Figure 2.

Cryptosporidium lysyl-tRNA synthetase (CpKRS) is an essential drug target. (A) Cryptosporidium sporozoites (wild type) were transfected with CRISPR/Cas9 constructs designed to target the 3´ region of the KRS gene for tagging with mNeonGreen (KRS-mNG, teal) or for gene knockout (Δkrs; two independent experiments, magenta and black). IFN-γ KO mice (4 per cage) were infected by gavage; fecal samples were collected from each cage and analyzed for expression of NanoLuciferase (RLUs, relative luminescence units; RLUs/2 mg of fecal material plotted; mean ± SD of three technical replicates). RLU >1000 indicate successful infection of transgenic parasites. (B) Mice were infected with a conditional CpKRS Cryptosporidium strain (KRS-DDD). KRS is stabilized in the presence of TMP (trimethoprim delivered in the drinking water, black), and degraded in the absence of TMP (water only, magenta). Degradation of KRS in the absence of TMP prevents infection, as measured by RLU. Representative graph of 2 biological replicates performed, each with 3 technical replicates; mean ± SD at each time point. (C) Transcript levels of CpKRS quantified by RT-qPCR during in vitro infection (transcript abundance normalized to 18S rRNA levels at each timepoint and reported relative to KRS levels at 3 HPI). Mean ± SD with individual technical repeats indicated. Representative graph of 3 biological replicates performed, each with 3 technical replicates. (D) Immunofluorescence microscopy of intestines from mice infected with KRS-mNG from A (tissue collected day 13 post infection when infection peaked; actin stained with phalloidin, white; DNA stained with DAPI, cyan; KRS-mNG, yellow; cytoplasmic localized protein Cryptosporidium tryptophan synthase B, TrpB; magenta). KRS is expressed in asexual (single nuclei trophozoite, Troph; 8-nuclei Meront) and sexual stages. Scale bar 5 µm.