The Role of REEL in Pasteurella multocida toxin on Swiss 3T3 cells
Pasteurella multocida is a small gram-negative coccobacillus that causes infections associated with atrophic rhinitis in pigs. Ti produces Pasteurella multocida toxin (PMT) as one of the major virulet factors. PMT is an intracellularly acting toxin that utilized membrane-receptor endocytosis to enter into mammalian cells. Through the use of green florescent protein (GFP), recent studies in Dr. Wilson's laboratory have shown that once PMT enters a cell it moves to the endoplasmic reticulum (ER) where it enters the cytosol and reaches its target. The main goal of a bacterial protein toxin is to get into the cytosol where it can react with its target. Bacterial protein toxins generally have two ways of getting to the cytosol. The route that a bacterial protein toxin takes depends on whether the protein toxin reacts to the low pH of endosomes. If the protein toxin does react to the low pH, it enters the cytosol via early endosomes. Many do not use pre-existing proteins for translocation. This process is called retrograde transport pathway. Proteins that reside in the ER have a KDEL-like sequence that enables them toe move back to the ER. There is a KDEL-like ER-retention sequence in PMT, REEL. Dr. Wilson's lab is currently interested in finding out the importance of the REEL sequence in toxin translocation.
Through the use of a PCR method, a construct coding for a fragment of PMT contains a mutated REEL sequence has been made pPCRTopo II. A Spe I/ Kpn I fragment of pPcr Topo II (4,3) will then be inserted into a vector (pTHB GFP ToxA) by ligation. PTHB-GFPToxA (REEL) will express the REEL mutant protein of PMT in E. Coli. This mutant protein will be purified by His-Tag affinity chromatography and the REEL mutant will be used in tissue culture experiment.
Once the experiments are done I expect to find that the REEL retrieval signal is important for PPMT to enter into the cytosol by translocation.
Through the use of a PCR method, a construct coding for a fragment of PMT contains a mutated REEL sequence has been made pPCRTopo II. A Spe I/ Kpn I fragment of pPcr Topo II (4,3) will then be inserted into a vector (pTHB GFP ToxA) by ligation. PTHB-GFPToxA (REEL) will express the REEL mutant protein of PMT in E. Coli. This mutant protein will be purified by His-Tag affinity chromatography and the REEL mutant will be used in tissue culture experiment.
Once the experiments are done I expect to find that the REEL retrieval signal is important for PPMT to enter into the cytosol by translocation.
School:
St. Mary's University
Department:
Biochemistry
Research Advisor:
Brenda Wilson
Department of Research Advisor:
Microbiology
Year of Publication:
2003