Regulatory RNAs in the pathogenic Epsilonproteobacteria, Helicobacter pylori and Campylobacter jejuni


Post-transcriptional regulation represents a central level of gene expression control in the cell. Non-coding RNAs and associated RNA-binding proteins have been described as key players in this regulatory process. The 50-200 nt long small RNAs (sRNAs) from bacteria are a heterogenous class of molecules which regulate bacterial gene expression in response to adverse growth and environmental stress conditions. A large number of sRNAs have been identified in a wide range of bacteria in the last two decades. However, most studies on the functional characterization of sRNAs focused on enterobacteria, such as Escherichia coli and Salmonella. In contrast, almost nothing is known about riboregulation in Epsilonproteobacteria, including major human pathogens such as Helicobacter pylori, the causative agent of gastric cancer, and Campylobacter jejuni, the most common cause of food-borne gastroenteritis. Our overall research goal is to establish Helicobacter and Campylobacter as new model organisms for riboregulation in pathogenic bacteria. Specifically, we focus on the identification of sRNAs and associated RNA-binding proteins as well as their functions and mechanisms in stress response and virulence of these prevalent human pathogens. Furthermore, we apply and develop deep sequencing-based approaches (RNA-seq) for transcriptome analyses and identification of novel RNAs in both host and pathogen.


Figure1: Colourized scanning electron micro-graph of Helicobacter pylori and human gastric epithelium cells. (Kindly provided by Dr. Volker Brinkmann, Max Planck Institute for Infectionbiology, Berlin)

Identification of sRNAs and global transcriptome analyses of H. pylori

The Gram-negative Epsilonproteobacterium Helicobacter pylori colonizes the stomachs of about 50% of the world's population and leads to gastritis, ulcer, and even gastric cancer. Genome sequencing of several strains revealed its potential proteins and a high genetic diversity. However, only little is known about post-transcriptional regulation in this pathogen. Moreover, H. pylori was even regarded as an organism that lacks riboregulation. However, we have recently developed a novel generic differential RNA-sequencing (dRNA-seq) approach based on next-generation sequencing technology which revealed a high transcriptome complexity of the small H. pylori genome, a massive antisense transcription as well as an unexpected number of more than 60 sRNAs (Sharma et al., 2010, Nature).

Functional characterization of sRNAs in H. pylori

Now we aim at the functional characterization of the newly identified sRNAs in H. pylori and to understand their potential roles during virulence. Besides the identification of factors and conditions which control sRNA expression and additional partners involved in riboregulation in H. pylori, we are especially interested in the molecular mechanisms of sRNAs.  Many of our sRNAs are potential candidates for trans-encoded antisense RNA regulators. For example, the very abundant sRNA, HPnc5490 represses expression of the chemotaxis receptor TlpB in H. pylori. Moreover, we have constructed deletion and overexpression mutants of other abundant sRNAs to identify their target genes and functional roles during stress response and virulence.

Figure 2: Colorized scanning electron micrograph of Campylobacter jejuni strain NCTC11168.
Figure 3: Infection of an intestinal tissue model with Campylobacter jejuni. (Left) Overview of the set-up of the intestinal 3D tissue model by reseeding the SISmuc (small intestinal submucosa) scaffold with the intestinal Caco-2 cell line, cultivation for 21 days, and subsequent infection with C. jejuni. (Right) Immunohistochemical staining of Caco-2 cells grown on SISmuc infected with C. jejuni strain 81-176 for 72 to 120 hours depicting the accumulation of bacterial cells in intestinal crypts. E-cadherin (magenta, adherence junctions), DAPI (blue, nuclei), C. jejuni (green, bacteria).

Comparative transcriptome analysis of multiple Campylobacter strains

Our above described dRNA-seq approach allowed us to define a genome-wide map of transcriptional start sites (TSS) and operons in H. pylori. However, this study was limited to one bacterial strain. We have now performed a comparative dRNA-seq analysis of multiple isolates of the related pathogen, Campylobacter jejuni to understand how transcriptome differences could contribute to phenotypic changes among closely related strains. Campylobacter is currently the most common cause of bacterial gastroenteritis in humans and has also been associated with several autoimmune disorders. Our comparative study reveals that the majority of TSS is conserved among strains but that there are also several strain-specific TSS, indicating divergent transcriptional output. Moreover, Northern blot analysis confirmed expression of several conserved and strain specific sRNA repoertires in C. jejuni, which could contribute to strain-specific gene regulation (Dugar et al, 2013, PLoS Genetics, in press).

Identification of RNA binding proteins in H. pylori and C. jejuni

Most of the functionally characterized sRNAs in enterobacteria require the RNA chaperone Hfq for their stability and function. In addition, Hfq is required for virulence in many bacterial pathogens. However, 50% of all bacteria, including Epsilonproteobacteria, lack Hfq. Our goal is to identify and functionally characterize auxiliary protein factors involved in riboregulation in Helicobacter and Campylobacter. To this end, we have started to isolate ribonucleoprotein (RNP)-complexes, followed by proteomics and RNA-seq analyses of protein and RNA partners and subsequent biochemical analyses. This will facilitate to address the question whether these bacteria use a different RNA-binding protein which replaces Hfq or whether their sRNAs act by novel mechanisms independent of a protein chaperone.

New three-dimensional (3D) infection models based on tissue engineering:

One of our goals is to study the impact of sRNAs in host-pathogen interactions. However, infection studies of human pathogens, such as Helicobacter or Campylobacter, are often impeded by the use of artificial in-vitro cell culture or animal models which are limited in their ability to comprehensively mimic the infection situation and disease in the human host. In a collaborative project with the Chair of Tissue Engineering in Würzburg (PI Heike Walles) we have been applying and developing new 3D infection models based on tissue engineering. Such complex multicellular 3D tissue-cultures, which employ an extracellular matrix scaffold, can mimic the complexity and microenvironment of human tissues. We have successfully established infection conditions with Campylobacter and a small-intestine model and have started to develop a new stomach model for Helicobacter. We are using these models to study new virulence genes such as sRNAs and for Dual RNA-seq analyses to globally identify host and pathogen genes relevant during infection. Using infections with C. jejuni transposon mutant libraries combined with deep sequencing analysis to track transposon insertions (Tn-seq) we aim to identify novel factors required for host-pathogen-interactions. The 3D tissue models will also be useful to study other pathogens and the development of new therapeutic and diagnostic tools.

Conclusion and Perspective

Our overall research goal is to establish Helicobacter and Campylobacter as new model organisms for RNA research in virulent bacteria and bacteria that lack Hfq. Using genetic, molecular biological, and biochemical methods we will obtain detailed insights into gene regulation in these bacterial pathogens. Moreover, we aim to facilitate deep sequencing-based approaches in a broader set of pro- and eukaryotic pathogens and in mixed pathogen-host communities. Overall, our research will not only help shed light on sRNA-mediated regulation in H. pylori and C. jejuni, but also in other bacterial pathogens. This is important for our understanding of virulence mechanisms and could provide new targets for antimicrobial therapies.