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. 2020 Dec:14:1-7.
doi: 10.1016/j.ijpddr.2020.07.003. Epub 2020 Jul 25.

A conserved coccidian gene is involved in Toxoplasma sensitivity to the anti-apicomplexan compound, tartrolon E

Gregory D Bowden  1 , Patricia M Reis  1 , Maxwell B Rogers  1 , Rachel M Bone Relat  1 , Kelly A Brayton  1 , Sarah K Wilson  2 , Bruno Martorelli Di Genova  2 , Laura J Knoll  2 , Felix J Nepveux V  3 , Albert K Tai  4 , Timothy R Ramadhar  5 , Jon Clardy  5 , Roberta M O'Connor  6
Affiliations

A conserved coccidian gene is involved in Toxoplasma sensitivity to the anti-apicomplexan compound, tartrolon E

Gregory D Bowden et al. Int J Parasitol Drugs Drug Resist. 2020 Dec.

Abstract

New treatments for the diseases caused by apicomplexans are needed. Recently, we determined that tartrolon E (trtE), a secondary metabolite derived from a shipworm symbiotic bacterium, has broad-spectrum anti-apicomplexan parasite activity. TrtE inhibits apicomplexans at nM concentrations in vitro, including Cryptosporidium parvum, Toxoplasma gondii, Sarcocystis neurona, Plasmodium falciparum, Babesia spp. and Theileria equi. To investigate the mechanism of action of trtE against apicomplexan parasites, we examined changes in the transcriptome of trtE-treated T. gondii parasites. RNA-Seq data revealed that the gene, TGGT1_272370, which is broadly conserved in the coccidia, is significantly upregulated within 4 h of treatment. Using bioinformatics and proteome data available on ToxoDB, we determined that the protein product of this tartrolon E responsive gene (trg) has multiple transmembrane domains, a phosphorylation site, and localizes to the plasma membrane. Deletion of trg in a luciferase-expressing T. gondii strain by CRISPR/Cas9 resulted in a 68% increase in parasite resistance to trtE treatment, supporting a role for the trg protein product in the response of T. gondii to trtE treatment. Trg is conserved in the coccidia, but not in more distantly related apicomplexans, indicating that this response to trtE may be unique to the coccidians, and other mechanisms may be operating in other trtE-sensitive apicomplexans. Uncovering the mechanisms by which trtE inhibits apicomplexans may identify shared pathways critical to apicomplexan parasite survival and advance the search for new treatments.

Keywords: Anti-apicomplexan; CRISPR/Cas9; Drug discovery; Natural products; Tartrolon E; Toxoplasma.

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Conflict of interest statement

Please declare any financial or personal interests that might be potentially viewed to influence the work presented. Interests could include consultancies, honoraria, patent ownership or other. If there are none state ‘there are none’.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
CRISPR/Cas9 mediated deletion of trg. (A) The plasmid used to provide T. gondii-optimized expression of the Cas9 enzyme and sgRNA for targeting trg. (B) The plasmid containing a mCherry expression cassette (red, pSAG1-mCherry-DHFR 3′ UTR) surrounded by 1 kb areas of sequence identity to the flanking regions of trg (grey) and a GFP expression cassette (green, pTUB1-eGFP-SAG1 3′ UTR) used to provide a repair template for homologous recombination of disrupted trg locus. (C) Expression of Cas9 with sg272370 guide RNA (pink) targeted trg and induced a double-stranded break. Linearized pBC-mCherry-272370-GFP plasmid provided a template for double homologous recombination, which is designed to replace the entire trg gene with mCherry and lose GFP for sorting. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2
Fig. 2
T. gondii responds to trtE treatment by upregulating trg in a rapid dose-dependent manner. (A) Changes in transcript abundance of trg in T. gondii parasites treated with 24.2 nM trtE or DMSO carrier control. (B) Expression of trg quantified by RT-qPCR of RNA extracted from T. gondii parasites treated with trtE (24.2 nM), known inhibitory compound pyrimethamine (Pyr, 10 μM), or 0.1% DMSO carrier control at 1, 2, 4 h post treatment. Three technical replicates were performed. Treatment of parasites with trtE significantly increased the expression of trg at 4 h post treatment (p ≤ 0.0001). (C) Expression of trg quantified by RT-qPCR of RNA extracted from T. gondii parasites treated with various concentrations of trtE or 0.1% DMSO carrier control. RNA was isolated from each condition 4 h post treatment and RT-qPCR samples were run in triplicate. Fold-change in gene expression for RT-qPCR experiments was calculated using the 2−ΔΔCt method and normalized by the expression of parasite actin. The relationship between expression of trg and concentration of the trtE treatment was determined to be linear (R2 = 0.7776, p ≤ 0.0001).
Fig. 3
Fig. 3
Schematic of identified homologs of Trg in coccidian parasites. Protein homologs to Trg (TGME49_272370) with predicted transmembrane domains (green) in order of descending sequence similarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4
Fig. 4
Deletion of trg increases parasite resistance to trtE treatment. (A) PCR detecting trg in gDNA isolated from T. gondii ME49ΔHPT:LUC parental line (P) and ME49 ΔHPT:LUC Δtrg clones. The anticipated 172 bp amplicon is detected in the parental line (white triangle) but was absent in the Δtrg clones. (B) Southern blot analysis of gDNA from the parental line and Δtrg clones digested with Bsu36I using mCherry probe to detect insertion events. The single anticipated 7.6 kb fragment present in all Δtrg clones (black triangle) was absent from the parental sample. (C) Parental (ME49 ΔHPT:LUC) and three Δtrg gene deletion mutants infected host cells for 24 h before being treated with varying concentrations of trtE. The growth of each strain was evaluated 24 h post treatment by LUC expression. Samples were run in triplicate, and three independent experiments performed. Estimation of the EC50 for each strain was accomplished using a four-parametric logistic regression in GraphPad Prism. The curve generated for the parental strain (R2 = 0.9797) estimated the EC50 to be 3.027 ng/ml (2.309–3.968; 95% CI). One curve adequately fit all the data from the three Δtrg clones tested (R2 = 0.9215, p = 0.16) with an estimated average EC50 of 5.088 ng/ml (4.144–6.247; 95% CI), which was significantly different from the EC50 of the parental line by extra sum-of-squares F test (p = 0.0004).
None
figs1RNA-Seq results. Differentially expressed genes of trtE-treated T. gondii RH parasites at 4, 8, and 12 h post treatment as determined by RNA-seq.
None
figs2RT-qPCR verification of RNA-Seq results. Expression of trg, TGGT1_311100, and bag1 transcripts in RNA extracted from T. gondii parasites treated with trtE (24.2 nM, red) or DMSO carrier control (0.1%, black) at 1, 2, and 4 h post treatment. Fold-change in gene expression was calculated using the 2−ΔΔCt method with three replicates. Treatment of parasites with trtE significantly increased the expression of trg at all time points (p = 0.04, 0.01, and 0.004 at 1, 2, 4 h, respectively). Expression of TGGT1_311100 during trtE treatment was not significantly different from treatment with DMSO (p = 0.4, 0.5, and 0.7 at 1, 2, 4 h respectively). At 4 h post treatment with trtE, expression of bradyzoite-specific gene bag1 was significantly lower (p = 0.001) than DMSO-treated parasites. Three technical replicates were performed on all samples.
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