Examination of Secondary Metabolite Biosynthesis in Apicomplexa

Abstract

Natural product discovery has traditionally relied on the isolation of small molecules from producing species, but genome-sequencing technology and advances in molecular biology techniques have expanded efforts to a wider array of organisms. Protists represent an underexplored kingdom for specialized metabolite searches despite bioinformatic analysis that suggests they harbor distinct biologically active small molecules. Specifically, pathogenic apicomplexan parasites, responsible for billions of global infections, have been found to possess multiple biosynthetic gene clusters, which hints at their capacity to produce polyketide metabolites. Biochemical studies have revealed unique features of apicomplexan polyketide synthases, but to date, the identity and function of the polyketides synthesized by these megaenzymes remains unknown. Herein, we discuss the potential for specialized metabolite production in protists and the possible evolution of polyketide biosynthetic gene clusters in apicomplexan parasites. We then focus on a polyketide synthase from the apicomplexan Toxoplasma gondii to discuss the unique domain architecture and properties of these proteins when compared to previously characterized systems, and further speculate on the possible functions for polyketides in these pathogenic parasites.

Exploring the Chain Release Mechanism from an Atypical Apicomplexan Polyketide Synthase

“…Among Apicomplexa, PKS gene clusters are restricted to the cyst-forming coccidian subclass. This association as well as the upregulation of Tg PKS2 during the cyst-forming bradyzoite stage suggests a potential role of the metabolite(s) produced in the cyst lipid wall make up . In C.…”

“…This association as well as the upregulation of TgPKS2 during the cyst-forming bradyzoite stage suggests a potential role of the metabolite(s) produced in the cyst lipid wall make up. 22 In C. parvum, a related coccidian parasite, putative PKS and FAS enzymes are upregulated at the lipid-walled oocyst stage, further supporting this proposal, 58 but the structure and function of polyketide-specialized metabolites in apicomplexan remain unknown. Currently, treatments with efficacy toward the latent cyst stage of toxoplasmosis are lacking; therefore, TgPKS2 or other similar biosynthetic gene clusters in Coccidia may be interesting therapeutic targets.…”

“…Toxoplasma gondii type I strain RH tachyzoite cultures, a kind gift from Prof. Sebastian Lourido, were propagated in Vero cells (ATCC, CCL-81) as described previously in a standard tissue culture incubator at 37 °C. , Parasites were grown for 72 h in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) (v:v), 1% antibiotic-antimycotic (Thermo Fisher Scientific) and subsequently released from Vero cell monolayers by passage through a 20-gauge needle and filtered through a 3 μm polycarbonate membrane (Thermo Fisher Scientific). Parasites were collected by centrifugation (4000× g , 10 min), and mRNA was extracted using a Quick-RNA Miniprep kit (NEB) according to the manufacturer’s protocol.…”

“…For example, the apicomplexan parasite Toxoplasma gondii encodes two type I PKSs, TgPKS1 and TgPKS2. 22 We recently elucidated the domain architecture of the 18.8 kb TgPKS2 (Figure 1A). 23 TgPKS2 has an intriguing putative substrate release containing a KS and TR domain, an arrangement that is rarely observed.…”

“…Apicomplexans, which include the parasites responsible for malaria, toxoplasmosis, and cryptosporidiosis, encode polyketide synthases with predicted TR domains that have yet to be characterized. For example, the apicomplexan parasite Toxoplasma gondii encodes two type I PKSs, Tg PKS1 and Tg PKS2 . We recently elucidated the domain architecture of the 18.8 kb Tg PKS2 (Figure A) …”