T3SS-mediated injection of CapG nanobodies in MDA-MB-231 cells. (A) Schematic depicting injection of a VHH (nanobody) fused to a T3-secretion signal (T3s) and β-lactamase (Bla) reporter into a mammalian cell by the T3SS of enteropathogenic E. coli (EPEC) strains. The nonfluorescent Bla substrate (CCF2/AM) is transformed by cellular esterases to the green fluorescent substrate CCF2. CCF2 hydrolysis by Bla results in blue fluorescence. (B) Blue fluorescence is observed on infection with attenuated EPEC bacteria (quad) carrying T3s-Bla fusions of GFPNb (positive control), CAPNb2 and CAPNb4. Green fluorescence is observed in MDA-MB-231 cells infected with ∆escN strains (lacking the essential ATPase of the T3SS) or a Bla construct lacking the T3s signal sequence (pCX340). (C) Quantification of the fluorescence intensity ratio 447/520 nm of cultures of MDA-MB-231 cells infected with attenuated EPEC quad strains, or EscN-deficient strains, carrying pCX340 (negative control) and T3s-Bla fusions of GFPNb, CAPNb2, and CAPNb4. Results are expressed as mean ± SEM of three independent infection experiments for every bacterial strain. Data were analyzed by using one-way analysis of variance (ANOVA) and Bonferroni Multiple Comparison Test with the PRISM software (Graphpad) to calculate the P values of each data set with the control (quad pCX340). Asterisks (***) indicate P < 0.001.