Recent Submissions

  • An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Infection.

    Schulte, Leon N; Schweinlin, Matthias; Westermann, Alexander J; Janga, Harshavardhan; Santos, Sara C; Appenzeller, Silke; Walles, Heike; Vogel, Jörg; Metzger, Marco; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (American Society for Microbiology (ASM), 2020-02-18)
    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.
  • Methods for characterizing, applying, and teaching CRISPR-Cas systems.

    Beisel, Chase L; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier, 2020-01-16)
    New drugs are desperately needed to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report screening commercial kinase inhibitors for antibacterial activity and found the anticancer drug sorafenib as major hit that effec-tively kills MRSA strains. Varying the key structural features led to the identification of a potent analogue, PK150, that showed antibacterial activity against several pathogenic strains at submicromolar concentrations. Furthermore, this antibiotic eliminated challenging persisters as well as established biofilms. PK150 holds promising therapeutic potential as it did not induce in vitro resistance, and shows oral bioavailability and in vivo efficacy. Analysis of the mode of action using chemical proteomics revealed several targets, which included interference with menaquinone biosynthesis by inhibiting demethylmenaquinone methyltrans-ferase and the stimulation of protein secretion by altering the activity of signal peptidase IB. Reduced endogenous menaquinone levels along with enhanced levels of extracellular proteins of PK150-treated bacteria support this target hypothesis. The associ-ated antibiotic effects, especially the lack of resistance development, probably stem from the compound’s polypharmacology.
  • Eleven grand challenges in single-cell data science.

    Lähnemann, David; Köster, Johannes; Szczurek, Ewa; McCarthy, Davis J; Hicks, Stephanie C; Robinson, Mark D; Vallejos, Catalina A; Campbell, Kieran R; Beerenwinkel, Niko; Mahfouz, Ahmed; et al. (BMC, 2020-02-07)
    The recent boom in microfluidics and combinatorial indexing strategies, combined with low sequencing costs, has empowered single-cell sequencing technology. Thousands-or even millions-of cells analyzed in a single experiment amount to a data revolution in single-cell biology and pose unique data science problems. Here, we outline eleven challenges that will be central to bringing this emerging field of single-cell data science forward. For each challenge, we highlight motivating research questions, review prior work, and formulate open problems. This compendium is for established researchers, newcomers, and students alike, highlighting interesting and rewarding problems for the coming years.
  • CRISPR-Cas Systems and the Paradox of Self-Targeting Spacers.

    Wimmer, Franziska; Beisel, Chase L; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Frontiers, 2019-01-01)
    CRISPR-Cas immune systems in bacteria and archaea record prior infections as spacers within each system's CRISPR arrays. Spacers are normally derived from invasive genetic material and direct the immune system to complementary targets as part of future infections. However, not all spacers appear to be derived from foreign genetic material and instead can originate from the host genome. Their presence poses a paradox, as self-targeting spacers would be expected to induce an autoimmune response and cell death. In this review, we discuss the known frequency of self-targeting spacers in natural CRISPR-Cas systems, how these spacers can be incorporated into CRISPR arrays, and how the host can evade lethal attack. We also discuss how self-targeting spacers can become the basis for alternative functions performed by CRISPR-Cas systems that extend beyond adaptive immunity. Overall, the acquisition of genome-targeting spacers poses a substantial risk but can aid in the host's evolution and potentially lead to or support new functionalities.
  • The chlamydial deubiquitinase Cdu1 supports recruitment of Golgi vesicles to the inclusion.

    Auer, Daniela; Hügelschäffer, Sophie D; Fischer, Annette B; Rudel, Thomas; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Wiley, 2019-11-01)
    Chlamydia trachomatis is the main cause of sexually transmitted diseases worldwide. As obligate intracellular bacteria Chlamydia replicate in a membrane bound vacuole called inclusion and acquire nutrients for growth and replication from their host cells. However, like all intracellular bacteria, Chlamydia have to prevent eradication by the host's cell autonomous system. The chlamydial deubiquitinase Cdu1 is secreted into the inclusion membrane, facing the host cell cytosol where it deubiquitinates cellular proteins. Here we show that inactivation of Cdu1 causes a growth defect of C. trachomatis in primary cells. Moreover, ubiquitin and several autophagy receptors are recruited to the inclusion membrane of Cdu1-deficient Chlamydia. Interestingly, the growth defect of cdu1 mutants is not rescued when autophagy is prevented. We find reduced recruitment of Golgi vesicles to the inclusion of Cdu1 mutants indicating that vesicular trafficking is altered in bacteria without active deubiquitinase (DUB). Our work elucidates an important role of Cdu1 in the functional preservation of the chlamydial inclusion surface.
  • Competitive exclusion is a major bioprotective mechanism of lactobacilli against fungal spoilage in fermented milk products.

