The Lyme disease spirochete, Borrelia burgdorferi, utilizes a complex enzootic life cycle to exist in nature. The bacterium occupies both an arthropod tick vector and a vertebrate reservoir, and to persist in these two very distinct environments, the bacterium undergoes significant adaptive changes in global gene expression. This adaptive response is regulated by an enhancer-binding protein, Rrp2, which activates the RpoN/RpoS alternative sigma factor cascade. While the role of Rrp2 in activating the RpoN/RpoS pathway is very well-established, a recent study has also shown that Rrp2 also functions independent of RpoN/RpoS and that this branch of Rrp2 regulation is required for viability in B. burgdorferi. To date, the identities of the genes within this Rrp2-dependent, RpoN/RpoS-independent regulatory branch have not been determined. Experiments in Specific Aim 1 will identify genes in B. burgdorferi that are regulated by Rrp2. The complementary techniques of high-throughput sequencing of cDNA libraries (RNA-Seq) and in-gel digestion followed by liquid chromatography gel-tandem mass spectrometry (GeLC/MS-MS) will be used to define global changes in the B. burgdorferi transcriptome and proteome, respectively. Samples will be generated from in vitro grown cultures of an rrp2 conditional mutant, as well as a clone that can overexpress rrp2.
In Specific Aim 2, genes and/or proteins identified in the prior Aim will be targeted for mutagenesis and phenotypic analyses. Directed allelic exchange will be used to create mutants in wild type B. burgdorferi. Because this branch of Rrp2-dependent, RpoN/RpoS-independent gene regulation is required for bacterial growth, a conditional mutation approach might be required to generate one or more of these mutants. B. burgdorferi mutants will first be assessed for in vitro growth defects, but it is possible that one or more of these mutants might not exhibit a significant in vitro defect. To test the viability of these latter mutats within the mammal, we will characterize the B. burgdorferi mutants for their abilities to infect an elicit disease in mice. The knowledge gained from the Aims presented in this proposal will help further our understanding of the biology of B. burgdorferi. Defining the physiological contributions of these genes to bacterial viability and/or mammalian infectivity will be the focus of at least one future R01 proposal, with the ultimate intention of identifying potential bacterial targets against which new therapeutics could be developed to prevent or treat Lyme disease.

Public Health Relevance

In nature, the bacterium that causes Lyme disease, Borrelia burgdorferi, colonizes either an arthropod tick vector or a vertebrate reservoir host. The ability f the bacterium to adapt to these two very diverse environments is critical and this adaptive response is controlled by the Rrp2/RpoN/RpoS regulatory cascade. This project focuses on a previously unstudied branch of gene Rrp2-dependent gene regulation that is independent of RpoN/RpoS and absolutely essential for viability of B. burgdorferi. By identifying genes in this unique branch of Rrp2-dependent regulation and determining their contribution(s) to growth and/or mammalian infection, we can gain insight into the basic biology of B. burgdorferi, as well as identify potential bacterial targets against which novel therapeutics could be developed to prevent and/or treat Lyme disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI119532-01
Application #
8951367
Study Section
Special Emphasis Panel (ZRG1-IDM-B (80))
Program Officer
Perdue, Samuel S
Project Start
2015-06-15
Project End
2017-05-31
Budget Start
2015-06-15
Budget End
2016-05-31
Support Year
1
Fiscal Year
2015
Total Cost
$219,923
Indirect Cost
$69,923
Name
University of Arkansas for Medical Sciences
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
122452563
City
Little Rock
State
AR
Country
United States
Zip Code
72205
Latham, Jacob I; Blevins, Jon S (2018) Generation of Conditional Mutants in Borrelia burgdorferi. Methods Mol Biol 1690:225-239
Yin, Yanping; Yang, Youyun; Xiang, Xuwu et al. (2016) Insight into the Dual Functions of Bacterial Enhancer-Binding Protein Rrp2 of Borrelia burgdorferi. J Bacteriol 198:1543-52