nisms by which this organism causes disease remain 
undefined. >X'lien EPEC adheres to epithelial cells, it 
causes the loss of microvilli. Accompanying this loss 
is the accumulation of host actin beneath the adher- 
ent organism, forming a pedestal upon which the 
organism rests. Several other epithelial cytoskeletal 
proteins were identified that also accumulate 
beneath this organism. These include the actin- 
crosslinking protein a-actinin and the two proteins 
talin and ezrin, which are involved in linking the 
cytoskeleton to membrane receptors. However, the 
cellular distribution of tropomyosin, tubulin, vin- 
culin, and the intermediate filament proteins kera- 
tin and vimentin are not affected by EPEC. 
Salmonella typhimurium is another diarrhea- 
causing organism that rearranges host cytoskeletal 
proteins. However, the rearrangements caused by 
the two organisms appear different. Although both 
alter actin, the actin beneath EPEC is tightly local- 
ized and outlines the adherent organism. In con- 
trast, actin rearrangement triggered by S. typhimur- 
ium appears stringier, but still surrounds the 
invading organism. 5. typhimurium triggers rear- 
rangement of a-actinin, talin, and ezrin. In contrast 
to EPEC, S. typhimurium also causes accumulation 
of tubulin and tropomyosin. It appears that the 
EPEC-induced rearrangements are stable and form a 
structure on which the adherent organism remains, 
while S. typhimurium transiently induces changes 
that are associated with actin-myosin-mediated up- 
take of the organism into the epithelial cell. 
Given EPEC's role in diarrhea, the effect of EPEC 
on the impermeability of polarized epithelial cells 
was also examined. EPEC causes a significant de- 
crease in transepithelial electrical resistance of po- 
larized Madin-Darby canine kidney (MDCK) and hu- 
man intestinal Caco-2 epithelial monolayers. This 
decrease occurs ~ 1 0 h after bacterial addition and 
appears to be mediated by intracellular organisms. 
Mutations in EPEC that affect actin accumulation 
and bacterial invasion (c/m and eae) do not cause 
the decrease in transepithelial resistance. These mu- 
tants also do not cause diarrhea in human volun- 
teers. Collectively this information suggests that an 
increase in transepithelial permeability may be in- 
volved in EPEC-mediated diarrhea. 
The cytoskeletal rearrangement that accumulates 
beneath adherent EPEC stains brightly with mono- 
clonal antibodies directed against phosphotyrosine. 
When HeLa cells are infected with EPEC, a 90-kDa 
HeLa cell protein becomes heavily tyrosine phos- 
phorylated. This tyrosine-phosphorylated protein is 
associated with the cytoskeleton and membrane. Ty- 
rosine protein kinase inhibitors block tyrosine phos- 
phorylation of the 90-kDa protein. These inhibitors 
also block EPEC uptake into HeLa cells. Addition- 
ally, a mutant strain of EPEC, cfm, which adheres 
normally but cannot cause actin accumulation, does 
not trigger tyrosine phosphorylation of the 90-kDa 
protein. Thus it appears that the EPEC cfm locus is 
involved in triggering host cell tyrosine kinase activ- 
ity, while the product of eae nucleates and orga- 
nizes actin into an ordered structure beneath the 
bacterium. When mixed in equal amounts, cfm and 
eae mutants are capable of complementing cytoskel- 
etal rearrangement and invasion of the cfm mutants 
but not the eae mutants. 
Host Tyrosine Kinase Activity Is Necessary 
for Yersinia and Listeria Uptake 
Into Epithelial Cells 
Several pathogenic bacteria have the capacity to 
be internalized into nonphagocytic cells, such as ep- 
ithelial cells. Yersinia species have several prod- 
ucts that are involved in invasion. The best charac- 
terized is invasin, a bacterial protein that interacts 
with /3j-integrins to mediate uptake. Several tyrosine 
kinase inhibitors, including staurosporine, genis- 
tein, and a tyrphostin, block invasin-mediated up- 
take into epithelial cells without affecting bacterial 
adherence or viability. The effect of staurosporine 
on invasion is rapidly reversible, with bacteria being 
internalized within 2 min after drug removal. These 
results indicate that invasin-mediated internaliza- 
tion triggers host tyrosine kinase activity, pre- 
sumably through a /Jj-integrin linkage with the 
bacterium. 
Gram-positive Listeria monocytogenes is another 
organism that is capable of entering into nonphago- 
cytic cells. Its uptake into cultured epithelial cells 
is also significantly blocked by host tyrosine kinase 
inhibitors. In contrast, invasion of S. typhimurium 
is not affected by these inhibitors. 
Collectively it appears that pathogenic bacteria 
have the capacity to adhere to host cell surfaces 
through defined receptors and then to pirate host 
cell events, such as signal transduction and cytoskel- 
etal rearrangement, for bacterial benefit, and that 
these actions potentiate disease. 
Dr. Finlay is Assistant Professor in the Biotech- 
nology Laboratory and the Departments of Bio- 
chemistry and Microbiology at the University of 
British Columbia, Vancouver. 
INTERNATIONAL RESEARCH SCHOLARS 507 