    Siedler, Solvej; Rau, Martin Holm; Bidstrup, Susanne; Vento, Justin M; Aunsbjerg, Stina Dissing; Bosma, Elleke F; McNair, Laura M; Beisel, Chase L; Neves, Ana Rute; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (American Society of Microbiology, 2020-01-31)
    A prominent feature of lactic acid bacteria (LAB) is their ability to inhibit growth of spoilage organisms in food, but hitherto research efforts to establish the mechanisms underlying bioactivity focused on the production of antimicrobial compounds by LAB. We show in this study, that competitive exclusion, i.e, competition for a limited resource by different organisms, is a major mechanism of fungal growth inhibition by lactobacilli in fermented dairy products. The depletion of the essential trace element manganese by two Lactobacillus species was uncovered as the main mechanism for growth inhibition of dairy spoilage yeast and molds. A manganese transporter (MntH1), representing one of the highest expressed gene products in both lactobacilli, facilitates the exhaustive manganese scavenging. Expression of the mntH1 gene was found to be strain-dependent, affected by species co-culturing and growth phase. Further, deletion of the mntH1 gene in one of the strains resulted in loss of bioactivity, proving this gene to be important for manganese depletion. The presence of a mntH gene displayed a distinct phylogenetic pattern within the Lactobacillus genus. Moreover, assaying the bioprotective ability in fermented milk of selected lactobacilli from ten major phylogenetic groups identified a correlation between the presence of mntH and bioprotective activity. Thus, manganese scavenging emerges as a common trait within the Lactobacillus genus, but differences in expression result in some strains showing more bioprotective effect than others.In summary, competitive exclusion through ion depletion is herein reported a novel mechanism in LAB to delay growth of spoilage contaminants in dairy products.IMPORTANCE In societies that have food choices, conscious consumers demand natural solutions to keep their food healthy and fresh during storage, simultaneously reducing food waste. The use of "good bacteria" to protect food against spoilage organisms has a long successful history, even though the molecular mechanisms are not fully understood. In this study, we show that depletion of free manganese is a major bioprotective mechanism of lactobacilli in dairy products. High manganese uptake and intracellular storage provides a link to the distinct non-enzymatic manganese catalyzed oxidative stress defense mechanism, previously described for certain lactobacilli. The evaluation of representative Lactobacillus species in our study identifies multiple relevant species groups for fungal growth inhibition via manganese depletion. Hence, through the natural mechanism of nutrient depletion, the use of dedicated bioprotective lactobacilli constitutes an attractive alternative to artificial preservation.
  • Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27.

    Wang, Xiuye; Hennig, Thomas; Whisnant, Adam W; Erhard, Florian; Prusty, Bhupesh K; Friedel, Caroline C; Forouzmand, Elmira; Hu, William; Erber, Luke; Chen, Yue; et al. (Nature publishing group, 2020-01-15)
    Infection by viruses, including herpes simplex virus-1 (HSV-1), and cellular stresses causewidespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) inhost genes. However, the underlying mechanisms remain unclear. Here, we demonstrate thatthe HSV-1 immediate early protein ICP27 induces DoTT by directly binding to the essentialmRNA 3’processing factor CPSF. It thereby induces the assembly of a dead-end 3’processing complex, blocking mRNA 3’cleavage. Remarkably, ICP27 also acts as a sequence-dependent activator of mRNA 3’processing for viral and a subset of host transcripts.Our results unravel a bimodal activity of ICP27 that plays a key role in HSV-1-induced hostshutoff and identify CPSF as an important factor that mediates regulation of transcriptiontermination. Thesefindings have broad implications for understanding the regulation oftranscription termination by other viruses, cellular stress and cancer.
  • An educational module to explore CRISPR technologies with a cell-free transcription-translation system

