-14)
    Autophagy is an evolutionary conserved catabolic pathway that ensures the degradation of intracellular components. The autophagic pathway is regulated by autophagy-related (Atg) proteins that govern formation of double-membraned vesicles called autophagosomes. Autophagy deficiency in regulatory T (Treg) cells leads to increased apoptosis of these cells and to the development of autoimmune disorders, predominantly characterized by intestinal inflammation. Recently, RORγt-expressing Treg cells have been identified as key regulators of gut homeostasis, preventing intestinal immunopathology. To study the role of autophagy in RORγt+ Foxp3+ Treg cells, we generated mice lacking the essential component of the core autophagy machinery Atg5 in Foxp3+ cells. Atg5 deficiency in Treg cells led to a predominant intestinal inflammation. While Atg5-deficient Treg cells were reduced in peripheral lymphoid organs, the intestinal RORγt+ Foxp3+ subpopulation of Treg cells was most severely affected. Our data indicated that autophagy is essential to maintain the intestinal RORγt+ Foxp3+ Treg population, thereby protecting the mice from gut inflammatory disorders.
  • RNA Structures and Their Role in Selective Genome Packaging.

    Ye, Liqing; Ambi, Uddhav B; Olguin-Nava, Marco; Gribling-Burrer, Anne-Sophie; Ahmad, Shazeb; Bohn, Patrick; Weber, Melanie M; Smyth, Redmond P; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (MDPI, 2021-09-08)
    To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution.
  • Impact of healthy aging on active bacterial assemblages throughout the gastrointestinal tract.

    Schütte, Kerstin; Schulz, Christian; Vilchez-Vargas, Ramiro; Vasapolli, Riccardo; Palm, Frederike; Simon, Bianca; Schomburg, Dirk; Lux, Anke; Geffers, Robert; Pieper, Dietmar H; et al. (Taylor & Francis, 2021-08-30)
    The adaption of gut microbiota (GM) throughout human life is a key factor in maintaining health. Interventions to restore a healthy GM composition may have the potential to improve health and disease outcomes in the elderly. We performed a comprehensive characterization of changes in the luminal and mucosa-associated microbiota composition in elderly compared with younger healthy individuals. Samples from saliva and feces, and biopsies from the upper and lower gastrointestinal tract (UGIT, LGIT), were collected from 59 asymptomatic individuals grouped by age: 40-55, 56-70, and 71-85 years). All underwent anthropometric, geriatric, and nutritional assessment. RNA was extracted and reverse-transcribed into complementary DNA; the V1-V2 regions of 16S ribosomal RNA genes were amplified and sequenced. Abundances of the taxa in all taxonomic ranks in each sample type were used to construct sample-similarity matrices by the Bray-Curtis algorithm. Significant differences between defined groups were assessed by analysis of similarity. The bacterial community showed strong interindividual variations and a clear distinction between samples from UGIT, LGIT, and feces. While in saliva some taxa were affected by aging, this number was considerably greater in UGIT and was subsequently higher in LGIT. Unexpectedly, aging scarcely influenced the bacterial community of feces over the age range of 40-85 years. The development of interventions to preserve and restore human health with increased age by establishing a healthy gut microbiome should not rely solely on data from fecal analysis, as the intestinal mucosa is affected by more significant changes, which differ from those observed in fecal analyses.
  • SPI2 T3SS effectors facilitate enterocyte apical to basolateral transmigration of -containing vacuoles .

    Fulde, Marcus; van Vorst, Kira; Zhang, Kaiyi; Westermann, Alexander J; Busche, Tobias; Huei, Yong Chiun; Welitschanski, Katharina; Froh, Isabell; Pägelow, Dennis; Plendl, Johanna; et al. (Taylor & Francis, 2021-09-20)
    Salmonella pathogenicity island (SPI) 2 type three secretion system (T3SS)-mediated effector molecules facilitate bacterial survival in phagocytes but their role in the intestinal epithelium in vivo remains ill-defined. Using our neonatal murine infection model in combination with SPI2 reporter technology and RNA-Seq of sorted primary enterocytes, we demonstrate expression of SPI2 effector molecules by intraepithelial Salmonella Typhimurium (S. Typhimurium). Contrary to expectation, immunostaining revealed that infection with SPI2 T3SS-mutants resulted in significantly enlarged intraepithelial Salmonella-containing vacuoles (SCV) with altered cellular positioning, suggesting impaired apical to basolateral transmigration. Also, infection with isogenic tagged S. Typhimurium strains revealed a reduced spread of intraepithelial SPI2 T3SS mutant S. Typhimurium to systemic body sites. These results suggest that SPI2 T3SS effector molecules contribute to enterocyte apical to basolateral transmigration of the SCV during the early stage of the infection.
  • Concatemeric Broccoli reduces mRNA stability and induces aggregates.

    Rink, Marco R; Baptista, Marisa A P; Flomm, Felix J; Hennig, Thomas; Whisnant, Adam W; Wolf, Natalia; Seibel, Jürgen; Dölken, Lars; Bosse, Jens B; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (PLOS, 2021-08-04)
    Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol.

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