Plasmodium berghei-Mediated NRF2 Activation in Infected Hepatocytes Enhances Parasite Survival

Figure 2

NRF2 is activated in P. berghei infected cells. (a) Schematic of the OKD48 reporter [28]. NRF2 is composed of 7 functional domains NRF2-ECH homology (Neh) domains 1-7. The Neh1 domain (green) contains the conserved CNC-bZIP region which functions in DNA binding. The Neh2 domain (red) is involved in KEAP1 binding and ubiquitination-dependent degradation. The reporter consists of amino acids 1-433 of human NRF2 (blue) fused to a NanoLuc luciferase (yellow). The fusion protein is controlled by an NRF2 inducible promoter containing 3 ARE sequences (violet). Endogenous NRF2 activation leads to the expression of the reporter and stabilization of the NRF2(1-433)-NLuc fusion protein which can be measured by standard bioluminescence assay. Schematic created with BioRender.com. (b) HuH7 cells stably transduced with the OKD48 NRF2-reporter construct [28] were infected with sporozoites constitutively expressing mCherry (PbmCherry). 5 hours post infection (hpi) cells were sorted by FACS in an infected and a noninfected control population. As a positive control, cells were treated with 1 μM sodium arsenite (ASN) for 6 hours. 6 hpi, luciferase activity was measured, and the signal was normalized to the number of living cells in each sample. Sorted infected cells were compared to sorted noninfected cells, while ASN treated cells were compared to untreated cells. The graph shows the relative induction compared to the respective control. The experiment was carried out 3 times independently. Depicted are mean and SD. Significance was determined by -test ( and ). (c) Generation of NRF2^-/- cells. HEK293T cells were transfected with either a Cas9-encoding plasmid or a plasmid encoding a single guideRNA and plasmids needed for lentiviral packaging and envelope. The viruses were harvested, and HuH7 cells were transduced with both the lentivirus containing the Cas9 and a virus containing one of the two guideRNAs. 2 different guideRNAs were used separately. Cells were then cloned out by limiting dilution and screened for knockout clones by western blot and sequencing. For the western blot, HuH7 WT cells and four different NRF2 knockout clones were cultivated in growth medium containing 10 μM sodium arsenite and 10 μM MG-132 to accumulate NRF2 protein. After 8 hours, cells were lysed and western blot analysis was performed. Whole protein lysates were separated on a 10% acrylamide gel, blotted on a nitrocellulose membrane, and probed with an anti-NRF2 antibody. As a loading control α-tubulin was detected, the targeted sequence of NRF2 exon 4 is depicted. In green and blue, the regions recognized by the two different guideRNAs are highlighted. Sequence analysis showed three to four alleles in all four clones obtained. Schematic created with BioRender.com. (d) HuH7 NRF2^-/- cells (clone g2–5) stably transduced with the OKD48 NRF2-reporter construct [28] were treated with 1 μM sodium arsenite (ASN) for 6 hours. Subsequently, reporter activity was measured by luciferase assay and the signal was normalized to the number of living cells in each sample. The graph shows the relative induction compared to the nontreated control. Mean and SD of three independent experiments are depicted. The -value was calculated using a Student’s -test (ns: ).

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