Borrelia burgdorferi, the Lyme disease spirochete, possesses genetically-encoded responses to doxycycline, but not to amoxicillin

Abstract

Some species of bacteria respond to antibiotic stresses by altering their transcription profiles, in order to produce proteins that provide protection against the antibiotic. Understanding these compensatory mechanisms allows for informed treatment strategies, and could lead to the development of improved therapeutics. To this end, studies were performed to determine whether Borrelia burgdorferi, the spirochetal agent of Lyme disease, also exhibits genetically-encoded responses to the commonly prescribed antibiotics doxycycline and amoxicillin. After culturing for 24 h in a sublethal concentration of doxycycline, there were significant increases in a substantial number of transcripts for proteins that are involved with translation. In contrast, incubation with a sublethal concentration of amoxicillin did not lead to significant changes in levels of any bacterial transcript. We conclude that B. burgdorferi has a mechanism(s) that detects translational inhibition by doxycycline, and increases production of mRNAs for proteins involved with translation machinery in an attempt to compensate for that stress.

Fig 1. Effects of antibiotics on B. burgdorferi replication rates.

(A) Doxycycline was added to freshly inoculated cultures at concentrations of 0.1 μg/mL, 0.2 μg/mL, and 0.4 μg/ml. (B) Amoxicillin was added to freshly inoculated cultures at concentrations 0.1 μg/mL, 0.2 μg/mL, and 0.4 μg/ml. Bacterial numbers were determined by microscopical examination with a Petroff-Hauser counting chamber after 1, 2, 3, 4 and 7 days of culture.

Fig 2. Doxycycline induced gene expression changes associated with protein translation.

(A) Fold change versus expression strength for all detectable genes after 3 or 24 hours doxycycline treatment compared to untreated controls. Red (increased) and blue (decreased) dots represent genes with significantly different levels in treated vs. control bacteria (α = 0.05, log₂(fold-change) > 1). Yellow dots represent significantly different expression (α = 0.05) without meeting our fold-change cutoff for differential expression (“sigNC”). Gray dots represent genes that were not significantly different between treatment and control bacteria (“NS”). Numbers of significantly upregulated (up) and downregulated (down) genes are shown as proportions of all detectable genes. (B) Clusters of Orthologous Genes (COG) pathways displayed as proportion of all detectable genes (“Total”) compared to differentially expressed genes after 3h or 24h of doxycycline treatment [47]. (C) Stacked bar graph showing the number of increased (red) and decreased (blue) genes in each COG pathway at 3h and 24h timepoints. Percentage of genes in each pathway that were differentially expressed is stated within each bar. Note: Unclassified and general function prediction not shown.

Fig 3. Amoxicillin did not induce gene expression changes.

Fold change versus expression strength for all detectable genes after 3 or 24 hours amoxicillin treatment compared to untreated controls. No genes were significantly different between treatment and control groups (α = 0.05), as indicated by gray dots (“NS”).

Fig 4. Photomicrographs of representative B. burgdorferi from (A) control, or (B, C, and D) amoxicillin-treated cultures after 24h incubation.

All fields are shown at the same relative magnification. Imaged with a 40x objective lens and darkfield illumination.