Analysis of type III translocation signals of enteropathogenic and enterohemorrhagic Escherichia coli effectors using a new fluorescence-based reporter system

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are human and animal pathogens that inject a growing repertoire of effector molecules into the host cells via a type III secretion system (TTSS) encoded by the locus of enterocyte effacement (LEE). We have recently shown that Cif is the first phage-encoded effector molecule to be recognized by this TTSS. Upon injection into the host cell, Cif induces cell cycle arrest in G2/M and reorganization of the actin cytoskeleton. In this study, we analyzed the domain that targets Cif to the TTSS using a new reporter system. We compared the translocation in living cells of four effector molecules encoded or not by the LEE (Cif, Map, Tir and EspF). We confirmed the role of CesT as a chaperone for Tir and Map and the role of the first amino acids (aa) of Tir as a secretion and translocation signal (STS). We could then show that the first 16 aa of Cif were also sufficient and necessary to mediate translocation into HeLa cells. Similarly the first 20 aa of Map, EspF and Tir were also shown to be able to mediate secretion and translocation in a type III-dependent but CesT independent fashion. These different STS was then fused to a truncated Cif lacking its first 20 aa. The chimeric proteins were efficiently injected into HeLa cells via the TTSS and were able to trigger cell cycle arrest in G2/M and stress fibers formation. This result supports the idea that Cif is composed of a C-terminal effector domain and of an exchangeable N-terminal STS.