With an estimated 300,000 cases occurring each year, Lyme disease (LD) is the most prevalent arthropod-borne infection in the United States. Borrelia burgdorferi (Bb), the LD spirochete, is maintained in nature via an enzootic cycle typically involving a rodent reservoir and Ixodes ticks. To transit between these two different host environments, Bb must alter its transcriptome, proteome, and metabolome in response to arthropod- and mammalian host- derived signals. Historically, efforts to understand the Bb enzootic cycle have focused on the spirochete?s differentially expressed surface molecules, usually lipoproteins, and the signal transduction pathways that regulate them. Our characterization of the Bb oligopeptide transporter (BbOpp) sets the stage for an in-depth exploration of ?nutritional virulence? as a central facet of Bb?s strategy for persistence in the natural world and its incidental human host. Bb is auxotrophic for all amino acids and possesses few annotated amino acid transporters. Yet, surprisingly little is known about the mechanisms whereby Bb obtains these essential protein building blocks. Although BbOpp has been studied extensively over the years, its importance for Bb viability and LD pathogenesis has not been established. Using a novel mutagenesis approach to precisely control expression of the ATPase that powers the system, we found that BbOpp is indeed essential for growth, viability and virulence. Unexpectedly, we also discovered that BbOpp plays a key role in regulating cell division and morphogenesis. Evidence to date suggests that the striking morphological abnormalities caused by peptide-starvation arise from interference with or inhibition of septum formation and cell separation. In many bacteria, amino acid starvation elicits a global reprogramming of the transcriptome, known as the stringent response, mediated by the effector nucleotide (?alarmone?) (p)ppGpp. However, analysis of (p)ppGpp levels indicates that the stringent response is not responsible for this unprecedented morphotype. Collectively, these results lead to the two-pronged central hypothesis of our proposal: (i) Bb must import peptides via the Opp system to maintain adequate intracellular amino acid pools for normal growth and (ii) progressive peptide deprivation elicits a Rel-independent growth inhibitory response that culminates during peptide starvation in dysregulation of the divisome. To address this hypothesis we will characterize the effects of peptide deprivation on intracellular amino acid pools (Specific Aim One); assess the effects of peptide nutrient stress on the Bb transcriptome and proteome (Specific Aim Two); and elucidate dysregulation of the Bb divisome caused by peptide starvation (Specific Aim Three).
Bb is unable to synthesize any amino acids and possesses few annotated transporters for single amino acids. Using a novel genetic approach to precisely control expression of the ATPase that powers its oligopeptide (Opp) transporter, we found that the Opp system, and, therefore, peptide uptake, are essential for growth, viability and virulence. Experiments outlined in our proposal will explore the two-pronged central hypothesis emanating from these findings: (i) Bb must import peptides via the Opp system to maintain adequate intracellular amino acid pools for normal growth and (ii) progressive peptide deprivation elicits a novel growth inhibitory response that culminates during peptide starvation in dysregulation of cell division.
