Gene Expression of F. tularensis LVS in hepatocytic cell line FL83B vs LVS cultured in MH broth
ABSTRACT: Transcriptional profiling of LVS grown in hepatocytic cell line FL83B was compared to that of LVS cultued in MH broth to discover genes important for intracellular growth. Two-condition experiment, Intracellular vs. Broth. Biological replicates: 4 Broth, 4 intracellular, independently grown and harvested. One replicate per array.
Project description:Transcriptional profiling of LVS grown in hepatocytic cell line FL83B was compared to that of LVS cultued in MH broth to discover genes important for intracellular growth. Two-condition experiment, Intracellular vs. Broth. Biological replicates: 4 Broth, 4 intracellular, independently grown and harvested. One replicate per array.
Project description:Francisella tularensis, the bacterial cause of tularemia, infects the liver and replicates in hepatocytes in vivo and in vitro. However, the factors that govern adaptation of F. tularensis to the intrahepatocytic niche have not been identified. Using cDNA microarrays, we determined the transcriptional profile of the live vaccine strain (LVS) of F. tularensis grown in the FL83B murine hepatocytic cell line compared to that of F. tularensis cultured in broth. The fslC gene of the fsl operon was the most highly upregulated. Deletion of fslC eliminated the ability of the LVS to produce siderophore, which is involved in uptake of ferric iron, but it did not impair its growth in hepatocytes, A549 epithelial cells, or macrophages. Therefore, we sought an alternative means by which F. tularensis might obtain iron. Deletion of feoB, which encodes a putative ferrous iron transporter, retarded replication of the LVS in iron-restricted media, reduced its growth in hepatocytic and epithelial cells, and impaired its acquisition of iron. Survival of mice infected intradermally with a lethal dose of the LVS was slightly improved by deletion of fslC but was not altered by loss of feoB. However, the ?feoB mutant showed diminished ability to colonize the lungs, liver, and spleen of mice that received sublethal inocula. Thus, FeoB represents a previously unidentified mechanism for uptake of iron by F. tularensis. Moreover, failure to produce a mutant strain lacking both feoB and fslC suggests that FeoB and the proteins of the fsl operon are the only major means by which F. tularensis acquires iron.
Project description:Francisella tularensis LVS was grown to mid log phase and culture split into two. One culture served as control and other added H2O2 to final concentration of 0.03%. After 20 min at 37 C, RNA was isolated from both cultures
Project description:These microarray studies are part of a larger study characterizing a deletion mutant of the putative transcriptional regulator IclR in Francisella tularensis LVS and SchuS4 strains. The microarrays were performed using RNA isolated from wild-type LVS and a LVS iclR deletion mutant after growing in Chamberlain?s defined media pH 6.3 to early mid-log phase. Results suggest that IclR affects expression of several genes after determining statistically significant differences by SAM. Comparison of gene expression between LVS iclR deletion mutant vs. wild-type LVS.
Project description:Bacterial attenuation is typically thought of as reduced bacterial growth in the presence of constant immune pressure. Infection with Francisella tularensis elicits innate and adaptive immune responses. Several in vivo screens have identified F. tularensis genes necessary for virulence. Many of these mutations render F. tularensis defective for intracellular growth. However, some mutations have no impact on intracellular growth, leading us to hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS (live vaccine strain) clpB (FTL_0094) mutant because this strain was attenuated in pneumonic tularemia yet induced a protective immune response. The attenuation of LVS clpB was not due to an intracellular growth defect, as LVS clpB grew similarly to LVS in primary bone marrow-derived macrophages and a variety of cell lines. We therefore determined whether LVS clpB induced an altered immune response compared to that induced by LVS in vivo. We found that LVS clpB induced proinflammatory cytokine production in the lung early after infection, a process not observed during LVS infection. LVS clpB provoked a robust adaptive immune response similar in magnitude to that provoked by LVS but with increased gamma interferon (IFN-?) and interleukin-17A (IL-17A) production, as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of a nonessential gene(s) that is involved in bacterial immune evasion, like F. tularensis clpB, can serve as a model for the rational design of attenuated vaccines.
Project description:Francisella tularensis is the causative agent of tularemia and a category A bioterrorism agent. The molecular basis for the extreme virulence of F. tularensis remains unclear. Our recent study found that capBCA, three neighboring genes, are necessary for the infection of F. tularensis live vaccine strain (LVS) in a respiratory infection mouse model. We here show that the capBCA genes are necessary for in vivo growth of F. tularensis LVS in the lungs, spleens, and livers of BALB/c mice. Unmarked deletion of capBCA in type A strain Schu S4 resulted in significant attenuation in virulence although the level of the attenuation in Schu S4 was much less profound than in LVS. We further demonstrated that CapB protein is produced at a low level under the in vitro culture conditions, and capB alone is necessary for in vivo growth of F. tularensis LVS in the lungs of BALB/c mice. Finally, deletional mutations in capB alone or capBCA significantly impaired intracellular growth of F. tularensis LVS in cultured macrophages, thus suggesting that the capBCA genes are necessary for intracellular adaptation of F. tularensis. The requirement of this gene locus in intracellular adaption at least in part explains the significant attenuation of F. tularensis capBCA mutants in virulence.
Project description:MicroRNA expression profiling in human liver progenitor cells following hepatocytic differentiation identified miR-122 and miR-194 as the microRNAs most strongly upregulated during hepatocytic differentiation of progenitor cells. MiR-194 was also highly upregulated following hepatocytic differentiation of human embryonic stem cells (hESCs). Overexpression of miR-194 in progenitor cells accelerated their differentiation into hepatocytes, as measured by morphological features such as canaliculi and expression of hepatocytic markers. Overexpression of miR-194 in hESCs induced their spontaneous differentiation, a phenotype accompanied with accelerated loss of the pluripotent factors OCT4 and NANOG and decrease in mesoderm marker HAND1 expression. We then identified YAP1 as a direct target of miR-194. Inhibition of YAP1 strongly induced hepatocytic differentiation of progenitor cells and YAP1 overexpression reversed the miR-194-induced hepatocytic differentiation of progenitor cells. In conclusion, we identified miR-194 as a potent inducer of hepatocytic differentiation of progenitor cells and further identified YAP1 as a mediator of miR-194's effects on hepatocytic differentiation and liver progenitor cell fate. Stem Cells 2016;34:1284-1296.