• 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.
    • 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.
    • A high-resolution transcriptome map identifies small RNA regulation of metabolism in the gut microbe Bacteroides thetaiotaomicron.

      Ryan, Daniel; Jenniches, Laura; Reichardt, Sarah; Barquist, Lars; Westermann, Alexander J; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (NPG, 2020-07-16)
      Bacteria of the genus Bacteroides are common members of the human intestinal microbiota and important degraders of polysaccharides in the gut. Among them, the species Bacteroides thetaiotaomicron has emerged as the model organism for functional microbiota research. Here, we use differential RNA sequencing (dRNA-seq) to generate a single-nucleotide resolution transcriptome map of B. thetaiotaomicron grown under defined laboratory conditions. An online browser, called 'Theta-Base' ( www.helmholtz-hiri.de/en/datasets/bacteroides ), is launched to interrogate the obtained gene expression data and annotations of ~4500 transcription start sites, untranslated regions, operon structures, and 269 noncoding RNA elements. Among the latter is GibS, a conserved, 145 nt-long small RNA that is highly expressed in the presence of N-acetyl-D-glucosamine as sole carbon source. We use computational predictions and experimental data to determine the secondary structure of GibS and identify its target genes. Our results indicate that sensing of N-acetyl-D-glucosamine induces GibS expression, which in turn modifies the transcript levels of metabolic enzymes.
    • MAPS integrates regulation of actin-targeting effector SteC into the virulence control network of Salmonella small RNA PinT.

      Correia Santos, Sara; Bischler, Thorsten; Westermann, Alexander J; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier (Cell Press), 2021-02-02)
      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.
    • How Insertion of a Single Tryptophan in the N-Terminus of a Cecropin A-Melittin Hybrid Peptide Changes Its Antimicrobial and Biophysical Profile.

      Ferreira, Ana Rita; Teixeira, Cátia; Sousa, Carla F; Bessa, Lucinda J; Gomes, Paula; Gameiro, Paula; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (MDPI, 2021-01-12)
      n the era of antibiotic resistance, there is an urgent need for efficient antibiotic therapies to fight bacterial infections. Cationic antimicrobial peptides (CAMP) are promising lead compounds given their membrane-targeted mechanism of action, and high affinity towards the anionic composition of bacterial membranes. We present a new CAMP, W-BP100, derived from the highly active BP100, holding an additional tryptophan at the N-terminus. W-BP100 showed a broader antibacterial activity, demonstrating a potent activity against Gram-positive strains. Revealing a high partition constant towards anionic over zwitterionic large unilamellar vesicles and inducing membrane saturation at a high peptide/lipid ratio, W-BP100 has a preferential location for hydrophobic environments. Contrary to BP100, almost no aggregation of anionic vesicles is observed around saturation conditions and at higher concentrations no aggregation is observed. With these results, it is possible to state that with the incorporation of a single tryptophan to the N-terminus, a highly active peptide was obtained due to the π-electron system of tryptophan, resulting in negatively charged clouds, that participate in cation-π interactions with lysine residues. Furthermore, we propose that W-BP100 action can be achieved by electrostatic interactions followed by peptide translocation.
    • Improved bacterial RNA-seq by Cas9-based depletion of ribosomal RNA reads.

      Prezza, Gianluca; Heckel, Tobias; Dietrich, Sascha; Homberger, Christina; Westermann, Alexander J; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Cold Spring Harbor Laboratory Press, 2020-04-28)
      A major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative nonribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH (depletion of abundant sequences by hybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH uses the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56%-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron DASH works robustly, even with subnanogram amounts of input RNA. Its efficiency, high sensitivity, ease of implementation, and low cost (∼$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.
    • Dual RNA-seq of Orientia tsutsugamushi informs on host-pathogen interactions for this neglected intracellular human pathogen.

      Mika-Gospodorz, Bozena; Giengkam, Suparat; Westermann, Alexander J; Wongsantichon, Jantana; Kion-Crosby, Willow; Chuenklin, Suthida; Wang, Loo Chien; Sunyakumthorn, Piyanate; Sobota, Radoslaw M; Subbian, Selvakumar; et al. (Nature Publishing Group, 2020-07-03)
      Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning to study the transcriptional architecture of Ot, we find evidence for wide-spread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.
    • 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.
    • Salmonella persisters undermine host immune defenses during antibiotic treatment.

      Stapels, Daphne A C; Hill, Peter W S; Westermann, Alexander J; Fisher, Robert A; Thurston, Teresa L; Saliba, Antoine-Emmanuel; Blommestein, Isabelle; Vogel, Jörg; Helaine, Sophie; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (American Association for the Advancement of Science, 2018-12-07)
      Many bacterial infections are hard to treat and tend to relapse, possibly due to the presence of antibiotic-tolerant persisters. In vitro, persister cells appear to be dormant. After uptake of Salmonella species by macrophages, nongrowing persisters also occur, but their physiological state is poorly understood. In this work, we show that Salmonella persisters arising during macrophage infection maintain a metabolically active state. Persisters reprogram macrophages by means of effectors secreted by the Salmonella pathogenicity island 2 type 3 secretion system. These effectors dampened proinflammatory innate immune responses and induced anti-inflammatory macrophage polarization. Such reprogramming allowed nongrowing Salmonella cells to survive for extended periods in their host. Persisters undermining host immune defenses might confer an advantage to the pathogen during relapse once antibiotic pressure is relieved.