Elucidation of the role of
PtdIns(4,5)
P(2) in epithelial function has been hampered by the inability to selectively manipulate the cellular content of this
phosphoinositide. Here we report that SigD, a
phosphatase derived from Salmonella, can effectively hydrolyze
PtdIns(4,5)
P(2), generating
PtdIns(5)P. When expressed by microinjecting
cDNA into epithelial cells forming confluent monolayers, wild-type SigD induced striking morphological and functional changes that were not mimicked by a
phosphatase-deficient SigD mutant (C462S). Depletion of
PtdIns(4,5)
P(2) in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of
phospholipase Cdelta, used as a probe for the
phosphoinositide by conjugation to
green fluorescent protein. Single-cell measurements of cytosolic pH indicated that the Na(+)/H(+) exchange activity of epithelia was markedly inhibited by depletion of
PtdIns(4,5)
P(2). Similarly,
anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of
PtdIns(4,5)
P(2) was associated with marked alterations in the actin cytoskeleton and its association with the plasma membrane. The junctional complexes surrounding the injected cells gradually opened and the PtdIns(4,5)P(2)-depleted cells eventually detached from the monolayer, which underwent rapid restitution. Similar observations were made in intestinal and renal epithelial cultures. In addition to its effects on
phosphoinositides, SigD has been shown to convert
inositol 1,3,4,5,6-pentakisphosphate (IP(5)) into
inositol 1,4,5,6-tetrakisphosphate (IP(4)), and the latter has been postulated to mediate the
diarrhea caused by Salmonella. However, the effects of SigD on epithelial cells were not mimicked by microinjection of IP(4). In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing
PtdIns(4,5)
P(2) with a membrane-targeted 5-phosphatase or by occluding the inositide using high-avidity tandem PH domain constructs. We therefore suggest that opening of the tight junctions and inhibition of Na(+)/H(+) exchange caused by
PtdIns(4,5)
P(2) hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced
diarrhea.