As per the recent report from the U.S. Centers for Disease Control and Prevention, more than 500,00 Lyme cases are reported in the United States each year. This accounts for a heavy economic burden on their healthcare system. Lyme disease or Lyme borreliosis is an infectious condition associated with the bite of an Ixodes genus black-legged tick, caused mainly by the spirochete bacterium Borrelia burgdorferi in the United States and by other bacteria, with few exceptions. The predominant cause in Europe and Asia of Lyme disease is B. burgdorferi.
Patients with Lyme disease are generally reported in the Northeastern and Upper Midwestern US regions. Connecticut, Delaware, Maine Maryland, and other locations are the primary states with endemic Lyme disease. Early localized phases of Lyme disease could include erythema, migrans rash, and low-grade fever. It usually occurs within 1 to 28 days after the tick bite. Between 7 and 14 days after a tick bite, 70% of patients develop the erythema migrans rash, typically at the site of the bite. The rash is usually uniform and develops at the site of the tick bite (it may burn, itch, or be asymptomatic at the tick bite area). This rash will spread over a few days, and concentric rings may be seen. Leaving the rash untreated can go on for 2 to 3 weeks and can extend up to 20 cm or larger.
Some patients have recurrent episodes of rash in about 20% of cases, and multiple lesions are not infrequent. But flu-like symptoms may be present at the same time. Broad-spectrum antibiotics are standard treatment for Lyme disease and may be given for long intervals, with immense ramifications for the patient.Â
Lyme disease progresses in three stages, which include localized early disease, late disease followed by disseminated early disease. The first symptoms are often such like erythema migrans, a characteristic ‘bull’s eye’ rash, fever, and tiredness. If untreated, the infection continues to progress, affecting several systems, and can result in neurological symptoms, cardiac complications, or arthritis. Chronic arthritis is common at the late stage, and encephalopathy may also be present; thus, early diagnosis and treatment are important.
The causative agent of Lyme disease was discovered in the early 1980s. Since then, Lyme disease has become the leading vector-borne illness in America. Global climate change has further expanded the distribution of arthropod vectors, driving geographic spread. B. burgdorferi is a spirochete with a sidearm and a reduced genome characterized by uniform fragmentation. Its genome consists of a single linear chromosome and approximately 20 linear and circular plasmids. To date, B. burgdorferi is the only known bacterium that depends on peptide uptake for basic viability.
While antibiotic courses do not distinctively target themselves to treat B. burgdorferi, patients suffer the loss of their microbiome, as well as off-target effects following therapy. Additionally, from a subset of patients who receive treatment, symptoms persist long after treatment, resulting in debilitation and PTLDS. What remains unknown is exactly what drives the continued symptoms in PTLDS. Possibilities include persistent bacteria, residual immunoreactive protein, autoimmune responses, protracted immune dysregulation, or off-target effects from antibiotic treatments lasting a long time.
The current study used Escherichia coli strains grown in Luria–Bertani (LB) broth or on LB plates containing appropriate antibiotics at 37°C. The first step to developing a high throughput screening tool for identifying inhibitors was to develop a binding assay that would readily identify OppA binding. This study used all strains of B. burgdorferi grown in a modified Barbour–Stoenner–Kelly–II (BSK–II) medium.Â
To serve as a pilot screen, 10 mM stock solutions of 2,240 compounds from the compound library in DMSO were screened using TSA against 50 μM of each compound in 384-well plates, employing reduced concentrations of OppA2 and the RFA assay. Z′-factors generated from all screened plates ranged from 0.58 to 0.78, indicating strong statistical confidence. The hit threshold for each plate was defined as values exceeding twice the standard deviation of the OppA2 reference control, a widely accepted standard for TSA screens.
To investigate a possible mechanism of inhibition, the research team also tested the effect of the inhibitor in a strain of B. burgdorferi carrying a functional insertional mutation in oppA2 (oppA2tn) to determine whether loss of OppA2 attenuates its effects. The inhibitor at 150 ÎĽM or vehicle control was applied, and the experiments were repeated to confirm the results.
In a pilot screen, 2,240 compounds from the 10 mM compound library stock solutions in DMSO were tested against a 50 ÎĽM concentration using the OppA2 and RFA assays at optimized conditions. Statistical confidence was high, with Z′ factors ranging from 0.58 to 0.78 across all screened plates. The ‘hitters’ were defined as values exceeding twice the standard deviation of the OppA2 reference control, adhering to best practices for TSA screens. Overall, the team established a drug discovery pipeline targeting previously unexplored and unique bacterial mechanisms.
However, cultivating B. burgdorferi in BSK-II at 37°C does not accurately replicate the mammalian host environment, as RpoS expression is only observed under high-density growth, and c-di-GMP accumulation occurs during cultivation. OppA inhibition is likely most effective during the metabolically active stage, such as host engagement (OppA1–4 active) or proliferation within the mammal (primarily OppA2 and OppA5).
For a comprehensive overview of Lyme disease symptoms, treatment options, and preventive measures, visit our detailed Lyme Disease page.
Reference: Skar GL, Blum MA, Simonsen KA. Lyme disease. eBook. StatPearls Publishing; 2024. Accessed from https://www.ncbi.nlm.nih.gov/books/NBK431066/
Holly KJ, Kataria A, Flaherty DP, Groshong AM. Unguarded liabilities: Borrelia burgdorferi’s complex amino acid dependence exposes unique avenues of inhibition. Front Antibiot. 2024;3:1395425. doi:10.3389/frabi.2024.1395425
