The use of GFP Gene Reporter System to Monitor Expression of genes hly and actA in Listeria monocytogenes inside host cells
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Authors
Jacobs, Kathleen
Issue Date
1997
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
Listeriosis is a disease caused by Listeria monocytogenes and infects mainly
immuno-compromised people, pregnant women, and the elderly. L. monocytogenes is a
gram-positive, non-spore-forming, food-borne pathogen that is more commonly found in
the environment than Salmonella. L. monocytogenes is difficult for a host to fight off
because the bacteria spends all but the first hour of infection inside the cell cytoplasm, a
place where the humoral immune system does not detect it. A gene from the Aequorea
victoria jellyfish, GFP, was inserted-into three L. monocytogenes -mutants (p357, p404,
and p411). The infection rate and gene expression ofhly and actA in L. monocytogenes
were viewed in vivo in various mouse cells. A plaque assay with L2 cells, a growth curve
and actin-tail stain with PtK2 cells, and extracellular fluorescent growth assay were
performed on wild-type and three mutants of L. monocytogenes. Cells were viewed
under both the GFP and actin filters in order to see the bacteria (green) and actin (red) in
the same image, and identify the overlapping area (yellow). The plaque assay and growth
curve had comparable growth rates, and confirmed that the number of bacteria double
every hour. In addition, the bacteria are first released into the cytoplasm from the cell
membrane vacuole when hly is expressed between 2.5 and 3 hours, as detected by the
fluorescence of GFP. actA is first expressed around 3 hours, and after 4 hours actin-tails
to move the bacteria into an adjacent cell are present. All bacteria in images were
intracellular as determined by the extracellular fluorescent growth assay. The bacteria do
not fluoresce in BHI or BHI at pH 7.5. However, GFP is expressed in DMEM, although
the brightness of fluorescence is much weaker to that seen intracellularly. The bacteria
could not continue to grow without the hly, expressed after two-and-a-half hours post-infection, or the actA, expressed after three hours post-infection, gene. As a result, the
infection does not spread to adjacent cells.
Description
vi, 41 p.
Citation
Publisher
Kalamazoo College
License
U.S. copyright laws protect this material. Commercial use or distribution of this material is not permitted without prior written permission of the copyright holder.