Fig. 2: PfCERLI2 is essential for blood-stage growth.
a Schematic representation of the selection linked integration targeted gene disruption (SLI-TGD) system used for the attempted gene knockout of Pfcerli2. b Schematic representation of the haemagglutinin (HA) tag, and GlmS ribozyme system used to identify and knockdown PfCERLI2. A plasmid that contained a 3′ Pfcerli2 flank (2154bp-2912bp) with an HA-tag and GlmS ribyzome was transfected into 3D7 parasites by single crossover recombination. Following mRNA production, glucosamine-6-phosphate (G6P) binds to the GlmS ribozyme, leading to the cleavage of Pfcerli2 mRNA and subsequent protein knockdown. c To confirm integration of the Pfcerli2HAGlmS plasmid, gDNA was harvested from transfected parasites. A Pfcerli2 specific forward primer, A, with either a Pfcerli2 specific, B (WT), or GlmS specific, C (integrated), reverse primer was used to confirm WT DNA sequence or integration of the Pfcerli2HAGlmS plasmid. d Western blot of ring, trophozoite, or schizont stage PfCERLI2HAGlmS lysates probed with either anti-HA (PfCERLI2) or anti-ERC (loading control) antibodies. N = 3 biological replicates. e Western blot of PfCERLI2HAGlmS schizont lysates either GLCN treated (+) or untreated (−) and probed with anti-HA (PfCERLI2) normalised to ERC (loading control) antibodies. A graph quantifying the relative level of expression between treatments. N = 3 biological replicates, error bars = SEM. f PfCERLI2HAGlmS or 3D7 WT parasites were treated with increasing concentrations of GLCN for 72 h, with the trophozoite-stage parasitaemia determined by flow cytometry. Growth is expressed as a percentage of an untreated media control. n = 4, box represents the 25th to 75th percentile, median and maximum value are shown. X-axis presented as a log 2 scale for viewing purposes.
