The secreted IpaB and IpaC invasins and their cytoplasmic chaperone IpgC are required for intercellular dissemination of Shigella flexneri

Abstract

Invasion of epithelial cells by Shigella flexneri involves entry and dissemination. The main effectors of entry, IpaB and IpaC, are also required for contact haemolytic activity and escape from the phagosome in infected macrophages. These proteins are stored in the cytoplasm in association with the chaperone IpgC, before their secretion by a type III secretion apparatus is activated by host cells. We used a Histagged IpgC protein to purify IpgC-containing complexes and showed that only IpaB and IpaC are associated with IpgC. Plasmids expressing His6-IpgC either alone or together with IpaB or IpaC under the control of an IPTG-inducible lac promoter were introduced into ipgC, ipaB or ipaC mutants. Induction of expression of the recombinant plasmid-encoded proteins by IPTG allowed bacteria to enter epithelial cells, and the role of these proteins in dissemination was investigated by incubating infected cells in either the absence or the presence of IPTG. The size of plaques produced by recombinant strains on cell mono-layers was regulated by IPTG, indicating that IpgC, IpaB and IpaC were each required for efficient dissemination. Electron microscopy analysis of infected cells indicated that these proteins were necessary for lysis of the membrane of the protrusions during cell-to-cell spread.