A selective antibiotic for Lyme disease

Abstract

Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment.

Keywords: B. burgdorferi; Lyme disease; Spirochetes; antibiotic; microbiome; transport.

Figure 1.. A screen for a selective anti-Borreliella compound.

A. Actinomycete strains were fermented in liquid culture. Concentrated culture supernatants were screened against B. burgdorferi Bb1286 expressing GFP, and against S. aureus HG003. Extracts showing specific activity against B. burgdorferi were fractionated by HPLC, and their activity was determined by bioassay. After LCMS and NMR analysis, the active compound from one of the producers was determined to be hygromycin A. B. MIC of hygromycin A against spirochaetes, human pathogens and commensals. Phylogenetic tree shows similarities between strains.

Figure 2.. The mechanism of action of Hygromycin A.

A. Measurement of protein synthesis inhibition using the GFP inducible (5 μg/ml anhydrotetraycycline, ATC) B. burgdorferi strain pCRW53 and flow cytometry. The median fluorescent intensity of 10⁵ analyzed cells is shown. Insert shows a histogram of FITC-A (GFP) distribution. B. Overview of the hygromycin A binding site in the T. thermophilus 70S ribosome viewed as a cross- cut section through the ribosome. Insert shows close-up view of hygromycin A bound in the PTC. The E. coli nucleotide numbering is used throughout. H-bond interactions are indicated with dashed lines. Note that by forming an H-bond with the base of nucleotide A2062 of the 23S rRNA (light blue), hygromycin A causes characteristic rotation of this nucleotide by approximately 160 degrees to form Hoogsteen base-pair with the m²A2503 of the 23S rRNA (red dashed arrow). The unrotated conformation of A2062 observed in the absence of the drug is shown in blue (PDB entry 4Y4P) (Polikanov et al., 2015). C. Schematic representation of the interactions of hygromycin A with the ribosome shown in panel B. Potential H-bond interactions are indicated with dashed lines, stacking interactions are shown with red arrows (upper panel). Secondary structure of the peptidyl transferase ring of the domain V of the 23S rRNA from Gram-negative bacterium Thermus thermophilus (lower panel). Nucleotides involved in HygA coordination are highlighted in blue and are conservative between T. thermophilus, E. coli, and B. burgdorferi. D. Toe-printing analysis of the HygA-induced translation arrest at the start codons of the ORFs. Cartoon representation of the toe-printing experiment illustrated with the ermBL gene (upper panel). Binding of hygromycin A (HygA) to the ribosomal A site arrests translation at the start codon of the gene.

Figure 3.. The basis for hygromycin A selectivity.

A. B. burgdorferi and E. coli cells were incubated at indicated concentrations of hygromycin A for 1 min or 40 min and intracellular accumulation was quantified by UHPLC/MS. Mean ± SD, N = 2. Linear regression is shown in dashed lines. Slopes are significantly different between B. burgdorferi and E. coli (p< 0.0001). B. RNAseq was performed on the B. burgdorferi hygromycin A resistant mutant in the presence (4μg/ml) or absence of hygromycin A. The volcano plot shows differential expression of genes in the presence of Hygromycin A relative to no drug control. Blue dots indicate statistically significant lower abundance, red dots indicate statistically significant higher abundance and black dots indicate that abundance is not significantly different. C. B. burgdorferi and E. coli cells were incubated at indicated concentrations of hygromycin A for 1 min and intracellular accumulation was quantified by UHPLC/MS. Mean ± SD, N = 2. (****=p< 0.0001, ***=p< 0.001, *=p< 0.05) D. Hygromycin A MIC fold-change of B. burgdorferi overexpressing bmpD as compared to B. burgdorferi wild-type (upper panel), of E. coli dTolC overexpressing bmpD as compared to E. coli dTolC under repressing (M9 + Glucose), inducing (M9 + Arabinose) and regular LB growth conditions (middle panel) and with addition of adenosine (A, lower panel).

Figure 4:. Animal efficacy of hygromycin A.

A. Quantification of B. burgdorferi in tissues (ear, muscle) of animals that ingested baits. The amount of 16S rRNA was converted into cell count as described in Materials and Methods. Infected mice were given hygromycin A bait (HygA) or doxycycline bait (Doxy); infected non-treated group (ND) and non-infected group (NI) were given drug-free bait for 5 days. B. Photograph of a C3H mouse eating a bait. C. The change in alpha diversity based on the Simpson index of the murine fecal microbiome from before to after treatment with hygromycin A (HygA) (per os), amoxicillin (Amox) (per os), or ceftriaxone (Cef) (subcutaneous). Mice were infected with B. burgdorferi N40 and treated twice a day for 5 days or were untreated (ND). Stool was collected before and after treatment and sequenced for the 16S rRNA gene and the alpha diversity was calculated using the Simpson Index metric. Each point represents the change in the Simpson index from before to after treatment for an individual mouse across three individual experiments. Bars represent the mean. Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparisons test (**=p<0.01, ***=p<0.001). D. The change in relative abundance (%) of the most abundant genera in the murine gut microbiome from before to after treatment with hygromycin A (per os), amoxicillin (per os), or ceftriaxone (subcutaneous). Mice were infected with B. burgdorferi N40 and treated twice a day for 5 days or were untreated. Stool was collected before and after treatment and was sequenced for the 16S rRNA gene. Each point represents the change in the relative abundance of the respective genus for an individual mouse across three individual experiments. Bars represent the mean.