The Tripartite Type III Secreton of Shigella flexneri Inserts Ipab and Ipac into Host Membranes

doi:10.1083/jcb.147.3.683

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© The Rockefeller University Press, 0021-9525/1999//683 $5.00
The Journal of Cell Biology, Volume 147, Number 3, , 1999 683-693

Original Article

The Tripartite Type III Secreton of Shigella flexneri Inserts Ipab and Ipac into Host Membranes

Ariel Blocker^a, Pierre Gounon^b, Eric Larquet^b, Kirsten Niebuhr^b, Véronique Cabiaux^c, Claude Parsot^a, and Philippe Sansonetti^a

^a Unité de Pathogénie Microbienne Moléculaire INSERM U389, Institut Pasteur, 75724 Paris Cedex 15, France
^b Station Centrale de Microscopie Electronique, Institut Pasteur, 75724 Paris Cedex 15, France
^c Laboratoire de Chimie Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, B-1050 Brussels, Belgium
Unité de Pathogénie Microbienne Moléculaire INSERM U389, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France.33-1-45-68-53-9833-1-40-61-37-71

ablocker{at}pasteur.fr

Bacterial type III secretion systems serve to translocate proteinsinto eukaryotic cells, requiring a secreton and a translocatorfor proteins to pass the bacterial and host membranes. We usedthe contact hemolytic activity of Shigella flexneri to investigateits putative translocator. Hemolysis was caused by formationof a 25-Å pore within the red blood cell (RBC) membrane.Of the five proteins secreted by Shigella upon activation ofits type III secretion system, only the hydrophobic IpaB andIpaC were tightly associated with RBC membranes isolated afterhemolysis. Ipa protein secretion and hemolysis were kineticallycoupled processes. However, Ipa protein secretion in the immediatevicinity of RBCs was not sufficient to cause hemolysis in theabsence of centrifugation. Centrifugation reduced the distancebetween bacterial and RBC membranes beyond a critical threshold.Electron microscopy analysis indicated that secretons were constitutivelyassembled at 37°C before any host contact. They were composedof three parts: (a) an external needle, (b) a neck domain, and(c) a large proximal bulb. Secreton morphology did not changeupon activation of secretion. In mutants of some genes encodingthe secretion machinery the organelle was absent, whereas ipaBand ipaC mutants displayed normal secretons.

Key Words: microbial pathogenesis • type III secretion • contact hemolysis • pore formation • membrane protein insertion

1.used in this paper: LPS, lipopolysaccharide; PIC, proteaseinhibitor cocktail

The first two authors contributed equally to this work.

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