Figure 2. Identification of EsxD as an interaction partner of EsxB.

(A) Mass spectrometry analysis of sample enriched for a protein of 11442.848 mass that co-purifies with EsxB in S. aureus USA300. (B) EsxB co-purifies with EsxD in S. aureus USA300. Wild type USA300 cultures carrying a plasmid expressing histidine tagged esxD (p_hisesxD) or control vector were spun and secreted proteins purified over Ni-NTA agarose. Bound proteins were eluted with imidazole, separated on SDS-PAGE and detected by immunoblotting with specific antibodies (α-EsxA, α-EsxB, α-EsxC, α-EsxD). (C) EsxD interacts with EsxB in vitro but not with EsxA or EsxC. Purified GST-EsxA, GST-EsxB and GST-EsxC were loaded on glutathione sepharose and incubated with purified His-EsxD. U and B denote unbound proteins in the flow through and bound proteins interacting with GST hybrids, respectively. Samples were separated by SDS-PAGE and stained with coomassie. (D) Protein interactions examined with the bacterial two-hybrid system. Bacterial cultures were spotted on solid medium containing Xgal, and the blue color indicates a positive interaction as seen for the control (Ctl, middle row left). Interactions were tested for the following pairs: (1) top row, EsxA:EsxA (A:A), EsxA:EsxB (A:B) and EsxB:EsxB (B:B); (2) middle row: positive control (Ctl), EsxD:EsxB (D:B), EsxD:EsxD (D:D), EsxD:EsxA (D:A); (3) lower row: EsxC:EsxA (C:A), EsxC:EsxC (C:C), EsxC:EsxB (C:B), EsxC:EsxD (C:D).