Bacterial pathogens,
such as Listeria, Shigella and
enteropathogenic E. coli, cause significant
human disease by subverting host cell functions.
To escape detection by the host immune system,
these particular pathogens commandeer the host
cell’s cytoskeleton to enhance both uptake
and invasion of neighboring uninfected cells.
By understanding how pathogens co-opt cellular
processes, we might better understand normal
host cell functions as well as exploit them for
cell/tissue engineering and gene therapy. Combining
novel optical tools with established molecular
and cellular techniques, research projects focus
on two broad questions (all bacterial strains,
including mutants, provided by collaborators):
1) How
do pathogenic bacteria enhance their uptake? Pathogenic
bacteria have evolved exquisite mechanisms to
enhance their uptake and subsequent vacuolar/lysosomal
escape into cytoplasm. Understanding these tricks
could aid therapeutic delivery of nanoparticles
containing drugs or DNA into cells. Using LTM
in the macrophage-like D. discoideum,
we are the first to resolve the fast kinetics
(<10s) and forces of nanoparticle uptake in
living cells. In subsequent studies, we plan
to use optical tweezers to micromanipulate both
bacteria and particles onto cells, thus precisely
initiating particle uptake. Subsequent monitoring
using both real-time fluorescence microscopy
and LTM will quantify their uptake kinetics and
forces. By comparing wild type and mutant bacteria,
we should be able to identify the factors that
control uptake. Because most candidate proteins
have been cloned, we can attach these factors
onto particles and determine if their uptake
is also enhanced. Listeria, Shigella,
and enteropathogenic E. coli all
use different mechanisms of entry and would provide
complementary information.
2) How
does actin polymerization push bacteria? Once
inside cellular cytoplasm, bacteria recruit
host factors to polymerize an actin “rocket” and
push bacteria into neighboring uninfected
cells. Mutants that cannot generate rockets
are no longer pathogenic. Even though
actin polymerization has been implicated
in many essential cellular functions,
its mechanism of force-generation remains
mysterious. Using Listeria monocytogenes,
our LTM data showed that existing molecular
models, including the Brownian ratchet
model, are flawed, if not wrong. In the
absence of credible models, we are in
a unique position to explain how actin
polymerization pushes the edges of cells,
as well as pushes bacteria. Additional
studies using LTM and mutant bacteria,
both in host cells and in biochemically
reconstituted systems, will provide new
data to form newer models.
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