Protein-protein and protein-membrane interactions of IpaC from Shigella flexneri

Shigella flexneri is a causative agent of shigellosis, a bacillary dysentery characterized by high fever, abdominal cramping and blood and mucous in the stool. During the disease process Shigella invades mucosal epithelial cells in the colon by using its effector proteins, the I&barbelow;nvasion p&barbelow;lasmid a&barbelow;ntigens (Ipa), which are delivered via the Type Three Secretion System. IpaC is responsible for subverting normal host cell signaling pathways to trigger Shigella uptake, thus it is essential for Shigella pathogenesis. This work is composed of three separate investigations. In the first investigation, a series of N-terminal deletion mutants were generated to define the boundaries of an IpaC functional region. Binding studies were then used to show that IpaB and IpgC bind specifically to this region. This work suggests that that IpaC contains a distinct region downstream of it N-terminal secretion signals that is required for the IpaC-IpgC intracellular and IpaC-IpaB extracellular interaction and probable IpaC translocation to epithelial cells. In vitro IpaC is able to disrupt fluorescein-containing liposomes and penetrate Langmuir phospholipid membranes. For the second investigation, to better understand membrane interactions along different regions of IpaC, amino acids at positions 40, 106, 136, 154, 314, 326, 336, 341, and 362 were individually replaced with Trp residues. Using functional assays and fluorescence techniques, characterization of the biological properties of IpaC were done. In addition to examining the properties of IpaC as an individual protein, the third investigation also observed IpaC in complex with its chaperone IpgC and with its translocon partner IpaB. This work suggests that the dissociation of the IpaC/IpgC complex can be triggered by a pH dependent mechanism. Using phospholipid membrane interaction techniques, this work demonstrates that IpgC can prevent IpaC interaction with membranes and that the IpaB/IpaC complex appears to have a distinct interaction with membrane that is different from individual IpaB or IpaC interactions. Overall, this work proposes a model that suggests a sequence of events for IpaB and IpaC interaction with the host cell membrane.