Characterization of the interaction of IpaB and IpaD, proteins required for entry of Shigella flexneri into epithelial cells, with a lipid membrane.
Entry of Shigella flexneri into epithelial cells and lysis of the phagosome involve the IpaB, IpaC, and IpaD proteins, which are secreted by type III secretion machinery. We report here the purification of IpaB and IpaD and the characterization of their lipid-binding properties as a function of pH. The interaction of IpaB with the membrane was quite independent of the pH whereas that of IpaD took place only at low pH. To support the data obtained with the purified proteins, we designed a system in which protein secretion by live bacteria was induced in the presence of liposomes, thereby allowing interaction of proteins with lipids directly after secretion and bypassing any purification step. In these conditions, both IpaB and IpaC, as well as minor amounts of IpaA and IpgD, were associated with the membrane and the ratio of IpaB to IpaC was modulated by the pH. The relevance of these results with respect to the dual roles of IpaB, IpaC and IpaD in induction of membrane ruffles and lysis of the endosomal membrane is discussed.
De Geyter
C
C
Université Libre de Bruxelles, Laboratoire de Chimie Physique des Macromolécules aux Interfaces, Brussels, Belgium.
Wattiez
R
R
Sansonetti
P
P
Falmagne
P
P
Ruysschaert
J M
JM
Parsot
C
C
Cabiaux
V
V
D000942:Antigens, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050356:Lipid Metabolism; D008081:Liposomes / Q000378:metabolism; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D012763:Shigella flexneri / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D005452:Fluoresceins; C063664:IpaA protein, Shigella flexneri; C095770:IpaD protein, Shigella flexneri; D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; C087447:ipaB protein, Shigella; C092342:IpaC protein, Shigella; C024990:asolectin; C007740:fluorexon