(A) Chip-on-chip analysis of the genomic region surrounding srcA including the 5′ upstream region. In vivo binding of SsrB was positive for 10 syntenic probes in the intergenic region (IGR) upstream of srcA. Each data point is a unique probe with averaged data from three biological replicates. Direction of gene transcription is shown by arrows beneath the abscissa and includes the fold-change in mRNA levels in ssrB mutant cells. (B) Accumulation of SrcA protein in cells requires SsrB. Cells harboring an srcA-HA allele were grown under SsrB-activating conditions and whole cell lysates (pellet) were probed by western blot for SrcA-HA, which accumulated in wild type cells (wt) but not in ssrB mutant cells (ΔssrB). (C) SrcA is not secreted into the culture supernatant. Cell-free secreted protein fractions from wt and ΔsrcA cultures were probed for SrcA-HA. SrcA-HA was not found in the secreted protein fraction from any of the cultures, whereas the type III-secreted protein, SseC, was secreted into the medium in an SsrB-dependent manner. (D) SrcA is required for competitive fitness in vivo. The ability of srcA mutant cells to compete with wild type parent cells was quantified by competitive infections of mice. Mice were infected orally with an equal proportion of mutant and wild type cells and the competitive index in the spleen and liver was determined at three days after infection. Complementation of srcA in trans (psrcA) restored the ability of srcA mutants to compete equally with wild type cells. Each data point represents an individual animal.

(A) Structure-based sequence alignment of full-length SrcA with structural orthologs CesT from enteropathogenic E. coli and SicP from S. Typhimurium. Orange arrowheads denote residues conserved between all three proteins; black arrowheads denote residues that participate in the cargo-binding interface as defined in (PDB 1JY0). Conserved residues are colored as follows: hydrophobic, yellow; negative charge, red; positive charge, blue; threonine, serine, pink; cysteine, light blue; proline, glycine, green. (B) Stereo image of a SrcA dimer with α-helices and β-strands in blue and red respectively.

(A) Crystal structures of SrcA, CesT, and SicP dimers highlighting the dimer interface. The two monomers of each chaperone dimer are colored in light and dark colors (green, SrcA; yellow, CesT; purple, SicP; orange, Spa15). (B) Space-filling model of SrcA bound to the SicP effector, SptP, based on a structural alignment. Negatively charged patches are red, positively charged patches are blue, and hydrophobic patches are white. Black arrows indicate the N-terminus.

Gel filtration chromatography was used to analyze (A) purified SrcA, (B) purified SsaN, and (C) a mixture of SrcA with SsaN. (D) Peak fractions (black arrows) in the elution volumes were collected and analyzed by western blot with an anti-His antibody to identify protein compositions. Pre-column samples of SrcA migrated as monomer and dimer species in SDS-PAGE gels (verified by LC-MS/MS) while SsaN was prominently a monomer. Incubation of SsaN with SrcA chaperone induced the formation of a high-molecular weight oligomeric species comprised of both proteins, which was only present in SsaN-SrcA mixtures.

(A) SILAC was used in conjunction with quantitative mass spectrometry-based proteomics for systematic identification of effector cargos bound by SrcA in vivo. (B) Mass:charge spectra for representative peptides (sequences shown) identified from SseL and OmpF, where normal isotope abundance-labeled peptides are indicated with open triangles and ²H₄-Lys or ¹³C₆-Arg labeled peptides are indicated by filled triangles. Protein abundance of SseL and PipB2 was examined by western blot in wild type and ΔsrcA mutant cells from secreted (C) and cytosolic protein fractions (D). Data is representative of 4 to 6 experiments.

(A) Intracellular positioning of SCVs containing wild type Salmonella or ΔpipB2 and ΔsrcA mutants after 10 and 24 h post-infection. HeLa cells were immunostained for bacteria (red) and LAMP1 (green), and the nucleus stained using DAPI (blue). Arrowheads indicate SCVs. Bar represents 10 µm. (B) HeLa cells were infected with wild type or ΔpipB2 and ΔsrcA mutants of S. Typhimurium. Cells were fixed at the indicated times, immunostained and analyzed as described in . Means with average deviation for two separate experiments are shown.