BioMi404 Prelim 2

BioMi404 Prelim 2 - Isabela Wieczorek BIOMI404 Prelim 2...

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Isabela Wieczorek BIOMI404 Prelim 2 Question I A. Type IV secretion systems are also called conjugal transfer systems. The system has genes that are similar to those involved in the transfer of DNA by conjugation systems. They are Sec independent and are composed of a large number of proteins. The formation of piluslike structure occurs in periplasm and transits both membranes. The system is found only in gram-negative bacteria. The system can transfer monomeric proteins, multimeric proteins, or single-stranded DNA complexed to proteins. In most systems, the protein is transferred directly to the host cell, after cell-to-cell contact. In some cases, the protein may be excreted into the external environment. This secretion system is present in the Legionella pneumophila Dot/Icm system. The Dot/Icm complex forms a pore that leads from the bacterial cytoplasm to the macrophage cytoplasm, allowing the direct transfer of effector proteins from the bacteria to the host. B. The Type IV secretion system of Bartonella henselae is called VirB, and it translocates various effector proteins into human endothelial cells (HEC). One component of the VirB system is the virB4 gene, and mutants with a deletion of virB4 do not have a functioning excretion system. The authors demonstrated that the effector protein BepD is not translocated into human cells in the absence of virB4 . The authors knew that BepD is phosphorylated by host cell tyrosine kinases once it enters the host cell. Additionally, tyrosine phosphorylation coincides with a “prominent shift in electrophoretic mobility”, which can be seen in an electrophoresis gel. Additionally, the authors tagged BepD with FLAG. Figure 2 pictures the two experiments the authors ran. In experiment 2A, “Ea.hy926 cells were infected with Bh strains expressing FLAG-tagged BepD. Cells were lysed, and the FLAG-tagged BedD was immunoprecipitated with anti-FLAG agarose and probed with antiphosphotyrosine antiblot in a Western blot” (858). Lane 5, with wild-type bacteria and FLAG-BepD shows that BepD shifts upward, and that it is the only lane that probes anti-phosphotyrosine, showing that BepD only becomes phosphorylated after VirB4-dependent translocation. Lane 4, with a ΔvirB4 and FLAG-BepD strain, did not show an electrophoretic shift and did not tag anti-phosphotyrosine. Figure 2B and 2C shows an immunocytochemical detection of FLAG-BepD in the infected cell. Cells infected with a wild-type strain (B) are the only ones showing the green color of the FLAG epitope. Panel B, which were cells infected with a ΔvirB4 strain, show the presence of bacteria in blue, but the absence of the green FLAG-epitope in the cell, indicating that BepD was not translocated. C. To identify proteins that enter the host cell dependent on the Type IV secretion system, one can fuse those proteins to calmodulin-activated adenylate cyclase (cyaA).
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