Metabolic Control of Virulence Attributes of the Agent of Lyme Disease

Helm, Sarah Marie
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Lyme disease (LD) is the most common vector-borne illness in the U.S. with approximately 300,000 new cases reported to the CDC each year. The etiologic agent of Lyme disease, Borrelia burgdorferi (Bb), is a spirochetal pathogen that is transmitted to vertebrate hosts via the bite of an infected Ixodes spp. tick. Bb is an extreme auxotroph, acquiring several critical nutrients from its tick vector or vertebrate hosts. Bb does not require iron for growth and hence responds to oxidative stress in a unique manner. While the genome of Bb encodes for homologs of Superoxide dismutase A (SodA), Borrelia oxidative stress regulator (BosR), Neutrophil activating protein A, (Dpr/NapA), and Coenzyme A disulphide reductase (CoADR), there are no conventional peroxidases or catalases involved in mediating oxidative stress response in Lyme spirochetes. Moreover, there are two homologs with similarity to Thioredoxin (Trx) and Thioredoxin reductase (TrxB) that could potentially influence the redox status of Bb and provide an additional mechanism to help protect itself against endogenous and exogenous reactive oxygen species. In vitro growth of Bb was inhibited by Auranofin at a concentration of 4 µg/mL, an FDA-approved organogold drug shown to inhibit thiol-based redox metabolism in various Gram-positive and some drug-resistant strains of bacteria. Oral administration of Auranofin to C3H/HeN mice for 14 days following mouse model of infection resulted in spirochetal burden slightly higher than in untreated animals although this increase was not found to be statistically significant. In vivo and in vitro analysis of the efficacy of Auranofin’s bactericidal capacities will aid in our understanding of the importance of the thiol-based redox metabolism in B. burgdorferi.

Since Bb is a nutrient auxotroph, it either has to acquire nutrients from its host or recycle its cellular stores of proteins and other key bio-molecules. To further understand the patho-physiology of Bb, we also plan to determine the role of various hypothetical proteins (bb0381, bb0032, bb0555, and bb0556) in its host-specific adaptation. To define the role of these various genes, we cloned and expressed each as a recombinant protein and have generated specific antibodies. Analysis of the in vitro and in vivo phenotypes of mutants of Bb will help determine their role in the metabolic and virulence capabilities of the agent of Lyme disease. Lastly, Borrelia host adaptation Regulator (BadR) is a major regulator of gene expression and contributes to the lifecycle of Bb due to its ability to bind to and repress rpoS, a “gatekeeper” which regulates many factors required for transmission and vertebrate infection. Incorporation of 15N-ammonium chloride and 13C-glucose by E. coli would allow for structural studies of the recombinant BadR protein using nuclear magnetic resonance spectroscopy. Incorporation of 13C6 15N2 L-lysine HCl and 13C6 15N4 L-arginine HCl by Bb and mutant strains would allow for further characterization by mass spectrometry. Protocols were developed consistent with the physiology of Bb to prepare such samples for analysis.

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Auranofin, Borrelia, Burgdorferi, Lyme, Spirochete, Tick
Integrative Biology