Growth of Legionella pneumophila 
in Cultured Phagocytes 
Dr. Isberg's laboratory has been analyzing the 
growth of L. pneumophila in monocytic cells, 
which normally kill invading microorganisms. 
L. pneumophila, the causative agent of Legion- 
naire's disease pneumonia, initiates infection in hu- 
man hosts by growing within lung macrophages. 
This process can be reproduced in cultured cell 
models, which indicates that this microorganism 
has a unique intracellular life style. Most bacteria 
internalized by macrophages are found in a phago- 
some that fuses with a lysosomal compartment, so 
that the microorganism is exposed to a variety of 
toxic factors. L. pneumophila, on the other hand, 
alters normal organelle traffic in the host cell, so 
that the phagosome does not fuse with lysosomes, 
allowing the bacterium to occupy a protected niche 
called the replicative phagosome. Once within this 
replicative phagosome, recruitment of host organ- 
elles to this site occurs, as mitochondria, ribosomes, 
and smooth vesicles are found surrounding this 
compartment. The bacterium then grows in this site. 
The nature of these various trafficking events is be- 
ing analyzed, and genetic approaches have been de- 
vised to determine the relative importance of these 
morphological changes in supporting intracellular 
growth of L. pneumophila. 
To analyze the nature of the organelle trafficking 
events that occur in the macrophage after an L. 
pneumophila infection. Dr. Michele Swanson has 
identified host cell markers that colocalize with the 
replicative phagosome. The results from these stud- 
ies indicate that the phagosome takes part in fusion 
with compartments that are part of the early stages 
of the macrophage secretory pathway. Surprisingly, 
she finds that growth of the microorganism occurs 
within the host endoplasmic reticulum. Recent stud- 
ies on a variety of other intracellular bacteria and 
viruses have indicated that this may be a common 
replication site and that there may be a poorly char- 
acterized pathway of fusion events that allows deliv- 
ery of endocytosed material to the endoplasmic re- 
ticulum. Current efforts that use a variety of mutant 
strains are devoted to identifying bacterial factors 
encoded by the bacterium that route the organism to 
this site. 
To analyze how L. pneumophila causes drastic 
alterations in organelle trafficking, Karen Berger has 
isolated bacterial mutations. The mutants fall into 
three phenotypic classes. Class I mutants enter via 
the normal receptor pathway used by the wild type, 
they inhibit phagolysosome fusion normally, but 
they are unable to fuse with the endoplasmic reticu- 
lum. Class II mutants, which are the most common. 
are defective for inhibiting phagolysosome fusion. 
Class III mutants, unlike the other classes, are only 
slightly defective for growth, are unaffected for fu- 
sion with the endoplasmic reticulum, but appear to 
be engulfed by both the normal route of receptor 
uptake as well as a second pathway for uptake. To 
characterize these mutants further, the laboratory 
isolated molecular clones that could genetically 
complement the defects in class I and class II mu- 
tants. A single 7-kb region of the L. pneumophila 
chromosome could complement these two classes, 
even though the phenotypes of these mutants are 
different. The mutations from both classes appear to 
lie in a single gene, now called dot (for defective 
organelle trafficking) , which encodes all 2-kDa hy- 
drophobic protein. Mutations that cause a defect in 
inhibiting phagolysosome fusion appear to make a 
totally defective Dot protein. Mutants that are able 
to inhibit phagolysosome fusion but are unable to 
cause organelle recruitment, on the other hand, 
seem to have partial Dot activity. Numerous strate- 
gies are currently being pursued to identify the bio- 
chemical activity of the Dot protein that affects 
these processes. 
Susannah Rankin developed a final approach to- 
ward analyzing intracellular growth of L. pneumo- 
phila, in which genetic loci are identified that are 
expressed exclusively when the organism is grow- 
ing in an intracellular environment but that are 
turned off when the organism is growing extracellu- 
larly. To this end, a clone bank was constructed in a 
vector harboring a promoterless gene encoding a 
protein involved in thymidine biosynthesis. The 
bank was then introduced into an auxotroph requir- 
ing expression of this gene to grow intracellularly. 
Clones that survived this enrichment were then 
grown extracellularly under conditions in which 
expression of this gene is lethal. Strains that sur- 
vived both enrichments contained molecular clones 
with loci that are expressed exclusively when the 
organism is growing intracellularly. With this strat- 
egy at least eight genes were cloned that are nor- 
mally only expressed by L. pneumophila when the 
microorganism is within a host cell. 
Yersinia pseudotuberculosis Entry 
Into Mammalian Cells 
To investigate the entry of Y. pseudotuberculosis 
into cultured mammalian cells, members of Dr. 
Isberg's group have been studying invasin, a 986- 
amino acid outer membrane protein encoded by this 
microorganism. Previous work from the laboratory 
had shown that the carboxyl terminus of invasin 
binds to four members of the i8,-chain integrin fam- 
ily of cell adhesion molecules, which are heterodi- 
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