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 microbi- 
ology and molecular genetics from Harvard Medical School. He conducted postdoctoral work on bacterial 
pathogenesis in the laboratory of Stanley Falkow at Stanford University. 
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 en- 
counter. Bacteria that establish infections in this 
manner are called intracellular microorgan- 
isms. Among the diseases they cause are tubercu- 
losis 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 mi- 
croorganisms 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 invade human cells that do not 
normally 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 entry into host cells, we have been analyz- 
ing the bacterium Yersinia pseudotuberculosis, 
an organism that causes an intestinal disease often 
accompanied by infection of multiple organ sys- 
tems. This microorganism is perhaps the most ef- 
ficient bacterium at entering into human cells 
grown in culture, and this has greatly facilitated 
analysis. 
To investigate intracellular growth, we have 
been analyzing Legionella pneumophila, the 
causative agent of Legionnaire's disease pneumo- 
nia. The intracellular growth process of this bac- 
terium 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 
103-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 the host cell's post-bind- 
ing uptake requires only a 20-kDa region of inva- 
sin. Host cells can internalize a variety of bacte- 
rial species or even inert latex particles that are 
coated with this relatively small region of the 
protein. Evidently invasin's sole function is to 
present this binding region to host cell receptors 
in order to permit entry into the cell. After the 
binding occurs, the host cells do most of the work 
in internalizing 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. It is also 
well known that members of this receptor family 
interact in some functional fashion with an im- 
portant host cell structure known as the 
cytoskeleton. 
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. 
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