Francisella tularensis is a pathogenic bacterium capable of replication within host monocytes and macrophages and is the causative agent of Tularemia. We utilized the attenuated live vaccine strain to model the infection and replication of F. tularensis in the mouse macrophage RAW 264.7 cell line. Our lab has previously identified three genes predicted to encode transcriptional regulators that are upregulated in the presence of human blood cells: FTL_0671, FTL_1199, and FTL_1665. The objective of this project is to determine if these genes play a role in the pathogenesis of F. tularensis. Wild type F. tularensis LVS and mutant bacteria with FTL_0671, FTL_1199, and FTL_1665 gene deletions were constructed and engineered to express an extremely bright variant of Emerald Green Fluorescent Protein (encoded in the plasmid, pKHEG). These strains were used to infect RAW 264.7 macrophages and fluorescence was monitored over time using a plate reader. Our results suggest that FTL_1199 and FTL_1665 are attenuated for intracellular replication indicating that the products of the mutated genes may be important for pathogenesis.
Francisella tularensis is a gram-negative intracellular pathogen that produces a severe infection known as Tularemia. RNA-Seq data revealed that in the presence of erythrocytes, several genes encoding putative transcriptional regulators were modulated: FTL_0671, FTL_1199, and FTL_1665. Gene deletion strains were constructed using Francisella tularensis LVS. The objective of this project was to screen these gene deletion strains for attenuation. A multifaceted approach was utilized to determine the level of replication within macrophages and overall attenuation in vivo. Transforming the bacteria with green fluorescent protein allowed a plate reader to visualize and quantify intracellular growth in macrophages. However, inconsistencies in the data from these experiments led to the utilization of a gentamicin protection assay. This protocol provided a more accurate and reliable method of determining intracellular replication. The results of this experiment revealed a significant increase in the replication of FTL_1665 within macrophages. We sought to determine if these results would translate to hypervirulence in a live model. A chicken embryo infection model confirmed that FTL_1665 was significantly hypervirulent in vivo. In the future, we plan to experiment with the upregulation of the target gene to produce an attenuated strain. This gene may also serve as a potential drug target.
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