Exploiting the Unique ATP-Binding Pocket of <i>Toxoplasma</i> Calcium-Dependent Protein Kinase 1 To Identify Its Substrates

Apicomplexan parasites rely on calcium as a second messenger to regulate a variety of essential cellular processes. Calcium-dependent protein kinases (CDPK), which transduce these signals, are conserved among apicomplexans but absent from mammalian hosts, making them attractive targets for therapeutic intervention. Despite their importance, the signaling pathways CDPK regulate remain poorly characterized, and their protein substrates are completely unknown. In <i>Toxoplasma gondii</i>, CDPK1 is required for calcium-regulated secretion from micronemes, thereby controlling motility, invasion, and egress from host cells. CDPK1 is unique among parasite and mammalian kinases in containing glycine at the key “gatekeeper” residue, which results in an expanded ATP-binding pocket. In the present study, we use a synthetic ATPγS analogue that displays steric complementarity to the ATP-binding pocket and hence allows identification of protein substrates based on selective thiophosphorylation. The specificity of this approach was validated by the concordance between the identified phosphorylation sites and the <i>in vitro</i> substrate preference of CDPK1. We further demonstrate that the phosphorylation of predicted substrates is dependent on CDPK1 both <i>in vivo</i> and <i>in vitro</i>. This combined strategy for identifying the targets of specific protein kinases provides a platform for defining the roles of CDPKs in apicomplexans

Representative fluorescence microscope images of dityrosine autofluorescence in sporulated and unsporulated oocysts of WT, <i>Δh1</i>, <i>Δh2</i>, and <i>Δh1Δh2</i> oocysts.

<p>All images were taken at 1000-1600ms exposure using a DAPI UV filter, but due to rapid photobleaching and differing levels of background signal in different oocyst fecal suspensions, direct comparison and quantification of fluorescence is not feasible. Scale bar = 10μm.</p

Distribution of parasite sexual developmental stages in cat intestinal ileums infected with WT, <i>Δh1</i> or <i>Δh2</i> mutant parasites.

<p>(A) Pooled counts from four (WT, <i>Δh2</i>) or six (<i>Δh1</i>) independent intestinal sections from an infected cat are summarized. (B) The relative proportion of each stage observed across the four or six samples are shown +/- SD. No significant differences in the distribution of parasite stages were seen (<i>P></i> 0.9999, Two-way ANOVA).</p

Development of bradyzoites <i>in vitro</i> and <i>in vivo</i>.

<p>(A) There was no significant difference in bradyzoite differentiation <i>in vitro</i> across the parasite lines in either tachyzoite conditions (left) (<i>P</i>> 0.99) or bradyzoite conditions (right) (<i>P</i>> 0.99) (Two-way ANOVA).(B) Representative pictures of tachyzoites, partial cysts, and complete cysts produced <i>in vitro</i> as assessed by DBL staining. (C) Brain cyst yields in mice 1–2 months post-infection. All parasite lines produced similar numbers of tissue cysts <i>in vivo</i> (Kruskal-Wallis, ns).</p

Copy number analysis of <i>AAH2</i> in <i>T</i>. <i>gondii</i> strains.

<p>(A) Copy number variation (CNV) of Tg_ME49_212740 (<i>AAH2</i>) in five representative strains. (B) CNV at each base across the <i>AAH2</i> gene in ME49. Red lines indicate the start and stop codons of <i>AAH2</i>. (C) PCR using primers (Z28+ Z65) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s003" target="_blank">S3 Table</a>) common to both <i>AAH1</i> and <i>AAH2</i> were used against the ME49 genome to amplify both genes for Sanger sequencing and single nucleotide polymorphism (SNP) determination. (D) Sanger sequencing of PCR fragments against the <i>AAH</i> genes of the wild type ME49 <i>Δhxg</i>::<i>Luc</i> strain shows a 2:1 ratio of <i>AAH2</i> to <i>AAH1</i> SNPs, indicating a duplication of the <i>AAH2</i> gene. Each SNP in the chromatograph is marked by a red dot. In the <i>Δaah2</i>::<i>HXG</i> knockout, all <i>AAH2</i> SNPs are no longer visible, indicating loss of both copies of the <i>AAH2</i> gene.</p

Synthetic Chondramide A Analogues Stabilize Filamentous Actin and Block Invasion by <i>Toxoplasma gondii</i>

