Acquisition of exogenous fatty acids renders apicoplast-based biosynthesis dispensable in tachyzoites of Toxoplasma

Abstract

Toxoplasma gondii is a common protozoan parasite that infects a wide range of hosts, including livestock and humans. Previous studies have suggested that the type 2 fatty acid synthesis (FAS2) pathway, located in the apicoplast (a nonphotosynthetic plastid relict), is crucial for the parasite's survival. Here we examined the physiological relevance of fatty acid synthesis in T. gondii by focusing on the pyruvate dehydrogenase complex and malonyl-CoA-[acyl carrier protein] transacylase (FabD), which are located in the apicoplast to drive de novo fatty acid biosynthesis. Our results disclosed unexpected metabolic resilience of T. gondii tachyzoites, revealing that they can tolerate CRISPR/Cas9–assisted genetic deletions of three pyruvate dehydrogenase subunits or FabD. All mutants were fully viable in prolonged cultures, albeit with impaired growth and concurrent loss of the apicoplast. Even more surprisingly, these mutants displayed normal virulence in mice, suggesting an expendable role of the FAS2 pathway in vivo. Metabolic labeling of the Δpdh-e1α mutant showed reduced incorporation of glucose-derived carbon into fatty acids with medium chain lengths (C14:0 and C16:0), revealing that FAS2 activity was indeed compromised. Moreover, supplementation of exogenous C14:0 or C16:0 significantly reversed the growth defect in the Δpdh-e1α mutant, indicating salvage of these fatty acids. Together, these results demonstrate that the FAS2 pathway is dispensable during the lytic cycle of Toxoplasma because of its remarkable flexibility in acquiring fatty acids. Our findings question the long-held assumption that targeting this pathway has significant therapeutic potential for managing Toxoplasma infections.

Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma

“…Our quantitative proteomic analysis shows potential compensatory mechanisms may be used by the parasites in response this early perturbation of the apicoplast lipid metabolism that precedes organelle loss. Tachyzoites are indeed known to be able to use exogenous lipid sources to adapt metabolically (86, 87), and interestingly upon depletion of TgSufS we observed a pattern of overexpression for ER-located enzymes involved in the synthesis of several phospholipids and ceramides (Table S3). These lipids are usually synthesized in the ER from apicoplast-synthesized precursors, but this may clearly indicate a compensatory mechanism that would make use of precursors scavenged form the host instead.…”

“…Interestingly, we could show that perturbing the Suf pathway, which is supposedly important for Fe-S-containing enzyme LipA, impacts the lipoylation of E2 subunit of the apicoplast-located PDH (Figure 7C). The PDH complex catalyzes the production of acetyl-CoA, which is the first step of the FASII system, and perturbation of either the PDH or other steps of the FASII system lead to a loss of the organelle and severely impairs fitness of the parasites (38, 86). Our quantitative proteomic analysis shows potential compensatory mechanisms may be used by the parasites in response this early perturbation of the apicoplast lipid metabolism that precedes organelle loss.…”

Aminoglycerophospholipid flipping and P4-ATPases in Toxoplasma gondii

“…Specifically, internalized lysophospholipids are acylated to yield PtdEtn and PtdCho, and their transport rates are sufficient to support the membrane biogenesis and growth of S. cerevisiae . Similarly, T. gondii tachyzoites satisfy their lipid requirements by de novo synthesis as well as salvage from the environment ( 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ). Soon after the invasion, the PV membrane becomes intimately associated with the host mitochondria and endoplasmic reticulum ( 60 ), which are two major sites of phospholipid biosynthesis in mammalian cells ( 61 ).…”

“…After invasion, the parasite dwells in a parasitophorous vacuole (PV) that is originally derived from the host plasma membrane but profoundly modified to enable its asexual reproduction by endodyogeny ( 3 , 4 ). The formation of progeny and concurrent expansion of the PV require extensive membrane biogenesis that is satisfied by de novo synthesis as well as salvage of lipids from the parasite environment ( 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ).…”

The NTP generating activity of pyruvate kinase II is critical for apicoplast maintenance in Plasmodium falciparum

“…Pyruvate should be required for fatty acid (FASII) and isoprenoid precursor synthesis in the apicoplast of T. gondii . The FASII pathway was thought to be essential for apicoplast maintenance and parasite survival ( Mazumdar et al, 2006 ; however, recent studies show that several PDH subunits and FASII enzymes can be deleted; Krishnan et al, 2020 ; Liang et al, 2020 ). These FASII-null parasite lines grow poorly in vitro and one would expect the PyrKII deletion line to share this phenotype unless the apicoplast has some other source of pyruvate.…”