Screen of Nuclear Localized Effecters Proteins in Coxiella burnetii
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In some pathogens such as Ehrlichia chaffeensis, Anaplasma phagocytophilum, Chlamydia, and Shigella, secreted effector proteins have been found to localize to the nucleus and alter the host’s immune response by modulating chromatin or binding to regulatory transcription molecules. By doing so, the pathogen is able to successfully grow and replicate within the host cell causing infection. Several of Coxiella burnetii’s secreted effector proteins have similarly been found to localize to the nucleus but have no confirmed role. The objective of this study is to confirm nuclear localization of previously identified nuclear effector proteins and pinpoint any interactions between host DNA and protein. Each GFP tagged effector protein of interest- CBU0129, CBU0794, CBU0393, CBU1314, and CBU1524, was ectopically expressed in HeLa cells to perform sub-cellular fractionation. These sub-cellular fractions were run on a SDS-Page gel and probed with anti-GFP to identify any association with specific fractions within the host. Nuclear localization signal (NLS) truncations were then constructed using splicing by overlap-extension PCR (SOEing PCR) to remove predicted nuclear localization signals from each effector. The ΔNLS constructs were also ectopically expressed in HeLa cells to visualize loss of localization. Chromatin Immunoprecipitation (ChIP) was performed to identify specific binding regions of each protein and further studies such as en Electrophoretic Mobility Shift Assay (EMSA) were attempted to confirm the binding of chromatin by the effector proteins. Sub-cellular fractionation confirmed the association of all five effector proteins to host chromatin, and the loss of nuclear localization by two effectors upon deletion of predicted NLS indicates that these effectors are not only specifically imported to the nucleus via NLS, but have some specialized role in the nucleus. Confirmation of these predicted protein-DNA interactions will allow Coxiella researchers to better understand how this organism survives and replicates. With more knowledge of these processes, scientists will be better equipped to prevent and treat future infections.
Galvan, Gloria (2013). Screen of Nuclear Localized Effecters Proteins in Coxiella burnetii. Honors and Undergraduate Research. Available electronically from