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Infect. Immun., May 1997, 1599-1605, Vol 65, No. 5
Copyright © 1997, American Society for Microbiology

Functional analysis of the Shigella flexneri IpaC invasin by insertional mutagenesis

S Barzu, Z Benjelloun-Touimi, A Phalipon, P Sansonetti and C Parsot
Unite de Pathogenie Microbienne Moleculaire, Unite U389 de l'Institut National de la Sante et de la Recherche Medicale, Institut Pasteur, Paris, France.

The ability of Shigella to enter epithelial cells, to escape from the phagocytic vacuole, and to induce apoptosis in macrophages requires the IpaB, IpaC, and IpaD proteins. An extracellular complex containing IpaB and IpaC can promote the uptake of inert particles by epithelial cells. To determine whether the function of IpaC is to act as an extracellular chaperone for IpaB in the Ipa complex or as an effector of entry involved in a direct interaction with the cell surface, we have constructed eight IpaC recombinant proteins by inserting the coding sequence for a 12- to 14-amino-acid fragment into restriction sites scattered within the ipaC gene. We have investigated the ability of recombinant proteins to bind IpgC in the bacterial cytoplasm and IpaB in the extracellular medium and to complement an ipaC null mutant for entry into HeLa cells, lysis of erythrocytes, and escape from the phagocytic vacuole in infected macrophages. Most recombinant proteins were produced and secreted at a level similar to that of wild-type IpaC and did not exhibit altered susceptibility to proteolysis by trypsin, and all were able to bind IpgC and IpaB. Some recombinant proteins did not complement the ipaC mutant for entry into HeLa cells, lysis of erythrocytes, or escape from the phagocytic vacuole, which indicates that IpaC plays an active role in these processes and does not act solely as a chaperone for IpaB. In addition, some insertions which were located outside of the hydrophobic region of IpaC differentially affected the abilities of Shigella to enter epithelial cells and to lyse cell membranes.

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