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takehome - Yanting Wang BIOMI 4040 March 8 2010 Prelim 1...

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Yanting Wang March 8, 2010 BIOMI 4040 Prelim 1 Question 1: Describe the methods used to demonstrate that Igll and VgrG proteins are secreted into macrophages. In the description be sure to explain how this method tells us these proteins are in the macrophages as opposed to being in extracellular milieu. To demonstrate that VgrG is secreted into macrophages, plasmids that fused VgrG to the Bordetella pertusis adenylate cyclase (CyaA) toxin were constructed. CyaA requires calmodulin, a calcium-binding protein expressed in all eukaryotic cells 1 , thus an increase in cAMP level would only when they are translocated into (intracellular) the cytosol of macrophages infected with F. tularenisis expressing the CyaA fusion proteins. Using CyaA as a reporter, VgrG activity can also be demonstrated. When VgrG proteins are secreted into the macrophages, CyaA will follow. Once in the cytosol of J774 macrophages, CyaA will induce an increase in cAMP. An increased level of cAMP will demonstrate both CyaA and VgrG presence in macrophage cytostol. The same principle was also applied to IglI. IglI was also fused to CyaA protein. As a positive control, PepO (known secreted protease protein) was fused to CyaA. The pepO-cyaA fusion caused an elevated cAMP level in infected J775 cells. For the negative control, Ftn expressing beta- lactamase, from which the N-terminal signal sequence was removed (modified for non- secretion), (ΔNBlaB) was fused to CyaA. There was no detection of cAMP elevation. If the proteins remained in the extracellular milieu, there would be no adenylate cyclase activity without the presence of ATP and cadonium. Plasmids used in the study were derived from Francisella plasmids pKK214 and the FTN1451 promoter was used for vgrG-cyaA, hcp-cyaA, pepO-cyaA, ΔNblaB-cyaA fusions. Quantification of cAMP levels was determined using the cAMP Biotrack Enzymeimmunoassay System. To further visualize VgrG and IglI within the infected macrophages, the vgrG and IglI strains were tagged using a FLAG-tag. A FLAG-tag is a polypeptide protein tag that is added to a protein of interest through recombination. The FLAG-tagged protein can then be recognized using an antibody 2 . Strains expressing FLAG-VgrG and FLAG-IglI were used to infect J774 macrophages. VgrG and IglI were visualized using a Cy3 (red)-
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labeled anti-FLAG monoclonal antibody. The bacteria was labeled with anti-Fn LPS (green) antibody. DAPI (blue) was also used for immunofluorescence activity. DAPI is a blue-fluorescent nucleic acid stain that stains preferentially to ds-DNA 3 . The secreted proteins (red) are visualized to be distinctly within the macrophage cytostol around and clearly distinct from the bacteria (green). The negative control, ΔNBlaB-FLAG do not have any detectable secreted (no red seen).
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