Identification of functional regions within invasion plasmid antigen C (IpaC) of Shigella flexneri

Shigella flexneri causes bacillary dysentery with symptoms resulting from t he inflammation that accompanies bacterial entry into the cells of the colo nic epithelium. The effectors of S. flexneri invasion are the Ipa proteins, particularly IpaB and IpaC, which are secreted at the host-pathogen interf ace following bacterial contact with a host cell. Of the purified Ipa prote ins, only IpaC has been shown to possess quantifiable in vitro activities t hat are related to cellular invasion. In this study, ipaC deletion mutants were generated to identify functional regions within the IpaC protein. From these data, we now know that the N-terminus and an immunogenic central reg ion are not required for IpaC-dependent enhancement of cellular invasion by S. flexneri. However, to restore invasiveness to an ipaC null mutant of S. flexneri, the N-terminus is essential, because IpaC mutants lacking the N- terminus are not secreted by the bacterium. Deletion of the central hydroph obic region eliminates IpaC's ability to interact with phospholipid membran es, and fusion of this region to a modified form of green fluorescent prote in converts it into an efficient membrane-associating protein. Meanwhile, d eletion of the C-terminus eliminates the mutant protein's ability to establ ish protein-protein contacts with full-length IpaC. Interestingly, the muta nt form of ipaC that restores partial invasiveness to the S. flexneri ipaC null mutant also restores full contact-mediated haemolysis activity to this bacterium. These data support a model in which IpaC possesses a distinct f unctional organization that is important for bacterial invasion. This infor mation will be important in defining the precise role of IpaC in S. flexner i pathogenesis and in exploring the potential effects of purified IpaC at m ucosal surfaces.