Molecular Genetics of Intracellular 
Microorganisms 
Ralph R. Isberg, Ph.D. — Assistant Investigator 
Dr. Isberg is also Assistant Professor of Molecular Biology and Microbiology at Tufts University School of 
Medicine. He received his A.B. degree in chemistry from Oberlin College and his Ph.D. degree in 
microbiology and molecular genetics from Harvard Medical School. He conducted postdoctoral work on 
bacterial pathogenesis in the laboratory of Stanley Falkow at Stanford University. His honors include a 
Searle Scholars Award and a National Science Foundation Presidential Young Investigator Award. 
MANY species of bacteria are capable of caus- 
ing diseases by colonizing and growing 
within human hosts, using tactics that avoid nor- 
mal immune responses. As part of a general strat- 
egy to establish an infectious niche, a variety of 
microorganisms cause diseases by entering and 
growing inside human cells soon after encounter. 
Bacteria that establish infections in this manner 
are called intracellular microorganisms. Among 
the diseases they cause are tuberculosis and the 
most common types of sexually transmitted and 
food-borne diseases found in the industrialized 
world. Despite the prevalence of such infections, 
there was little information until recent years on 
the factors expressed by these microorganisms 
that allow them to enter host cells and thrive. 
The objectives of our research are to investi- 
gate two important aspects of the life-style of in- 
tracellular microorganisms. First, we would like 
to determine at the molecular level how these 
organisms can enter human cells that do not nor- 
mally internalize bacteria. Second, we want to 
analyze factors they encode that allow them to 
survive and grow within the ordinarily hostile en- 
vironment of human cells. Our main approach 
has been to identify bacterial species that enter or 
grow particularly well within host cells and to 
develop genetic and biochemical techniques for 
analyzing their strategies. The primary rationale 
for this approach is that it provides insights into 
basic processes that are applicable to numerous 
intracellular microorganisms. 
To investigate the molecular mechanism of bac- 
terial binding and entry into host cells, we have 
been analyzing the bacterium Yersinia pseudo- 
tuberculosis, an organism that causes an intes- 
tinal disease often accompanied by infection of 
multiple organ systems. 
To investigate intracellular growth, we have 
been analyzing Legionella pneumophila, the 
causative agent of Legionnaire's disease pneumo- 
nia. The intracellular growth process of the bacte- 
rium is very similar to that of a wide range of 
intracellular microorganisms, and development 
of molecular strategies for analyzing it has been 
relatively straightforward. 
Yersinia pseudotuberculosis Entry Into 
Cultured Human Cells 
Y. pseudotuberculosis can enter host cells via 
three different paths. For each path the microor- 
ganism apparently encodes a unique set of pro- 
tein factors to be used at different tissue sites dur- 
ing the infection process. We have focused on the 
path that is promoted by the protein invasin, the 
product of the bacterial inv gene. Invasin is a 
108-kDa protein on the surface of the bacterium 
that allows it to enter human cells by binding 
receptor molecules on their surface. We have 
shown that a 20-kDa region of invasin binds the 
host cells, and this region is sufficient to promote 
uptake. Evidence indicates that after the binding 
occurs, the host cells do most of the work in in- 
ternalizing the bacterium. 
Invasin binds at least four different receptors. 
Called integrins, these had been previously iden- 
tified by investigators interested in a variety of 
mammalian cell adhesion processes. The particu- 
lar integrin receptors that bind invasin can adhere 
to a variety of mammalian proteins, such as fibro- 
nectin and molecules that allow adhesion of im- 
mune response cells to inflamed tissues. 
Although invasin binds these well-character- 
ized receptors, there is no obvious sequence simi- 
larity between invasin and other proteins that 
bind integrins, and mutations that eliminate the 
interaction between invasin and its receptors 
identify amino acid residues not previously 
shown to be involved in integrin binding. 
Integrins are clearly not the only host-encoded 
factors necessary for internalization. Analysis of 
this process has indicated that two additional fac- 
tors are required. Mutant studies of one of these 
integrin receptors indicate that a cell structure 
called a clathrin coat directly interacts with the 
integrin receptor during the internalization of 
the bacterium. If this interaction is eliminated, 
the bacterium cannot be internalized. A second 
structure, the host cell cytoskeleton, also is in- 
volved in the internalization, but we believe that 
this structure performs an indirect role and does 
not directly bind the integrin during all stages of 
bacterial uptake. 
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