Molecular Pathogenicity Studies of Enteric Bacteria 
Gary K. Schoolnik, M.D. — Associate Investigator 
Dr. Schoolnik is also Associate Professor of Medicine and of Microbiology and Immunology at Stanford 
University School of Medicine. He received his M.D. degree from the University of Washington. He was an 
intern, resident, and chief resident in internal medicine at Massachusetts General Hospital, a fellow in 
infectious diseases with King Holmes and Thomas Buchanan at the University of Washington, and a post 
doctoral fellow with Emil Gotschlich at the Rockefeller University. He founded the Division of Geographic 
Medicine at Stanford University and established a research center for the study of infectious diseases in 
southern Mexico. 
BACTERIAL, viral, and parasitic infections of 
the gastrointestinal tract cause an estimated 
500 million illnesses and 5 million deaths each 
year among children living in the developing 
countries. The principal mission of my laboratory 
is to discover how these infectious agents cause 
disease, how they are spread between individ- 
uals, and how this information can lead to new 
tactics for prevention and treatment. This effort 
has entailed work in two settings: molecular stud- 
ies in our laboratory at Stanford University, and 
epidemiological investigations at a field labora- 
tory in southeastern Mexico, where infections of 
this kind are common. 
In the first setting, the unit of analysis is the 
organism itself. We try to determine how the or- 
ganism attaches to, invades, and damages human 
cells. In the second setting, the unit of analysis is 
a household or a village. In this context we seek 
to understand how the organism is transmitted 
within the community, where its reservoirs are, 
and how it manages to survive as a viable entity in 
the real world. 
From studies of the latter kind, we have learned 
that the survival of a pathogenic microorganism 
correlates with its ability to occupy different habi- 
tats. Within the context of a Third World village, 
these habitats include contaminated food and 
well water, sewage, and the gastrointestinal tracts 
of people and animals. From an experimental 
point of view, we have tried in the last year to 
identify molecular and genetic events that tran- 
spire within an infectious agent as it enters a new 
habitat and adapts. 
The results of these studies have led to the rec- 
ognition that pathogenic bacteria seldom attack 
the gastrointestinal tract as individual, indepen- 
dent, well-separated organisms. Instead, they first 
seem to form infectious units composed of multi- 
ple organisms and then become established as ad- 
herent colonies on the surface of the intestine. 
While our studies have focused on one kind of 
microbe — enteropathogenic strains of Esche- 
richia coli (EPEC) — we have discovered that this 
phenomenon is exhibited by many other bacte- 
rial species that selectively infect mucosal 
surfaces. 
Accordingly, we now believe that the capacity 
to form infectious units confers a selective advan- 
tage on bacterial species infecting mucous mem- 
branes, that it is very probably an essential deter- 
minant of virulence, and that it offers a logical 
point of attack for the prevention of disease by 
vaccination. This phenomenon is discussed be- 
low as it pertains to EPEC and its ability to cause 
gastroenteritis. 
Formation of Infectious Units 
EPEC are a common cause of infantile diarrhea 
in Third World children. When small bowel 
biopsies of infected children are performed, colo- 
nies of EPEC are found attached to the underlying 
epithelia. Similar findings are also seen in cul- 
tured epithelial cells, to which EPEC readily ad- 
here as circumscribed clusters of bacteria, a phe- 
nomenon known as localized adherence. From 
these observations we know that the bacteria 
within the colonies not only interact with host 
cells to which they are bound, but also with each 
other. Scanning electron micrographs revealed 
multiple, ribbon-like structures coursing be- 
tween the bacteria, and fibers also appeared to 
tether individual bacteria to the epithelial cell 
surface. 
Transmission electron micrographs of these in- 
terbacterial ribbons showed that they are com- 
prised of many individual filaments that, having 
laterally aggregated and intertwined, formed 
bundles 50-500 nm wide and 15-20 ^lm long. 
These tended to twist, curl, and form loops. More- 
over, bundles expressed by different organisms 
appeared to form three-dimensional arrays re- 
sembling a fishing net or a meshwork, in which 
individual bacteria were embedded. 
EPEC did not express these bundles while 
growing on most laboratory culture media. How- 
ever, when transferred to chambers containing 
cultured epithelial cells, EPEC evinced bundles 
within 15 minutes, and by 30 minutes colonies 
composed of tightly adherent bacteria appeared 
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