RNA-mediated gene regulation in the Lyme disease pathogen

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, an emerging infectious disease and the foremost vector-borne bacterial infection in the world. Given that B. burgdorferi inhabits tick and mammalian hosts, environments with very different temperatures, immune responses and sources of metabolites, the bacterium must harbor robust gene regulatory mechanisms in order to survive. The major focus of the group has been to discover and characterize B. burgdorferi genes important for infectivity and stress response. We recognized the importance of mapping RNA boundaries (their 5' and 3' ends), which is critical for gene annotation, the discovery of novel transcripts, and mechanistic characterization of genes. Therefore, we performed 5'RNA-seq on total RNA isolated from B. burgdorferi grown in culture. This method identified RNA 5' ends and distinguished transcription starts sites (TSSs) from 5' processed ends. These data were then compared to a previous genetic screen that identified transcriptionally active sequences on the B. burgdorferi genome during a mouse infection. This resulted in a dataset of putative mammalian-specific B. burgdorferi transcriptional events and identified numerous genes for future study. One gene identified by our approach, bb0562, was an annotated hypothetical protein. Targeted deletion of bb0562 revealed the gene encodes a protein important for disseminated infection in mice by needle inoculation and tick bite transmission. The gene bb0562 was also found to be important for spirochete growth in low serum media, leading to a growth defect which could be rescued with the addition of various long chain fatty acids. We identified two canonical lipase motifs within BB0562 and demonstrated in vitro lipolytic activity with purified BB0562 protein. Collectively, this work established bb0562 as a novel B. burgdorferi nutritional virulence determinant (1). Ongoing work in our lab has been focused on the characterization of other genes, particularly regulatory RNAs. This is an unstudied area of B. burgdorferi gene regulation. To expand upon our previous study, we augmented our RNA-seq approach and now combine three techniques to further refine RNA boundaries: 5'RNA-seq, 3'RNA-seq (which captures termination events and identifies RNA 3' ends), and total RNA-seq (which sequences genes in their entirety). As proof of concept, we applied this approach to the model organism Escherichia coli (2). In doing so we identified numerous RNA fragments derived from 5' regions of mRNAs and internal to open reading frames (ORFs). We documented regulation for multiple transcripts and identified a function for a small RNA encoded internal to an essential cell division gene. This work revealed that regulatory transcripts are derived from a wide range of locations in bacterial genomes (3). Further studies to identify B. burgdorferi RNA regulators and characterize their physiological roles are ongoing.