The Tripartite Type III Secreton of Shigella flexneri Inserts IpaB and IpaC into Host Membranes Ariel Blocker,* Pierre Gounon,‡ Eric Larquet,‡ Kirsten Niebuhr,‡ Véronique Cabiaux,§ Claude Parsot,* and Philippe Sansonetti* *Unité de Pathogénie Microbienne Moléculaire INSERM U389 and ‡Station Centrale de Microscopie Electronique, Institut Pasteur, 75724 Paris Cedex 15, France; §Laboratoire de Chimie Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, B-1050 Brussels, Belgium Abstract. Bacterial type III secretion systems serve to translocate proteins into eukaryotic cells, requiring a secreton and a translocator for proteins to pass the bacterial and host membranes. We used the contact hemolytic activity of Shigella ﬂexneri to investigate its putative translocator. Hemolysis was caused by forma- tion of a 25-Å pore within the red blood cell (RBC) membrane. Of the ﬁve proteins secreted by Shigella upon activation of its type III secretion system, only the hydrophobic IpaB and IpaC were tightly associated with RBC membranes isolated after hemolysis. Ipa pro- tein secretion and hemolysis were kinetically coupled processes. However, Ipa protein secretion in the imme- diate vicinity of RBCs was not sufﬁcient to cause hemolysis in the absence of centrifugation. Centrifuga- S HIGELLA demic form flexneri of bacillary is the etiological dysentery. agent This gram-nega- of the en- tive bacterium causes disease by invading the co- lonic mucosa (Hale, 1998). All bacterial genes necessary for entry into host cells have been identified. They are clustered within a 30-kb region of a large virulence plas- mid (Parsot, 1994). This region carries two types of genes: the ipa and ipg genes encoding the entry-mediating pro- teins and their individual intrabacterial chaperones, and the mxi and spa genes which code for proteins forming a type III secretion apparatus (secreton) required for secre- tion of the invasins (IpaA-D and IpgD) Type III secretons are found in many other pathogenic gram-negative bacterial species that have developed di- verse survival strategies within their hosts. Several compo- nents of type III secretons share sequence similarities with The first two authors contributed equally to this work. Address correspondence to Ariel Blocker, Unité de Pathogénie Micro- bienne Moléculaire INSERM U389, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Tel.: 33-1-40-61-37-71. Fax: 33-1-45- 68-53-98. E-mail: ablocker@pasteur.fr tion reduced the distance between bacterial and RBC membranes beyond a critical threshold. Electron mi- croscopy analysis indicated that secretons were consti- tutively assembled at 378C before any host contact. They were composed of three parts: (a) an external needle, (b) a neck domain, and (c) a large proximal bulb. Secreton morphology did not change upon activa- tion of secretion. In mutants of some genes encoding the secretion machinery the organelle was absent, whereas ipaB and ipaC mutants displayed normal secretons. Key words: microbial pathogenesis • type III secre- tion • contact hemolysis • pore formation • membrane protein insertion components of flagellar basal bodies (Hueck, 1998). Ku- bori et al. (1998) recently identified and biochemically iso- lated the macromolecular structure formed by the Salmo- nella type III secreton. They described this organelle as a cylindrically symmetrical object composed of two parts: (a) a 7–8-nm-wide and 60-nm-long needle emanating from the bacterial surface and (b) a shorter cylinder, formed by a succession of plates (20–40 nm in diameter) presumed to traverse the inner and outer bacterial membranes and the peptidoglycan. A major function of the type III secretons is to transport, upon contact with host cells, proteins from the bacterial cytoplasm into the host cell cytoplasm or its limiting membrane (Cornelis and Wolf-Watz, 1997; Kenny et al., 1997; Galan and Collmer, 1999). In S. flexneri, the mxi and spa operons and the ipa and ipg operons are expressed at 378C, and Ipa proteins re- main in the bacterial cytoplasm until the secretion machin- ery is activated by host contact or external signals such as serum or the small amphipatic dye molecule Congo red (Ménard et al., 1994a; Bahrani et al., 1997). Physical con- tact of the bacterium with the host cell induces formation of a localized macropinocytic-like ruffle on the host cell surface that internalizes the bacterium (Clerc and San- ª The Rockefeller University Press, 0021-9525/99/11/683/11 $5.00 The Journal of Cell Biology, Volume 147, Number 3, November 1, 1999 683–693 http://www.jcb.org 683