|
I am conducting a generalized
search to identify bacterial factors that are required for efficient
growth in
the cytosol using a FACS-based selection strategy. ActA-negative L. monocytogenes replicate within
the cytosol with similar efficiency as wild-type bacteria, but are
unable to move via actin-based motility or spread to neighboring host
cells. A transposon library of an ActA-negative L. monocytogenes
strain expressing GFP was used to infect murine bone marrow-derived
macrophages (BMM). Six hours post-infection, the infected macrophages
were sorted using FACS. Macrophages infected with L. monocytogenes
harboring transposon insertions that resulted in decreased replication
efficiency in the cytosol were less fluorescent than macrophages
infected with ActA-negative L.
monocytogenes. Intracellular L.
monocytogenes were isolated from these macrophages and
transposon-disrupted genes identified. Using this technique, candidate
genes have been identified whose products may be involved in altering
the metabolism of L. monocytogenes
to fulfill nutrient requirements necessary for optimal intracellular
replication.
View Larger Image
Figure. Flow cytometry analysis of
infected bone marrow-derived macrophages (BMM). Exponential phase
bacterial cultures were used to infect BMM at an MOI of 1. After
6 hr, the infected macrophages were analyzed by flow cytometry. The
black line represents the fluorescence intensity of macrophages
infected with an ActA-negative strain of L. monocytogenes expressing GFP.
The blue line represents the fluorescence intensity of macrophages
infected with a transposon mutant of the GFP-expressing ActA-negative
strain that results in a defect in intracellular replication.
|
