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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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.