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. Yet, fundamental aspects of gene expression have not been studied extensively B. burgdorferi. We have recognized the importance of mapping RNA boundaries (their 5' and 3' ends) in bacteria, which is critical for gene annotation, the discovery of novel transcripts, and mechanistic characterization of genes. Several RNA-seq approaches have been developed to globally determine RNA boundaries: 5'RNA-seq identifies and distinguishes transcription starts sites (TSSs) from 5' processed ends; total RNA-seq sequences genes in their entirety; 3'RNA-seq captures termination events and identifies RNA 3' ends. As proof of concept, we first applied these sequencing approaches to the model organism Escherichia coli (1). In doing so we identified numerous E. coli 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 an E. coli small RNA encoded internal to an essential cell division gene. We performed 5'-, total and 3'RNA-seq on RNA isolated from B. burgdorferi grown in culture (2). This identified complex gene arrangements and operons, untranslated regions and small RNAs. Remarkably, 63% of RNA 3' ends mapped upstream or internal to open reading frames (ORFs), including genes involved in the unique infectious cycle of B. burgdorferi. We hypothesize these RNAs result from premature termination and regulatory events such as cis-acting RNA regulation. Our findings uncovered an abundance of potential RNA regulators for future study in B. burgdorferi. In a separate study, we compared identified 5' ends to transcriptionally active sequences on the B. burgdorferi genome during a mouse infection. 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. 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 (3). Ongoing work in our lab has been focused on the characterization and physiological roles of other B. burgdorferi genes, particularly regulatory RNAs, and regulatory networks, particularly regulation of spirochete motility.