2018-03-10

a, Schematic overview. C. parvum sporozoites were prepared from oocysts purified from infected calves and electroporated in the presence of plasmid DNA prior to infection of HCT-8 cells. Luminescence measurements (the means of 3 technical replicates, s.d. shown as error bar) of T. gondii (f), human HCT-8 cells (g), or C. parvum (all other panels, blue) transfected with nanoluciferase (Nluc) expression plasmids. C. parvum transfection requires electroporation (b) of DNA (c) into parasites (d). Transfection also requires plasmids to carry parasite specific promoter sequences (e, f, testing C. parvum and T. gondii promoters in both parasites), and is susceptible to the Cryptosporidium drug nitazoxanide (h). Lipofection of HCT-8 cells with the original Nluc plasmid pNL1.1 (Promega), but not derived parasite vectors results in luciferase activity in the host alone (g). Choice of promoter (i, Eno, enolase; Aldo, aldolase; Tub, alpha-tubulin 5′ regions, the 3′ UTR was uniformly from the enolase gene) or codon composition (j, oNluc, optimized to 35% GC) influences expression level in C. parvum. Note automatic gain adjustment of luminescence measurements, units are not comparable between panels. Independent experiments were repeated 3 times, and representative data are shown.