The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes.
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 formation of a 25-A 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 protein secretion and hemolysis were kinetically coupled processes. However, Ipa protein secretion in the immediate vicinity of RBCs was not sufficient to cause hemolysis in the absence of centrifugation. Centrifugation reduced the distance between bacterial and RBC membranes beyond a critical threshold. Electron microscopy analysis indicated that secretons were constitutively assembled at 37 degrees C 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 activation of secretion. In mutants of some genes encoding the secretion machinery the organelle was absent, whereas ipaB and ipaC mutants displayed normal secretons.
Blocker
A
A
Unité de Pathogénie Microbienne Moléculaire INSERM U389, Institut Pasteur, 75724 Paris Cedex 15, France. ablocker@pasteur.fr
Gounon
P
P
Larquet
E
E
Niebuhr
K
K
Cabiaux
V
V
Parsot
C
C
Sansonetti
P
P
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001386:Azides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002498:Centrifugation; D003224:Congo Red / Q000494:pharmacology; D019286:Endopeptidase K / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism* / Q000382:microbiology / Q000648:ultrastructure; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism / Q000382:microbiology* / Q000648:ultrastructure; D005798:Genes, Bacterial; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D008854:Microscopy, Electron; D015394:Molecular Structure; D009154:Mutation; D009994:Osmolar Concentration; D012756:Sheep; D012763:Shigella flexneri / Q000737:chemistry / Q000378:metabolism* / Q000472:pathogenicity / Q000648:ultrastructure; D013696:Temperature
D000942:Antigens, Bacterial; D001386:Azides; D001426:Bacterial Proteins; C095770:IpaD protein, Shigella flexneri; C087447:ipaB protein, Shigella; C092342:IpaC protein, Shigella; D003224:Congo Red; D019286:Endopeptidase K