Figure 1. Secretion of sdAbs to the extracellular medium with T3SS of EPEC.

(A) Schematic representation of the T3SS-complex encoded by EPEC, labeling the essential ATPase EscN, the extracellular EspA filament and the secreted EspB and EspD translocators. The secretion of T3sNbs from the cytoplasm of the bacteria to the extracellular medium is indicated. (B) Coomassie staining of TCA-precipitated proteins found in the extracellular media of cultures of wt EPEC or ΔescN strains carrying plasmids pSA10, pT3sVamy, or pT3sVgfp, as indicated. Cultures were grown at 37°C in DMEM and induced with 0.1 mM IPTG for 4 h. The protein bands of EspA, EspB, EspD, and that of the Sec-dependent autotransporter EspC, are labeled. Size in kDa of protein standards for SDS-PAGE are shown on the right. (C) Western blot analysis of the proteins found in extracellular media (Culture supernantants; lanes 1–6) and cells (bacterial lysates; lanes 7–12) from the same cultures as in (B). WB developed with mAbs anti-E-tag (top panels) or anti-GroEL (bottom panels) to control the absence of cytoplasmic proteins in the extracellular media. (D) Binding activity of the secreted sdAbs. ELISA with His-tag purified T3sVgfp (left) and T3sVamy (right), at the indicated concentrations (nM), against their cognate antigens (GFP or Amy) and BSA (negative control). Bound T3sV_HHs developed with anti-E-tag mAb-POD and their Optical Density (O.D.) determined at 490 nm.