Francisella tularensis is a bacterium that induces the zoonotic disease tularemia. In the course of infection, F. tularensis bacteria invade erythrocytes, a phenomenon that heightens the colonization of ticks after a blood meal. To better understand the mechanism of erythrocyte invasion, we hypothesized that transcription of bacterial genes significant in erythrocyte invasion would be upregulated upon exposure to these host cells. An RNA-seq unveiled that transcription of 7% of F. tularensis genes augment when in erythrocyte presence. Of these, we pinpointed three putative transcriptional regulators, namely FTL_0671, FTL_1199, and FTL_1665. The goal was to delete FTL_1199 in F. tularensis LVS. Splicing by overlap extension PCR amplified and duplicated the up and downstream (~500 bp each) regions of the target gene in tandem into a shuttle vector that is insecure within F. tularensis. This newly generated plasmid, pDEL1199, was mobilized inside of F. tularensis by conjugation. Merodiploid strains generated by homologous recombination were isolated and transformed with pGUTS – a stable plasmid that encodes a homing endonuclease (I-SceI) and a kanamycin resistance cassette. Expression of I-SceI within the merodiploid produces a double-stranded break in pDEL1199 that had previously integrated in the chromosome. This breakage resulted in a second recombination that either ensued to wild-type or deletion of FTL_1199 deduced through a PCR. Finally, in DFTL_1199 strains, pGUTS was cured by successive cultivation in the absence of selection followed by replica-plating on chocolate II agar ± kanamycin. Gentamicin protection assays involving F. tularensis DFTL_1199 suggest that FTL_1199 is important in erythrocyte invasion. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence, R15HL14735 from NHLBI, and funds from the NASA West Virginia Space Grant Consortium).
Francisella tularensis is a bacterium that induces the zoonotic disease tularemia. In the course of infection, F. tularensis bacteria invade erythrocytes, a phenomenon that heightens the colonization of ticks after a blood meal. To better understand the mechanism of erythrocyte invasion, we hypothesized that transcription of bacterial genes significant in erythrocyte invasion would be upregulated upon exposure to these host cells. An RNA-seq unveiled that transcription of 7% of F. tularensis genes augment when in erythrocyte presence. Of these, we pinpointed a putative transcriptional regulator, FTL_1199. The goal was to delete FTL_1199 in F. tularensis LVS. SOE PCR amplified and duplicated the up and downstream regions of the target gene in tandem into a shuttle vector that is insecure within F. tularensis. This newly generated plasmid, pDEL1199, was mobilized inside of F. tularensis by conjugation. Merodiploid strains generated by homologous recombination were isolated and transformed with pGUTS. Expression of I-Sce1 within the merodiploid produces a double-stranded break. This breakage resulted in a second recombination that either ensued to wild-type or deletion of FTL_1199 deduced through a PCR. Finally, in DFTL_1199 strains, pGUTS was cured by successive cultivation in the absence of selection followed by replica-plating on chocolate II agar ± kanamycin. Gentamicin protection assays showed reduced levels of erythrocyte invasion for F. tularensis DFTL_1199 compared to wild type bacteria. However, complementation of FTL_1199 to the deletion mutant restored this strain’s ability to invade red blood cells. These findings demonstrate that FTL_1199 is important for erythrocyte invasion by F. tularensis. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence, R15HL14735 from NHLBI, and funds from the NASA West Virginia Space Grant Consortium).
Francisella tularensis, a highly infectious bacterium, is the causative agent of Tularemia (rabbit fever). Categorized by the Center for Disease Control and Prevention as a Category A bioterrorism agent, Francisella tularensis is of the highest level of concern. Previously, we identified that dillapiole, a compound extracted from fennel, dampens F. tularensis virulence gene expression. While having no apparent effect on the viability of F. tularensis, treatment with this compound leads to reduced bacterial viability during in vitro infection of THP-1 monocytes and RAW 264.7 macrophages. In this study, we sought to determine if dillapiole exhibited a therapeutic effect in vivo, and to characterize the toxicity and pharmacology of this compound. In a murine tularemia model, female mice treated with dillapiole trended toward increased survival compared to those treated with the vehicle. However, dillapiole- or vehicle-treated male mice showed increased mortality compared to the females, suggesting gender-specific differences in the murine immune response to F. tularensis. Dillapiole was not toxic to HEK-293 cells in vitro, nor was this compound toxic to primary human hepatocytes when tested up to a concentration of 11 mg/ml (50 mM). Dillapiole was shown to be relatively stable in human, rat, and mouse plasma with a half-life greater than 120 minutes in all cases. However, this compound showed moderately high binding to plasma proteins (86% in human plasma and 75% in mouse plasma). In addition, while dillapiole showed moderate clearance by human and rat liver microsomes, mouse liver microsomes exhibited high clearance. Collectively, these data could explain the minimal efficacy observed in vivo. Therefore, future investigations should involve the rat infection model to determine the potential efficacy of dillapiole as a novel treatment for tularemia.
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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