IMIB - Institute for Molecular Infection Biology

Approaching cellular heterogeneity in the abundant microbiota member Bacteroides thetaiotaomicron

The human intestine is colonized by myriads of bacteria, commonly referred to as the intestinal microbiota. Gut bacteria are constantly exposed to their host and interact with co-colonizing commensal bacteria or invading pathogens. The cellular states of the interacting cells are probably decisive for the outcome of their encounters. For example, bacterial pathogens deploy cell-to-cell variability, with distinct sub-populations cooperating to establish infection. Microbiota species, too, exhibit phenotypic variation, with potentially important roles for the gut ecosystem. For example, Bacteroides thetaiotaomicron is a predominant member of the human gut microbiota and known to phase-variably express cell surface molecules. This results in phenotypically diverse bacterial sub-populations with varying susceptibility to phage infection, differential detection by secreted host IgA, and varying susceptibility to antibiotic treatment [1,2]. In addition, B. thetaiotaomicron forms metabolically diverse sub-populations, even when grown under homogeneous lab conditions ^[3]. The molecular sources and functional consequences of this metabolic heterogeneity are unknown.

In this project, we will apply bacterial single-cell RNA-seq (scRNA-seq) [4] to profile heterogeneous gene expression of B. thetaiotaomicron on genome-wide scale. Individual GFP-expressing Bacteroides cells will be collected using Fluorescence Activated Cell Sorting. Lysis conditions need to be optimized for efficient cell disruption, while retaining high-quality RNA. Sequencing libraries will be generated following the MATQ-Seq protocol, in combination with our sensitive ribosomal depletion procedure [5]. Once established, scRNA-seq will be applied to B. thetaiotaomicron grown in vitro under different stress conditions or in presence of defined carbohydrates sources.  Transcriptomics will be complemented with functional assays to reveal potential ‘bet-hedging’ or ‘division-of-labor’ strategies. The long-term goal is to record Bacteroides single-cell transcriptomes during host colonization and under infection conditions.

References:

1.         Porter, N. T. et al. Phase-variable capsular polysaccharides and lipoproteins modify bacteriophage susceptibility in Bacteroides thetaiotaomicron. Nat. Microbiol. 5, 1170–1181 (2020).

2.         Jiang, X. et al. Invertible promoters mediate bacterial phase variation, antibiotic resistance, and host adaptation in the gut. Science (80-. ). 363, 181–187 (2019).

3.         Hehemann, J.-H. et al. Single cell fluorescence imaging of glycan uptake by intestinal bacteria. ISME J. 13, 1883–1889 (2019).

4.         Imdahl, F., Vafadarnejad, E., Homberger, C., Saliba, A.-E. & Vogel, J. Single-cell RNA-sequencing reports growth-condition-specific global transcriptomes of individual bacteria. Nat. Microbiol. 5, 1202–1206 (2020).

5.         Prezza, G. et al. Improved bacterial RNA-seq by Cas9-based depletion of ribosomal RNA reads. Rna rna.075945.120 (2020) doi:10.1261/rna.075945.120.