Fig 7. Genetic and chemical induction of the methylfolate trap during Salmonella infection of macrophages.

(A) Survival of Salmonella strains in macrophages treated with SULFAs. Macrophages J774A.1 were infected for 1 h followed by 18 h chase, during which cells were untreated or treated with 1 mg/ml SMZ. Colony forming units (c.f.u.) were determined by serial dilution and plating method. (B) Cellular uptake and conversion of exogenous B₁₂ in mammalian cells requires transcobalamin (TC) and CblC proteins, respectively. Antivitamin B₁₂ molecules such as EtPhCbl inhibit transcobalamin and CblC, thereby restricting B₁₂ bioavailability to intracellular bacteria. (C) Depletion of CblC expression, detected by Western Blot using a specific antibody (top), caused B₁₂ starvation (middle) and increased SULFA sensitivity (bottom) of intracellular Salmonella. siRNA transfected THP-1 macrophages were infected with S. typhimurium cells expressing β-galactosidase from a B₁₂ starvation-responsive promoter for 1 h, followed by 18 h chase, during which the infected macrophages were treated without or with 1 mg/ml SMZ. B₁₂ starvation was estimated by determining β-galactosidase activity while Salmonella survival measured by c.f.u. counting. (D) Chemical restriction of B₁₂ sensitizes intracellular S. typhimurium to SULFA treatment. Macrophages J774A.1 were infected with S. typhimurium cells harboring a B₁₂ molecular probe for 1 h followed by 18 h chase, during which cells were untreated or treated with 1 mg/ml SMZ or/and 50 nM EtPhCbl. B₁₂ starvation was estimated through measuring enzymatic activity (top) and expression of β-galactosidase by Western Blot (middle). Salmonella survival from the corresponding macrophages was measured through c.f.u. counting (bottom). Error bars represent standard deviations from biological triplicates. ns, no significant difference compared to control groups.