    Collias, Daphne; Marshall, Ryan; Collins, Scott P.; Beisel, Chase L.; Noireaux, Vincent; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Oxford Academic, 2019-05-21)
    Within the last 6 years, CRISPR-Cas systems have transitioned from adaptive defense systems in bacteria and archaea to revolutionary genome-editing tools. The resulting CRISPR technologies have driven innovations for treating genetic diseases and eradicating human pests while raising societal questions about gene editing in human germline cells as well as crop plants. Bringing CRISPR into the classroom therefore offers a means to expose students to cutting edge technologies and to promote discussions about ethical questions at the intersection of science and society. However, working with these technologies in a classroom setting has been difficult because typical experiments rely on cellular systems such as bacteria or mammalian cells. We recently reported the use of an E. coli cell-free transcription-translation (TXTL) system that simplifies the demonstration and testing of CRISPR technologies with shorter experiments and limited equipment. Here, we describe three educational modules intended to expose undergraduate students to CRISPR technologies using TXTL. The three sequential modules comprise (i) designing the RNAs that guide DNA targeting, (ii) measuring DNA cleavage activity in TXTL and (iii) testing how mutations to the targeting sequence or RNA backbone impact DNA binding and cleavage. The modules include detailed protocols, questions for group discussions or individual evaluation, and lecture slides to introduce CRISPR and TXTL. We expect these modules to allow students to experience the power and promise of CRISPR technologies in the classroom and to engage with their instructor and peers about the opportunities and potential risks for society.
  • An RNA Surprise in Bacterial Effector Mechanisms

    Gerovac, Milan; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier BV, 2019-12)
    acterial pathogens secrete effector proteins to manipulate host signaling proteins and cellular structures. In this issue of Cell Host & Microbe, Pagliuso et al. (2019) propose an effector mechanism in Listeria monocytogenes whereby an RNA-binding protein associates with bacterial RNA that stimulates RIG-I (retinoic acid inducible gene I)-based innate immunity in the host cytosol.
  • The conserved 3' UTR-derived small RNA NarS mediates mRNA crossregulation during nitrate respiration.

    Wang, Chuan; Chao, Yanjie; Matera, Gianluca; Gao, Qian; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Oxford Academic, 2019-12-21)
    Small noncoding RNAs (sRNAs) from mRNA 3' UTRs seem to present a previously unrecognized layer of bacterial post-transcriptional control whereby mRNAs influence each other's expression, independently of transcriptional control. Studies in Escherichia coli and Salmonella enterica showed that such sRNAs are natural products of RNase E-mediated mRNA decay and associate with major RNA-binding proteins (RBPs) such as Hfq and ProQ. If so, there must be additional sRNAs from mRNAs that accumulate only under specific physiological conditions. We test this prediction by characterizing candidate NarS that represents the 3' UTR of nitrate transporter NarK whose gene is silent during standard aerobic growth. We find that NarS acts by Hfq-dependent base pairing to repress the synthesis of the nitrite transporter, NirC, resulting in mRNA cross-regulation of nitrate and nitrite transporter genes. Interestingly, the NarS-mediated repression selectively targets the nirC cistron of the long nirBDC-cysG operon, an observation that we rationalize as a mechanism to protect the bacterial cytoplasm from excessive nitrite toxicity during anaerobic respiration with abundant nitrate. Our successful functional assignment of a 3' UTR sRNA from a non-standard growth condition supports the notion that mRNA crossregulation is more pervasive than currently appreciated.
  • MetaMap: An atlas of metatranscriptomic reads in human disease-related RNA-seq data

    Simon, L. M.; Karg, S.; Westermann, A. J.; Engel, M.; Elbehery, A. H.A.; Hense, B.; Heinig, M.; Deng, L.; Theis, F. J.; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Oxford University Press, 2018-06-01)
    Background: With the advent of the age of big data in bioinformatics, large volumes of data and high-performance computing power enable researchers to perform re-analyses of publicly available datasets at an unprecedented scale. Ever more studies imply the microbiome in both normal human physiology and a wide range of diseases. RNA sequencing technology (RNA-seq) is commonly used to infer global eukaryotic gene expression patterns under defined conditions, including human disease-related contexts; however, its generic nature also enables the detection of microbial and viral transcripts. Findings: We developed a bioinformatic pipeline to screen existing human RNA-seq datasets for the presence of microbial and viral reads by re-inspecting the non-human-mapping read fraction. We validated this approach by recapitulating outcomes from six independent, controlled infection experiments of cell line models and compared them with an alternative metatranscriptomic mapping strategy. We then applied the pipeline to close to 150 terabytes of publicly available raw RNA-seq data from  more than 17,000 samples from more than 400 studies relevant to human disease using state-of-the-art high-performance computing systems. The resulting data from this large-scale re-analysis are made available in the presented MetaMap resource. Conclusions: Our results demonstrate that common human RNA-seq data, including those archived in public repositories, might contain valuable information to correlate microbial and viral detection patterns with diverse diseases. The presented MetaMap database thus provides a rich resource for hypothesis generation toward the role of the microbiome in human disease. Additionally, codes to process new datasets and perform statistical analyses are made available.
  • Thermodynamic control of -1 programmed ribosomal frameshifting.

