Host-Pathogen Interactions in Microbial Pathogenesis 
ium is one pathogen that requires host actin fila- 
ments for invasion. When S. typhimurium enters 
cukured epitheUal cells, there is a large rear- 
rangement in intracellular actin and the function- 
ally related cytoskeletal proteins a-actinin, tropo- 
myosin, and talin that surround the membrane 
engulfing the invading organism. This rearrange- 
ment, presumably needed for functional internal- 
ization, is triggered by bound extracellular bacte- 
ria and occurs in the region beneath adherent 
organisms. Once the pathogen is inside a vacu- 
ole, the epithelial cytoskeleton returns to its nor- 
mal distribution. 
We have been examining the signals trans- 
duced through the host cell membrane that are 
responsible for triggering this cytoskeletal rear- 
rangement, and host cell signals that are neces- 
sary for the uptake of other pathogenic bacteria 
into epithelial cells. Host tyrosine kinases appear 
to participate in the uptake of several pathogens, 
including Yersinia species (causative agent of 
gastrointestinal problems). Listeria monocyto- 
genes (a gram-positive organism associated with 
meningitis and serious infections of neonates), 
and enteropathogenic Escherichia coli (caus- 
ative agent of diarrhea). We are using various 
kinase inhibitors, monoclonal antibodies that 
recognize phosphotyrosine residues, and radiola- 
beling of host phosphate pools to begin to iden- 
tify the components of the signal transduction 
pathways that these bacteria pirate for their 
own use. 
An in Vitro Blood-Brain Barrier to Study 
Bacterial Meningitis 
Bacterial meningitis (infection of the brain lin- 
ing) is a common and serious disease in both 
children and adults. In an effort to define more 
clearly the molecular mechanisms mediating this 
disease, we developed a model in vitro blood- 
brain barrier (BBB) . This model uses primary iso- 
lates of microcapillary endothelial cells from bo- 
vine brain. Reseeded onto permeable substrates, 
these cells form impermeable monolayers with 
tight junctions and have several features charac- 
teristic of the BBB. 
When Haemophilus influenzae (the most 
common cause of pediatric meningitis) is added 
to these monolayers, they are completely 
disrupted by a cytotoxic mechanism — similar to 
the effects observed in vivo. We have found that 
this cytotoxicity is mediated by bacterial lipo- 
polysaccharide (LPS) in concert with a soluble 
serum factor. Current research has a twofold aim: 
first, to block this cytotoxicity with monoclonal 
antibodies against the serum factor in a primate 
meningitis model and second, to define the signal 
transduction pathways that are triggered in the 
endothelial cell by the LPS. In addition, the role 
of cytokines in this cytotoxic event is being 
studied. 
Collectively this work provides several insights 
as to how pathogenic bacteria manifest disease. 
Studies with 5. typhimurium have afforded new 
perceptions into the molecular biology of intra- 
cellular parasitism. Definition of the signal trans- 
duction pathways of invasive pathogenic bacteria 
is providing evidence that many pathogens ex- 
ploit existing pathways, often utilizing them to 
pirate the host cytoskeleton for their own advan- 
tage. Work with the BBB has offered new opportu- 
nities for therapeutic intervention in treating 
meningitis. 
494 
