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These proteins interfere with host cell functions, being main virulence factors of Gram-negative pathogens. Analyses of the process of translocation, the subcellular distribution, and the dynamics of effector proteins in host cells have been hampered by the lack of suitable tags and detection systems. Here we describe the use of self-labeling enzyme tags for generation of fusions with effector proteins that are translocated and functional in host cell manipulation. Self-labeling reactions with cell-permeable ligand dyes are possible prior to or after translocation. We applied the new approach to superresolution microscopy for effector protein translocation. For the first time, we show the dynamic properties of effector proteins in living host cells after translocation by intracellular bacteria. 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