    Bock, Lars V; Caliskan, Neva; Korniy, Natalia; Peske, Frank; Rodnina, Marina V; Grubmüller, Helmut; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Nature Research, 2019-10-10)
    mRNA contexts containing a 'slippery' sequence and a downstream secondary structure element stall the progression of the ribosome along the mRNA and induce its movement into the -1 reading frame. In this study we build a thermodynamic model based on Bayesian statistics to explain how -1 programmed ribosome frameshifting can work. As training sets for the model, we measured frameshifting efficiencies on 64 dnaX mRNA sequence variants in vitro and also used 21 published in vivo efficiencies. With the obtained free-energy difference between mRNA-tRNA base pairs in the 0 and -1 frames, the frameshifting efficiency of a given sequence can be reproduced and predicted from the tRNA-mRNA base pairing in the two frames. Our results further explain how modifications in the tRNA anticodon modulate frameshifting and show how the ribosome tunes the strength of the base-pair interactions.
  • CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9.

    Dugar, Gaurav; Leenay, Ryan T; Eisenbart, Sara K; Bischler, Thorsten; Aul, Belinda U; Beisel, Chase L; Sharma, Cynthia M; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier/ Cel Press, 2018-03-01)
    Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) to silence foreign double-stranded DNA. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. Here, we show that the Campylobacter jejuni Cas9 (CjCas9) can bind and cleave complementary endogenous mRNAs in a crRNA-dependent manner. Approximately 100 transcripts co-immunoprecipitated with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs. A subset of these RNAs was cleaved around or within the predicted binding site. Mutational analyses revealed that RNA binding was crRNA and tracrRNA dependent and that target RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage was PAM independent, improved with greater complementarity between the crRNA and the RNA target, and was programmable in vitro. These findings suggest that C. jejuni Cas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.
  • RNA target profiles direct the discovery of virulence functions for the cold-shock proteins CspC and CspE.

    Michaux, Charlotte; Holmqvist, Erik; Vasicek, Erin; Sharan, Malvika; Barquist, Lars; Westermann, Alexander J; Gunn, John S; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (National Academy of Sciences, 2017-06-27)
    The functions of many bacterial RNA-binding proteins remain obscure because of a lack of knowledge of their cellular ligands. Although well-studied cold-shock protein A (CspA) family members are induced and function at low temperature, others are highly expressed in infection-relevant conditions. Here, we have profiled transcripts bound in vivo by the CspA family members of Salmonella enterica serovar Typhimurium to link the constitutively expressed CspC and CspE proteins with virulence pathways. Phenotypic assays in vitro demonstrated a crucial role for these proteins in membrane stress, motility, and biofilm formation. Moreover, double deletion of cspC and cspE fully attenuates Salmonella in systemic mouse infection. In other words, the RNA ligand-centric approach taken here overcomes a problematic molecular redundancy of CspC and CspE that likely explains why these proteins have evaded selection in previous virulence factor screens in animals. Our results highlight RNA-binding proteins as regulators of pathogenicity and potential targets of antimicrobial therapy. They also suggest that globally acting RNA-binding proteins are more common in bacteria than currently appreciated.
  • scSLAM-seq reveals core features of transcription dynamics in single cells.

    Erhard, Florian; Baptista, Marisa A P; Krammer, Tobias; Hennig, Thomas; Lange, Marius; Arampatzi, Panagiota; Jürges, Christopher S; Theis, Fabian J; Saliba, Antoine-Emmanuel; Dölken, Lars; et al. (Springer-Nature, 2019-01-01)
    Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease1. However, current scRNA-seq approaches provide only a snapshot of gene expression and convey little information on the true temporal dynamics and stochastic nature of transcription. A further key limitation of scRNA-seq analysis is that the RNA profile of each individual cell can be analysed only once. Here we introduce single-cell, thiol-(SH)-linked alkylation of RNA for metabolic labelling sequencing (scSLAM-seq), which integrates metabolic RNA labelling2, biochemical nucleoside conversion3 and scRNA-seq to record transcriptional activity directly by differentiating between new and old RNA for thousands of genes per single cell. We use scSLAM-seq to study the onset of infection with lytic cytomegalovirus in single mouse fibroblasts. The cell-cycle state and dose of infection deduced from old RNA enable dose-response analysis based on new RNA. scSLAM-seq thereby both visualizes and explains differences in transcriptional activity at the single-cell level. Furthermore, it depicts 'on-off' switches and transcriptional burst kinetics in host gene expression with extensive gene-specific differences that correlate with promoter-intrinsic features (TBP-TATA-box interactions and DNA methylation). Thus, gene-specific, and not cell-specific, features explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations.
  • Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis.