Apicomplexan parasites such as <i>Toxoplasma gondii</i> rely on actin-based motility to cross biological barriers and invade host cells. Key structural and biochemical differences in host and parasite actins make this an attractive target for small-molecule inhibitors. Here we took advantage of recent advances in the synthesis of cyclic depsipeptide compounds that stabilize filamentous actin to test the ability of chondramides to disrupt growth of <i>T. gondii in vitro</i>. Structural modeling of chondramide A (<b>2</b>) binding to an actin filament model revealed variations in the binding site between host and parasite actins. A series of 10 previously synthesized analogues (<b>2b</b>–<b>k</b>) with substitutions in the β-tyrosine moiety blocked parasite growth on host cell monolayers with EC₅₀ values that ranged from 0.3 to 1.3 μM. <i>In vitro</i> polymerization assays using highly purified recombinant actin from <i>T. gondii</i> verified that synthetic and natural product chondramides target the actin cytoskeleton. Consistent with this, chondramide treatment blocked parasite invasion into host cells and was more rapidly effective than pyrimethamine, a standard therapeutic agent. Although the current compounds lack specificity for parasite vs host actin, these studies provide a platform for the future design and synthesis of synthetic cyclic peptide inhibitors that selectively disrupt actin dynamics in parasites

Disruption of the <i>AAH1</i> and <i>AAH2</i> genes.

<p>(A) Schematic of the <i>AAH2</i> knockout strategy in the wild-type ME49 <i>Δhxg</i>::<i>Luc</i> strain (referred to as WT). A CRISPR-Cas9 construct with guide RNAs targeted to the 5’ and 3’ UTRs of <i>AAH2</i> was co-transfected with the <i>pΔaah2</i>::<i>HXG</i> plasmid (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s002" target="_blank">S2 Table</a>) and selected for with MPA +Xanthine to delete <i>AAH2</i> to produce the clone <i>Δaah2</i>::<i>HXG</i> (<i>Δh2-HXG</i>). Subsequently, the <i>HXG</i> gene was replaced with either a clean fusion of the <i>AAH2</i> 5’ and 3’ UTRs (<i>pΔaah2</i>) or an <i>AAH2</i> cDNA construct (<i>pAAH2</i>). (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s002" target="_blank">S2 Table</a>) using 6-thioxanthine selection against the <i>HXG</i> locus to create the clean knockout clone <i>Δaah2</i> (<i>Δh2</i>) (upper panel) and the complement clone <i>Δaah2</i>::<i>AAH2</i> (<i>Δh2-H2</i>) (lower panel). Yellow Bars: CRISPR targeting sites. Black bars: PCR screening primer target regions (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s003" target="_blank">S3 Table</a>). (B) Schematic of the knockout strategy for <i>AAH1</i>. A CRISPR-Cas9 construct with guide RNAs targeted to the 5’ and 3’ UTRs of <i>AAH1</i> was co-transfected with the <i>pΔaah1</i>::<i>DHFR-Ts</i> repair construct (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s002" target="_blank">S2 Table</a>) into WT or <i>Δaah2</i> parasites to create the clones <i>Δaah1</i> (<i>Δh1</i>) and <i>Δaah1Δaah2</i> (<i>Δh1Δh2</i>). Transfectants were selected for via pyrimethamine resistance. Subsequently, using <i>pΔuprt</i>::<i>AAH1</i>::<i>HXG</i>, a cDNA copy of <i>AAH1</i> driven by its native 5’ and 3’ UTRs was complemented into the <i>UPRT</i> locus by means of the <i>HXGPRT</i> drug resistance marker selected for with MPA +Xanthine, negative selection against <i>UPRT</i> with FUDR, and a single-cutting CRISPR-Cas9 construct targeted to the <i>UPRT</i> gene (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s002" target="_blank">S2 Table</a>), creating the complement clones <i>Δaah1-AAH1</i> (<i>Δh1-H1</i>) and <i>Δaah1Δaah2-AAH1</i> (<i>Δh1Δh2-H1</i>). Brown & Yellow Bars: CRISPR targeting sites. Black bars: PCR screening primer target regions (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s003" target="_blank">S3 Table</a>). (C) PCR verification of successful ablation and complementation of knockouts. Expected product sizes: <i>Tubulin</i> (Tub): 0.378kb. <i>AAH1</i> (H1): 0.745kb (Native), 0.278kb (cDNA). <i>AAH2</i> (H2): 0.745kb (Native), 0.278kb (cDNA). (D) Growth assays of parasites seeded into 96-well plates and allowed to proliferate for 24 h, then quantified using a luciferase assay. The WT, <i>Δh1</i>, Δh2, <i>Δh1Δh2</i>, <i>Δh1-H1</i>, <i>Δh2-H2</i>, and <i>Δh1Δh2-H1</i> parasites showed no significant difference in total growth (Kruskal-Wallis test, <i>P</i> = 0.0672, N = 3 per strain).</p