Salmonella Typhimurium: Infection Protocol

All strains used in this study had a Salmonella enterica serovar Typhimurium SL1344 background (SB300; streptomycin resistant) (74 (link)). Besides the wild type (Salmonella Typhimurium WT), the previously described ΔinvG (64 (link)) and ΔsipA ΔsopBEE2 (referred to here as Salmonella Typhimurium Δ4) mutants (66 (link)) were used. The ΔmotA mutant was generated via transfer of a previously described deletion (75 (link)) from a Salmonella Typhimurium 14028 strain (C1172) to the SL1344 background by P22 transduction. Chloramphenicol-resistant, isogenically tagged Salmonella Typhimurium WT (tags A to C) and Salmonella Typhimurium ΔinvG (tags D to F) strains were used in an earlier study (65 (link)). The pFPV-mCherry (rpsM-mCherry; Addgene plasmid number 20956) (60 (link)), pM975 (pssaG-GFPmut2) (15 (link), 22 (link)), and pZ1400 (puhpT-GFP) (18 (link)) reporter plasmids were previously used and validated. For infections, Salmonella Typhimurium cultures were grown overnight for 12 h in LB–0.3 M NaCl (Sigma-Aldrich) with appropriate antibiotics, followed by subculturing in the same medium without antibiotics at a 1:20 dilution for 4 h. Prior to microinjection, the inoculum was reconstituted in antibiotic-free complete human or mouse IntestiCult medium (StemCell) at a concentration of 5 × 10⁸ to 1 × 10⁹ CFU/ml.