    Liao, Chunyu; Ttofali, Fani; Slotkowski, Rebecca A; Denny, Steven R; Cecil, Taylor D; Leenay, Ryan T; Keung, Albert J; Beisel, Chase L; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Springer-Nature, 2019-07-03)
    CRISPR-Cas systems inherently multiplex through CRISPR arrays—whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.
  • Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells as a Cellular Model to Study Infection.

    Martins Gomes, Sara F; Westermann, Alexander J; Sauerwein, Till; Hertlein, Tobias; Förstner, Konrad U; Ohlsen, Knut; Metzger, Marco; Shusta, Eric V; Kim, Brandon J; Appelt-Menzel, Antje; et al. (Frontiers, 2019-01-01)
    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.
  • Barriers to genome editing with CRISPR in bacteria.

    Vento, Justin M; Crook, Nathan; Beisel, Chase L; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Springer, 2019-06-05)
    Genome editing is essential for probing genotype-phenotype relationships and for enhancing chemical production and phenotypic robustness in industrial bacteria. Currently, the most popular tools for genome editing couple recombineering with DNA cleavage by the CRISPR nuclease Cas9 from Streptococcus pyogenes. Although successful in some model strains, CRISPR-based genome editing has been slow to extend to the multitude of industrially relevant bacteria. In this review, we analyze existing barriers to implementing CRISPR-based editing across diverse bacterial species. We first compare the efficacy of current CRISPR-based editing strategies. Next, we discuss alternatives when the S. pyogenes Cas9 does not yield colonies. Finally, we describe different ways bacteria can evade editing and how elucidating these failure modes can improve CRISPR-based genome editing across strains. Together, this review highlights existing obstacles to CRISPR-based editing in bacteria and offers guidelines to help achieve and enhance editing in a wider range of bacterial species, including non-model strains.
  • Rapid transcriptional responses to serum exposure are associated with sensitivity and resistance to antibody-mediated complement killing in invasive Typhimurium ST313.

    Ondari, Edna M; Klemm, Elizabeth J; Msefula, Chisomo L; El Ghany, Moataz Abd; Heath, Jennifer N; Pickard, Derek J; Barquist, Lars; Dougan, Gordon; Kingsley, Robert A; MacLennan, Calman A; et al. (F1000Research, 2019-01-01)
    Background: Salmonella Typhimurium ST313 exhibits signatures of adaptation to invasive human infection, including higher resistance to humoral immune responses than gastrointestinal isolates. Full resistance to antibody-mediated complement killing (serum resistance) among nontyphoidal Salmonellae is uncommon, but selection of highly resistant strains could compromise vaccine-induced antibody immunity. Here, we address the hypothesis that serum resistance is due to a distinct genotype or transcriptome response in S. Typhimurium ST313. Methods: Six S. Typhimurium ST313 bloodstream isolates, three of which were antibody resistant, were studied. Genomic content (single nucleotide polymorphisms and larger chromosomal modifications) of the strains was determined by Illumina and PACBIO sequencing, and functionally characterized using RNA-seq, transposon directed insertion site sequencing (TraDIS), targeted gene deletion and transfer of selected point mutations in an attempt to identify features associated with serum resistance.   Results: Sequence polymorphisms in genes from strains with atypical serum susceptibility when transferred from strains that were highly resistant or susceptible to a strain that exhibited intermediate susceptibility did not significantly alter serum killing phenotype. No large chromosomal modifications typified serum resistance or susceptibility. Genes required for resistance to serum identified by TraDIS and RNA-seq included those involved in exopolysaccharide synthesis, iron scavenging and metabolism. Most of the down-regulated genes were associated with membrane proteins. Resistant and susceptible strains had distinct transcriptional responses to serum, particularly related to genes responsible for polysaccharide biosynthesis. There was higher upregulation of wca locus genes, involved in the biosynthesis of colanic acid exopolysaccharide, in susceptible strains and increased expression of fepE, a regulator of very long-chain lipopolysaccharide in resistant strains. Conclusion: Clinical isolates of S. Typhimurium ST313 exhibit distinct antibody susceptibility phenotypes that may be associated with changes in gene expression on exposure to serum.
  • An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system.

    Wandera, Katharina G; Collins, Scott P; Wimmer, Franziska; Marshall, Ryan; Noireaux, Vincent; Beisel, Chase L; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier, 2019-05-21)
    The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. While Acrs have been identified against a few CRISPR-Cas systems, likely many more await discovery and application. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli-derived cell-free transcription-translation system. Using known Acrs against type II Cas9 nucleases as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors.

View more