Role of sipA in the early stages of Salmonella typhimurium entry into epithelial cells.
Salmonella virulence depends on an ability to invade host cells, which is in turn dependent on a type III protein secretion system encoded in Salmonella pathogenicity island 1 (SPI1). Several protein targets of the SPI1-encoded secretion system are translocated into host cells, where they subvert cellular processes that contribute to bacterial invasion, actin rearrangement, membrane ruffling and other aspects of virulence. We examined the role of sipA (encoding the translocated protein SipA) and found that a sipA mutant was significantly less invasive in Madin-Darby canine kidney (MDCK) cells than in its parental strain at the earliest stages of infection (5 min). The invasion defect associated with sipA was no longer apparent after 15 min of infection. Confocal microscopy of F-actin in tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin-stained MDCK cells revealed no difference in either the frequency or the morphology of membrane ruffles induced by wild-type and sipA mutant strains of S. typhimurium. Time-lapse phase-contrast microscopy of membrane ruffle propagation in live cells confirmed that the sipA mutant induced membrane ruffles as efficiently as the wild-type bacteria. These studies also revealed that, after ruffle propagation, individual sipA mutant S. typhimurium either invaded more slowly than wild-type bacteria or failed to invade at all. Furthermore, although wild-type S. typhimurium typically maintained a position central to the developing membrane ruffle, sipA mutant bacteria frequently moved initially to the periphery of the spreading ruffle and were sometimes observed to detach from it. A wild-type pattern of invasion was restored to the sipA mutant after the introduction of sipA on a plasmid. Together, these data indicate that loss of sipA significantly decreases the efficiency of S. typhimurium invasion at the early stages of infection without affecting its ability to induce membrane ruffles. It thus appears that the secreted effector protein SipA promotes invasion by a previously unrecognized mechanism separate from the induction of membrane ruffling per se.
Jepson
M A
MA
Cell Imaging Facility and Department of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK. m.a.jepson@bristol.ac.uk
Kenny
B
B
Leard
A D
AD
D000199:Actins / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D001426:Bacterial Proteins; D002462:Cell Membrane / Q000648:ultrastructure; D004285:Dogs; D004847:Epithelial Cells / Q000382:microbiology*; D008840:Microfilament Proteins; D009154:Mutation; D012486:Salmonella typhimurium / Q000472:pathogenicity*
D000199:Actins; D001426:Bacterial Proteins; D008840:Microfilament Proteins; C118287:SipA protein, Salmonella