Search

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.

Cryptosporidium sp. are apicomplexan parasites that cause significant morbidity and possible mortality in humans and valuable livestock. There are no drugs on the market that are effective in the population most severely affected by this parasite. This study is the first high-throughput screen for potent anti-Cryptosporidium natural products sourced from a unique marine compound library. The Harbor Branch Oceanographic Institute at Florida Atlantic University has a collection of diverse marine organisms some of which have been subjected to medium pressure liquid chromatography to create an enriched fraction library. Numerous active compounds have been discovered from this library, but it has not been tested against Cryptosporidium parvum. A high-throughput in vitro growth inhibition assay was used to test 3764 fractions in the library, leading to the identification of 23 fractions that potently inhibited the growth of Cryptosporidium parvum. Bioassay guided fractionation of active fractions from a deep-sea sponge, Leiodermatium sp., resulted in the purification of leiodolide A, the major active compound in the organism. Leiodolide A displayed specific anti-Cryptosporidium activity at a half maximal effective concentration of 103.5 nM with selectivity indexes (SI) of 45.1, 11.9, 19.6 and 14.3 for human ileocecal colorectal adenocarcinoma cells (HCT-8), human hepatocellular carcinoma cells (Hep G2), human neuroblastoma cells (SH-SY5Y) and green monkey kidney cells (Vero), respectively. The unique structure of leiodolide A provides a valuable drug scaffold on which to develop new anti-Cryptosporidium compounds and supports the importance of screening natural product libraries for new chemical scaffolds.

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.