• 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.
    • 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.
    • The minimal meningococcal ProQ protein has an intrinsic capacity for structure-based global RNA recognition.

      Bauriedl, Saskia; Gerovac, Milan; Heidrich, Nadja; Bischler, Thorsten; Barquist, Lars; Vogel, Jörg; Schoen, Christoph; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Nature Research, 2020-06-04)
      FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition.