Functional analysis of the Shigella flexneri IpaC invasin by insertional mutagenesis

Abstract

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.

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

“…Whether there is a relationship between IpaC oligomerization and IpaC-mediated signals to host cells is unclear, but this certainly warrants continued investigation. Barzu et al (1997) used insertional mutagenesis to examine the functional organization of IpaC. Small peptide insertions into the hydrophobic region of IpaC resulted in reduced synthesis in S. flexneri and abolished contact-mediated haemolysis for the bacterium.…”

“…This work also shows that region I is needed for secretion of IpaC by S. flexneri. None of the insertions by Barzu et al (1997) led to a defect in IpaC secretion, presumably because they were either not located at the precise site needed to disrupt secretion or Fig. 8.…”

Galectin-3, a marker for vacuole lysis by invasive pathogens

“…The ipaC-and ipaB-mutants were described previously (Menard et al, 1993). IpaC351 was described previously (Barzu et al, 1997) and ipaC/pHI3 was described in Page et al (1999). L. monocytogenes was previously described in Mounier et al (1990).…”

“…To discriminate between these two possibilities, we wanted to uncouple invasion and vacuole lysis. To this end, we utilized an ipaC null mutant recomplemented by recombinant IpaC containing a fragment of 14 amino acids inserted between residues N351 and S352 (Barzu et al, 1997). This ipaC/IpaC351 strain was shown to be non-invasive in epithelial cells and yet retained the ability to lyse the vacuole when phagocytosed by macrophages (Barzu et al, 1997).…”

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

“…The extracellular IpaBC complex is apparently suf-®cient for entry, as latex beads coated with this complex are internalized by epithelial cells (Me  nard et al, 1996). IpaB and IpaC are also needed for escape from the phagosome when bacteria are internalized by macrophages (High et al, 1992;Zychlinsky et al, 1994) and for contact haemolytic activity (Me  nard et al, 1993;Barzu et al, 1997). Each of these proteins is also endowed with speci-®c functions: microinjection of IpaB into macrophages led to apoptosis (Chen et al, 1996), and microinjection of IpaC into epithelial cells led to the formation of ®lopodial structures .…”