Fig. 3.

In vitro characterization of alanine racemase mutants. a, b Invasion assay of SEN ∆alr, SEN ∆dadX, SEN ∆3897, SEN ∆alr∆dadX, Alr complemented strain (pCH112-alr in SEN ∆alr∆dadX) and WT in HCT116 cell-lines. d-Alanine (100 µg/ml) was added in cell culture media 1 h before infection. c Confocal microscopy images of HCT116 cells infected with WT and SEN ∆alr∆dadX for 50 min at MOI 100, gentamicin treated for 2 h and fixed. Cells were stained for actin with Alexa Fluor 546 Phalloidin (Red) for 15 min at room temperature. Salmonella strains were GFP-tagged shown in green and actin in red. Scale bar: 10 μm. d Uptake Assay and e Survival Assay of SEN ∆alr, SEN ∆dadX, SEN ∆3897, SEN ∆alr∆dadX and WT in RAW264.7 in murine macrophages at 2 and 24 h time-points respectively. d-Alanine (100 µg/ml) was added in cell culture media 1 h before infection f SPI-1/SPI-2 gene expression of WT, SEN ∆alr and SEN ∆3897 through qRT-PCR analysis. All experiments were performed in triplicate with data represented as mean ± SD. RT room temperature. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001