Fig. 3: A system for inducible knockdown reveals an essential role for PKG, CDPK4, ASP2 and ASP3 in egress or invasion.
From: Babesia divergens host cell egress is mediated by essential and druggable kinases and proteases
a, CRISPR–Cas9 is used to introduce an HA-DD-glmS or HA-glmS tag to the 3′ end of pkg by homologous recombination. The same approach was used for all genes. The arrows represent primers used to validate integration as shown in Extended Data Fig. 3e. KD, knockdown. b, Western blot analysis showing DD and glmS-induced knockdown of PKG over time. c, Growth of knockdown parasites over 72 h. Each point represents the mean of an individual experiment performed in technical triplicate and n = 3. The mean ± s.d. is shown. Data are normalized so +Shld is 100% for each replicate. Statistical analysis was conducted using two-way ANOVA with Dunnett correction for multiple comparisons (two sided). All statistical values shown are relative to +Shld−GlcN parasites of the same line. ***P = 0.0003; ****P < 0.0001. d, The number of parasites per iRBC assessed by microscopy after knockdown (−Shld+GlcN) of PKG or CDPK4 for 40 h. A total of 100 iRBCs were counted per condition, and 3 biological replicates are shown. The mean ± s.d. is shown. Statistical analysis was conducted using two-way ANOVA with Dunnett correction for multiple comparisons (two sided). All statistical values shown are relative to WT +Shld−GlcN parasites with the same N compared between conditions. **Exact P values are provided for the following in −Shld+GlcN conditions: WT versus PKG 1N P = 0.0028, WT versus CDPK4 1N P = 0.0030, WT versus PKG >4N P = 0.0031, WT versus CDPK4 P = 0.0089. e, Stained micrographs showing phenotypes after 24 h (ASP2 and ASP3) or 40 h (PKG and CDPK4) of knockdown (KD). Scale bars, 3 µm. For b–e, +Shld was 500 nM, +GlcN was 1 mM and −Shld−GlcN was 0 nM.
