THERESA EVANGELISTA & JOSEPH HORZEMPA, Department of Biomedical Sciences, West Liberty University, West Liberty, WV, 26074. Use of Heliopsis and Epilobium extract to test for reduced viability of Francisella tularensis. Through a collaboration with the National Center for Natural Products Research, we identified multiple extracts from plants that exhibited novel antimicrobial activity. These extracts led to reduced viability of the pathogenic bacterium, Francisella tularensis during an in vitro infection but did not alter bacterial viability in the absence of host cells. Therefore, rather than inhibiting a central biological pathway of bacteria, these extracts either functioned through immune activation or dampening virulence factor expression of F. tularensis. Two of these extracts were obtained from plants of the genera Heliopsis and Epilobium. In this work, we sought to validate and extend the previous findings. Future experimentation will utilize bio-assay-guided fractionation to isolate and identify the compounds responsible for the previously observed antimicrobial activity. In addition, we will conduct mechanistic studies to determine whether the responsible compound activates host cells or downregulates virulence gene expression of F. tularensis.
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 causes the zoonotic disease tularemia. During infection, F. tularensis bacteria invade erythrocytes, a phenomenon that enhances the colonization of ticks after a blood meal. To gain insight into the mechanism of erythrocyte invasion, we hypothesized that transcription of bacterial genes important in red blood cell invasion would increase upon exposure to these host cells. An RNA-seq analysis revealed that transcription of 7% of F. tularensis genes increased when in the presence of erythrocytes. Of these, we identified three putative transcriptional regulators, namely FTL_0671, FTL_1199, and FTL_1665. The goal of this work was to determine the role of FTL_0671 in erythrocyte invasion by F. tularensis LVS. After successfully generating a DFTL_0671 strain by homologous recombination, we tested the ability of this mutant to invade erythrocytes using a gentamicin protection assay. As seemingly more DFTL_0671 bacteria invaded red blood cells than wild-type LVS, this preliminary experiment suggested that FTL_0671 may restrict erythrocyte invasion. In addition to being a potential transcriptional regulator, FTL_0671 is homologous to PanK, a protein required for CoA biosynthesis in some bacteria. To determine whether FTL_0671 functioned in this capacity, LVS or DFTL_0671 was cultured in a chemically defined medium, devoid of CoA. In this experiment, DFTL_0671 did not exhibit diminished growth, suggesting that either FTL_0671 does not encode an enzyme required for CoA biosynthesis, or that F. tularensis can utilize an alternative pathway to synthesize this critical coenzyme.
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).
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