Carlson College of Veterinary Medicine

Mycobacterium avium (MAC) within macrophages undergoes a phenotype change that allows for more efficient entry into surrounding host cells. We hypothesized that, by developing an in vitro system resembling the intra-vacuolar environment, one could generate insights into the mycobacterial intracellular phenotype. MAC was incubated in "elemental mixtures" that reproduce metal concentrations and pH in the vacuoles at different time points and then used to infect fresh macrophages. Incubation of MAC with mixture correspondent to the vacuole environment 24 h post-infection, infected macrophages at a significantly higher rate than bacteria that were incubated in 7H9 broth. Uptake occurred by macropinocytosis, similarly to the uptake of bacteria passed through macrophages. Genes reported to be upregulated in intracellular bacteria, such as Mav1365, Mav2409, Mav4487, and Mav0996, were upregulated in MAC incubated in the 24-h elemental. Like MAC obtained from macrophages, the vacuoles of bacteria from the 24-h elemental mixture were more likely to contain LAMP-1. A step-wise reduction scheme of the 24-h elemental mixture indicated that incubation in physiologically relevant concentrations of potassium chloride, calcium chloride, and manganese chloride was sufficient to induce characteristics of the intracellular phenotype. It was demonstrated that bacteria harboring the intracellular phenotype induced early-onset macrophage cell death more efficiently than broth grown bacteria. This new trace elemental mixture mimicking the condition of the vacuole at different time points has the potential to become an effective laboratory tool for the study of the MAC and M. tuberculosis disease process, increasing the understanding of the interaction with macrophages.