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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., Graphical abstract Image 1, Highlights • Tartrolon E is a broad-spectrum anti-apicomplexan compound. • T. gondii responds to tartrolon E treatment by upregulating expression of the coccidian gene, trg. • The gene product of trg has multiple transmembrane domains and is phosphorylated and thus may play a role in signaling. • Deletion of trg results in an increase in parasite resistance to tartrolon E treatment.

Cryptosporidium is a waterborne gastrointestinal parasite that causes outbreaks of diarrheal disease worldwide. Despite the impact of this parasite on human health there are no effective drugs or vaccines. Transcriptomic data can provide insights into host-parasite interactions that lead to identification of targets for therapeutic interventions. However, for Cryptosporidium, interpreting transcriptomes has been challenging, in part due to the presence of multiple life cycle stages, the lack of appropriate host cells and the inability to culture the parasite through its complete life cycle. The recent improvements in cell culture and the ability to tag and isolate specific life cycle stages will radically improve transcriptomic data and advance our understanding of Cryptosporidium host-parasite interactions.

Apicomplexan parasites cause severe disease in both humans and their domesticated animals. Since these parasites readily develop drug resistance, development of new, effective drugs to treat infection caused by these parasites is an ongoing challenge for the medical and veterinary communities. We hypothesized that invertebrate-bacterial symbioses might be a rich source of anti-apicomplexan compounds because invertebrates are susceptible to infections with gregarines, parasites that are ancestral to all apicomplexans. We chose to explore the therapeutic potential of shipworm symbiotic bacteria as they are bona fide symbionts, are easily grown in axenic culture and have genomes rich in secondary metabolite loci [1,2]. Two strains of the shipworm symbiotic bacterium, Teredinibacter turnerae, were screened for activity against Toxoplasma gondii and one strain, T7901, exhibited activity against intracellular stages of the parasite. Bioassay-guided fractionation identified tartrolon E (trtE) as the source of the activity. TrtE has an EC50 of 3 nM against T. gondii, acts directly on the parasite itself and kills the parasites after two hours of treatment. TrtE exhibits nanomolar to picomolar level activity against Cryptosporidium, Plasmodium, Babesia, Theileria, and Sarcocystis; parasites representing all branches of the apicomplexan phylogenetic tree. The compound also proved effective against Cryptosporidium parvum infection in neonatal mice, indicating that trtE may be a potential lead compound for preclinical development. Identification of a promising new compound after such limited screening strongly encourages further mining of invertebrate symbionts for new anti-parasitic therapeutics., Author summary Apicomplexans are intracellular protozoan parasites that cause significant disease in humans and the livestock we rely on for food. Because these parasites easily develop drug resistance, new drugs are always needed. To identify new anti-apicomplexan drugs, we investigated the compounds produced by symbiotic bacteria of shipworms, marine mollusks that burrow into and eat wood. We screened shipworm symbiotic bacteria for anti-parasitic activity and identified a compound, tartrolon E, with potent, rapid, highly selective and irreversible activity against parasites representing all branches of the apicomplexan tree. This compound was also highly effective in neonatal mice against infection with the intestinal apicomplexan parasite, Cryptosporidium. This study describes the first pan-anti-apicomplexan compound and unveils an unexplored source of anti-parasitic compounds.