Protein-protein interactions in the assembly of Shigella flexneri invasion plasmid antigens IpaB and IpaC into...

Shigella flexneri is a facultative intracellular bacterial pathogen that invades human colonic epithelial cells by a process called pathogen-induced phagocytosis. Pathogen entry requires three virulence plasmid-encoded proteins called invasion plasmid antigens (Ipa) B, C and D which are secreted upon bacterial contact with a host cell. Following their secretion, IpaB and IpaC are found within a complex of proteins that may also contain IpaA and IpaD. Previous work has shown that exogenously added recombinant IpaC is sufficient for promoting the uptake of S. flexneri in gentamicin-protection assays. It is shown here that purified recombinant Ipa proteins can also be used to investigate the formation of Ipa protein complexes in vitro. The protein-protein contacts involved in the formation of Ipa complexes appear to include previously undescribed IpaC-IpaC interactions in addition to a strong association between IpaB and IpaC. IpaD does not appear to interact with either IpaB or IpaC in vitro although it is possible that recombinant IpaD forms homodimers that are stabilized by disulfide bridges involving this protein's single cysteine residue. This investigation represents the first characterization of the biochemistry of Ipa complex assembly.