Fig 2. Identification of YhaO and YhaJ as potential virulence determinants.

(A) Screening of the yhaOMKJ locus for a role in virulence. SDS-PAGE profile of secreted proteins from TUV93-0, yhaO, yhaM, yhaK and yhaJ cultured in MEM-HEPES. Arrows indicate the location of the major LEE-encoded secreted effectors Tir, EspD and EspA as identified by mass-spectrometry. Samples were normalized according to cellular OD⁶⁰⁰ to normalize loading into each well. Immunoblot analysis of EspD levels from secreted (Sec) and whole cell lysate (WCL) fractions confirmed the SDS-PAGE results. Anti-GroEL was used to verify equal concentrations of WCL, which corresponded to OD⁶⁰⁰ normalized culture samples, loaded into each well (B) SDS-PAGE analysis highlighting complementation of the ΔyhaO and ΔyhaJ phenotypes by plasmids pyhaO and pyhaJ. SDS PAGE and immunoblot analysis of secreted protein profiles and EspD cytoplasmic expression confirmed the results. Protein secretion experiments were performed on multiple occasions.