RNA–Mediated Gene Regulation in the Lyme Disease Pathogen
- Philip P. Adams, PhD, Head, Group on Gene Regulation in Bacterial Pathogens
- Emily Petroni, BS, Postbaccalaureate Intramural Research Training Award Fellow
- Daniel Soliman, BS, Postbaccalaureate Intramural Research Training Award Fellow
- Daniel Tetreault, BA, Postbaccalaureate Intramural Research Training Award Fellow
The goal of this research is to identify and characterize gene regulation in the spirochete Borrelia burgdorferi, the causative agent of Lyme disease. We use genetics, RNA sequencing, and murine infection models to pinpoint genes for mechanistic study.
Lyme disease is 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. The data were then compared with a previous genetic screen that identified transcriptionally active sequences on the B. burgdorferi genome during a mouse infection, which 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 that the gene encodes a protein important for disseminated infection in mice by needle inoculation and tick bite transmission. The bb0562 gene was also found to be important for spirochete growth in low-serum media, leading to a growth defect that could be rescued by 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, the work established bb0562 as a novel B. burgdorferi nutritional virulence determinant [Reference 1].
Ongoing work in our lab focuses on the characterization of other genes, particularly of regulatory RNAs, 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 the approach to the model organism Escherichia coli [Reference 2]. 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 several transcripts and identified a function for a small RNA encoded internal to an essential cell-division gene. The work revealed that regulatory transcripts are derived from a wide range of locations in bacterial genomes.
Further studies to perform 3′RNA-seq in B. burgdorferi to better understand transcription termination and identify novel RNA regulators and their physiological roles are ongoing.
Figure 1. RNA-seq approach to map RNA ends
Click image to view.
5′RNA-seq utilizes Tobacco acid phosphatase (TAP) to distinguish transcription start sites (TSSs) from processed 5′ ends. In 3′RNA-seq, adaptors are added prior to RNA shearing. 5′ and 3′ adaptors are labeled in green and red.
Additional Funding
- NICHD Early Career Award (2022) to Philip Adams, concluded
Publications
- Kuhn HW, Lasseter A, Adams PP, Flores Avile C, Stone BL, Akins DR, Jewett TJ, Jewett MW. BB0562 is a nutritional virulence determinant with lipase activity important for Borrelia burgdorferi infection and survival in fatty acid deficient environments. PLoS Pathog 2021 17(8):e1009869.
- Adams PP, Baniulyte G, Esnault C, Chegireddy K, Singh N, Monge M, Dale RK, Storz G, Wade JT. Regulatory roles of Escherichia coli 5' UTR and ORF-internal RNAs detected by 3' end mapping. eLife 2021 10:e62438.
Collaborators
- Ryan Dale, PhD, Computer Support Services Core, NICHD, Bethesda, MD
- Mollie Jewett, PhD, University of Central Florida, Orlando, FL
- Gisela Storz, PhD, Section on Environmental Gene Regulation, NICHD, Bethesda, MD
- Joseph Wade, PhD, Wadsworth Center, New York State Department of Health, Albany, NY
Contact
For more information, email philip.adams@nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/adams.
