Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• Established Bacteroides as a model for RNA-based regulation in gut microbes.
• Identified functional sRNAs involved in antibiotic sensitivity, stress response, and metabolism.
• Defined the role of the RNA-binding protein RbpB in regulating polysaccharide utilization and colonization.
• Developed Thetabase, an online transcriptome browser for Bacteroides thetaiotaomicron, another model organism of the human gut microbiota (Thetabase: https://micromix.helmholtz-hiri.de/bacteroides/).

Opis badań

My lab investigates RNA-based regulation in the gut microbiota, with a focus on Bacteroides fragilis. As both a commensal and an opportunistic pathogen, it serves as a suitable model to study how regulatory RNAs influence microbe-host interactions.

We study small regulatory RNAs (sRNAs) and RNA-binding proteins (RBPs) that mediate bacterial responses to environmental stress, nutrient availability, and host signals. These post-transcriptional mechanisms are key to survival and adaptation in the gut, yet remain poorly defined in Bacteroides.

We are generating a transcriptomic framework for B. fragilis using RNA-seq methods optimized for sRNA identification and transcription start site mapping. Candidates are selected based on expression under specific conditions and evolutionary conservation. Predicted targets are validated biochemically. Since Bacteroides lack canonical RNA chaperones such as Hfq, we are also working to identify alternative RBPs with roles in sRNA function, RNA stability, and translational control.

A parallel focus is on host interaction. Using epithelial co-culture models, we assess how B. fragilis RNAs affect immune signaling and barrier function. This provides insight into how RNA regulation contributes to shifts between symbiosis and pathogenicity.

While B. fragilis is the primary system, we aim to expand to related species such as Akkermansia muciniphila, and Faecalibacterium prausnitzii, allowing comparative analysis of conserved and niche-specific regulation.

Long-term, we are exploring bacterial extracellular vesicles (EVs) as tools to deliver regulatory RNAs or antisense constructs to gut microbes—an approach with potential for targeted microbiome modulation.

Our work addresses a key gap in microbiome biology: understanding how RNA-level control enables gut bacteria to adapt, persist, and interact with the host.

Metodologia

We use a combination of RNA-sequencing, biochemical assays and molecular biology to study post-transcriptional regulation in gut microbes. For transcriptome mapping, we apply RNA-seq approaches such as dRNA-seq and Cappable-seq to identify transcript boundaries, operon structure, and sRNAs. These are performed under gut-relevant conditions (e.g. bile, anaerobiosis, host exposure) to capture context-specific regulation.

Predicted sRNAs are validated by northern blotting and qRT-PCR. Regulated targets are predicted using in silico tools (IntaRNA, CopraRNA), followed by validation using biochemical assays such as EMSAs, reporter fusions, and mutational analyses. For interaction mapping, we use MS2-affinity pulldowns coupled to RNA-seq.

To identify novel RNA-binding proteins, we use UV-crosslinking, RNA pulldown assays, and mass spectrometry. Candidate RBPs are further characterized for RNA binding and regulatory activity using electrophoretic methods and translational reporter assays.

Microbe-host interaction studies are performed using co-culture systems with colonic epithelial cells. These allow us to assess how regulatory RNAs affect immune signaling and barrier integrity. We monitor cytokine expression and epithelial responses using qPCR, ELISA, and microscopy.

We are also developing bacterial extracellular vesicle (EV) isolation protocols to test their use as delivery vehicles for RNA-based tools. Vesicle content is characterized by RNA-seq and proteomics.

Anaerobic culturing, genetic manipulation, and in vitro assays are carried out using standardized protocols we have optimized for Bacteroides species.

Wybrane publikacje

• Ruttiger AS, Ryan D, Spiga L, Lamm-Schmidt V, Prezza G, Reichardt S, Langford M, Barquist L, Faber F, Zhu W et al: The global RNA-binding protein RbpB is a regulator of polysaccharide utilization in Bacteroides thetaiotaomicron. Nat Commun 2025, 16(1):208.
• Ryan D, Bornet E, Prezza G, Alampalli SV, Franco de Carvalho T, Felchle H, Ebbecke T, Hayward RJ, Deutschbauer AM, Barquist L et al: An expanded transcriptome atlas for Bacteroides thetaiotaomicron reveals a small RNA that modulates tetracycline sensitivity. Nat Microbiol 2024, 9(4):1130-1144.
• Prezza G*, Ryan D*, Madler G, Reichardt S, Barquist L, Westermann AJ: Comparative genomics provides structural and functional insights into Bacteroides RNA biology. Mol Microbiol 2022, 117(1):67-85.
• Ryan D, Jenniches L, Reichardt S, Barquist L, Westermann AJ: A high-resolution transcriptome map identifies small RNA regulation of metabolism in the gut microbe Bacteroides thetaiotaomicron. Nat Commun 2020, 11(1):3557.
• Ryan D, Prezza G, Westermann AJ: An RNA-centric view on gut Bacteroidetes. Biol Chem 2020, 402(1):55-72.

Współpraca

• Alexander Westermann, University of Würzburg, Würzburg, Germany
• Carsten Kroger, Trinity College Dublin, Dublin, Ireland
• Mrutyunjay Suar, KIIT University, Bhubaneswar, India

Nagrody i wyróżnienia

• Daniel Ryan. PostDoc Plus Funding. 2022. University of Würzburg, Germany.
• Daniel Ryan. HZI Paper of the Month. 2020. Helmholtz Centre for Infection Research, Germany.
• Daniel Ryan. ASM Student-Postdoc Travel Grant. 2016. American Society for Microbiology, USA.