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The bacterial and host cell determiants required for intracellular
replication of Listeria monocytogenes
are not well
characterized. Forward chemical genetics provides a
powerful approach to identify genes of interest in this process. A
library of small molecules (of known bioactivity) was screened to
identify inhibitors that disrupt infection by L. monocytogenes. Murine bone
marrow-derived macrophages were incubated with various chemical
compounds (one compound per well) prior to infection with
GFP-expressing, wild-type L.
monocytogenes. Following infection, plates
were screened by automated microscopy and visual analysis was performed
to identify compounds which disrupt intracellular infection.
Preliminary data suggests that
small molecules which affect
host intracellular calcium levels inhibit L.
monocytogenes growth within
macrophages. Furthermore, synthetic compounds which
inhibit specific tyrosine kinases, also influence listerial
replication. Secondary screens are being performed to identify which
specific cellular pathways are affected by these
compounds to yield a better understanding of L. monocytogenes
pathogenesis. The ultimate goal of this research is to develop small
molecule inhibitors for therapeutic use during intracellular bacterial
infections.
View Larger Image
Figure. (A) Small molecule
screening procedure. (B) Murine bone marrow-derived macrophages were
plated at a density of 10,000 cells per well in 384 well plates. The
small
molecule library was pin transferred into the wells and the plates were
incubated with the compounds for 2 hr prior to infection. Cell
monolayers were infected with GFP-expressing L. monocytogenes
and the infection was later stopped by
the addition of paraformaldehyde. Host cell nuclei were stained with
Hoechst dye (blue). Bacteria appear green due to expression of GFP.
Magnification is 10X.
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