Molecular Genetics of Intracellular Microorganisms 
Our investigation of invasin/integrin has led to 
a model for Yersinia uptake into host cells. Bind- 
ing of invasin to its integrin receptor leads to rear- 
rangement of the cytoskeleton — rearrangement 
requisite to entry. A signal must be sent to cause 
the host cell to internalize the microorganism, 
and the internalization is facilitated by the ex- 
traordinary avidity with which invasin binds its 
receptors. Other proteins that bind the identical 
integrins cannot produce this signal so effi- 
ciently, because they do not bind the receptors 
tightly. 
Thus invasin appears to promote entry of the 
microorganism because it binds an important re- 
ceptor that interacts with clathrin coats and com- 
municates with the cell cytoskeleton, and be- 
cause it binds so tightly to this receptor. 
Legionella pneumophila Growth 
in Phagocytic Cells 
Z. pneumophila causes a variety of diseases in 
humans, including Legionnaire's disease pneumo- 
nia. The bacterium grows in lung tissues after en- 
counter with its human host. Its favorite habitat is 
within alveolar macrophages, cells that normally 
function to kill invading microorganisms. An im- 
portant mechanism for macrophages to kill or 
inhibit the growth of a microorganism is to inter- 
nalize it and sequester it in a compartment called 
a phagosome, which in turn fuses with a lyso- 
somal compartment filled with antibacterial fac- 
tors. L. pneumophila is able to grow within the 
phagosome, convert it into an organelle with a 
unique morphology, and prevent the introduc- 
tion of the antibacterial lysosomal components 
into this site. 
We have been interested in determining how L. 
pneumophila is able to establish and grow within 
this protective niche. We have been taking two 
tactics toward analyzing this process. Our first 
approach has been to isolate mutations in this bac- 
terium that prevent it from growing intracellu- 
larly. Our second approach has been to identify 
factors that are selectively synthesized by the bac- 
terium only during intracellular growth. 
Using the first approach, three easily distin- 
guishable classes of mutants have been isolated. 
The first class causes the bacterium to be internal- 
ized by a macrophage via a novel pathway, and 
this causes an extreme defect in bacterial growth. 
The second class, and most easily isolated, con- 
sists of mutants that are no longer able to prevent 
the lysosomal contents from being introduced 
into the phagosome. The third class appears nor- 
mal for uptake as well as for shutting out the lyso- 
somal components, but the phagosome contain- 
ing the mutant microorganism no longer exhibits 
the unique morphology usually found in a Le- 
gionella infection. 
These classes of mutants indicate that the mi- 
croorganism performs a distinct series of steps 
within the macrophage, each of which contrib- 
utes to the parasite's efficient growth. To investi- 
gate the factors missing in these mutants and ana- 
lyze the steps in growth performed by this 
bacterium, we have identified a small region of 
the Legionella chromosome that encodes the fac- 
tors missing in the latter two mutant classes. We 
are currently analyzing this region of the chromo- 
some intensively, with the hope of purifying the 
factors encoded by this region in order to de- 
scribe their functions in molecular detail. 
Our second approach involves using a novel 
scheme to identify L. pneumophila genes that are 
regulated in a fashion such that they are turned 
off when the bacterium is growing outside the 
host cell but are rapidly turned on during growth 
within macrophages. This tactic involves cloning 
fragments of L. pneumophila chromosomal DNA 
in front of a reporter gene and introducing these 
molecular clones into the bacterium. All molecu- 
lar clones constructed in this manner are killed 
unless they contain genes that are regulated in the 
desired fashion. Using this strategy, we have 
cloned at least eight genes that are normally only 
expressed by L. pneumophila when the microor- 
ganism is within a host cell. 
We hope that these two strategies will allow 
identification of most of the factors encoded by L. 
pneumophila that mediate the striking rearrange- 
ment of host cell organelles and facilitate groMT:h 
of the microorganism. 
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