Evaluation of the cytotoxicity and antibacterial activity of a synthetic tunicamycin derivative against Mycobacterium avium complex

Authors: COLOMBATTI OLIVIERI, MARIA - Oak Ridge Institute For Science And Education (ORISE); PRICE, NEIL - Retired ARS Employee; Jackson, Michael; Bannantine, John

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/29/2025
Publication Date: 5/14/2025
Citation: Colombatti Olivieri, M.A., Price, N.P., Jackson, M.A., Bannantine, J.P. 2025. Evaluation of the cytotoxicity and antibacterial activity of a synthetic tunicamycin derivative against Mycobacterium avium complex. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2025.1604400.
DOI: https://doi.org/10.3389/fmicb.2025.1604400

Interpretive Summary: Mycobacterium avium subspecies paratuberculosis causes Johne's disease in cattle and sheep. This bacterium has a high resistance to most antibiotics. In this study, we tested the capability of two new synthetic drugs to inhibit the bacteria that causes Johne's disease. We also tested these compounds against other closely-related mycobacteria. Finally, we tested the cytotoxicity of these new drugs on eukaryotic cells to determine if they would harm the infected host. We discovered that the bacterial pathogen is quite resistant to these sythnthetic drugs such that doses toxic to the host would need to be used if treating to eliminate the bacteria. This information is important to scientists and veterinarians working in the field.

Technical Abstract: Two synthetic derivatives of the tunicamycin antibiotic, TunR1 and TunR2, were previously developed that significantly reduced toxicity in eukaryotes but remained potent against Gram positive prokaryotes. The average minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) for TunR2 ranged from 16 to 32 µg/mL when tested on seven Mycobacterium avium subspecies paratuberculosis (Map) strains. MICs were higher for the closely related Mycobacterium avium subspecies hominissuis (>32 µg/mL), and lower for M. marinum (0.025 µg/mL) and M. smegmatis (3.2 µg/mL). Effects on the Map cell wall could be detected by electron microscopy at TunR2 concentrations above 128 µg/mL. The toxicity of TunR2 in eukaryotes was evaluated in vitro by hemolysis of bovine red blood cells (RBCs) and by MTT viability assay on a bovine epithelial cell line, cultured bovine peripheral blood mononuclear cells (PBMCs), and bovine monocyte-derived macrophages (bMDMs). The concentrations of the drug that produce 50% of inhibition (IC50) in each of these three cell types was lower than the MIC for Map. Hemolytic activity was demonstrated in 91% of RBCs when exposed to 31 µg/mL of TunR2. Also, low-dose TunR2 treatment of infected macrophages did not significantly decrease Map survival after 48h of infection. These results suggest that TunR2 is not a good candidate to treat Map infections.