Limits...
Phosphatidylthreonine and Lipid-Mediated Control of Parasite Virulence.

Arroyo-Olarte RD, Brouwers JF, Kuchipudi A, Helms JB, Biswas A, Dunay IR, Lucius R, Gupta N - PLoS Biol. (2015)

Bottom Line: The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum.The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion.Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Parasitology, Humboldt University, Berlin, Germany.

ABSTRACT
The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.

Show MeSH

Related in: MedlinePlus

Knockdown of PSS in the Δtgpts/TgPSS-2HA-DD strain does not rescue growth defect caused by the loss of PTS alone.(A) Autoradiography of TLC-resolved lipids after labeling of the indicated strains with radioactive serine. Parasites were cultured during the intracellular phase without or with Shield1 (0.5 μM, 24 hr) followed by labeling of extracellular parasites (5 x 107) with 14C-serine (2 μCi, 100 μM, 2 hr, 37°C). Lipids were resolved in chloroform/ethanol/water/triethylamine (30:35:7:35). For corresponding quantitative radiolabeling and phospholipid analysis, refer to S12 Fig. (B–C) Relative growth fitness of the parasite strains incubated with or without Shield1. Plaque assays were executed and quantified, as described in Materials and Methods. Note that growth of the Δtgpts/TgPSS-2HA-DD and Δtgpts strains are equivalent irrespective of Shield1 in cultures. Statistics was performed with respect to the untreated parental strain (mean ± SEM, n = 3 assays; *p < 0.05, **p < 0.01, ***p < 0.001).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4643901&req=5

pbio.1002288.g007: Knockdown of PSS in the Δtgpts/TgPSS-2HA-DD strain does not rescue growth defect caused by the loss of PTS alone.(A) Autoradiography of TLC-resolved lipids after labeling of the indicated strains with radioactive serine. Parasites were cultured during the intracellular phase without or with Shield1 (0.5 μM, 24 hr) followed by labeling of extracellular parasites (5 x 107) with 14C-serine (2 μCi, 100 μM, 2 hr, 37°C). Lipids were resolved in chloroform/ethanol/water/triethylamine (30:35:7:35). For corresponding quantitative radiolabeling and phospholipid analysis, refer to S12 Fig. (B–C) Relative growth fitness of the parasite strains incubated with or without Shield1. Plaque assays were executed and quantified, as described in Materials and Methods. Note that growth of the Δtgpts/TgPSS-2HA-DD and Δtgpts strains are equivalent irrespective of Shield1 in cultures. Statistics was performed with respect to the untreated parental strain (mean ± SEM, n = 3 assays; *p < 0.05, **p < 0.01, ***p < 0.001).

Mentions: To examine whether an elevated level of PtdSer underlies the observed growth phenotype in the PTS mutant, we created a double mutant (Δtgpts/TgPSS-2HA-DD; S10A Fig). The TgPSS gene was fused with a Shield1-regulated degradation domain (DD) and 2HA epitopes at 3’-end [18] to achieve a conditional expression of PSS protein. The PSS-2HA-DD fusion protein showed a predominant fluorescent signal in the ER (S10B Fig). We also observed apparent staining of PSS with the markers of mitochondrion (F1B) [19] and acidocalcisomes/plant-like vacuole (vacuolar proton pyrophosphatase 1; VP1) [20], whereas other organelles, micronemes, rhoptries, dense granules, and apicoplast did not show evident PSS staining (S11 Fig). Expression of PSS-2HA-DD could be regulated by exposure to Shield1 (S10B and S10C Fig). Metabolic labeling of parasite lipids with serine (PtdSer and nascent decarboxylated product PtdEtn) also confirmed that PSS activity was restored to the parental level in the absence of Shield1 (Fig 7A, S12A Fig). A knockdown of PSS activity reinstated PtdSer content in the Δtgpts strain (S12B Fig). Even a normal PtdSer pool, however, was unable to rectify the growth defect in the Δtgpts/TgPSS-2HA-DD double mutant, which mirrored plaques formed by the Δtgpts strain (Fig 7B and 7C). These results exclude the impact of amplified PtdSer in disrupting the lytic cycle, while strengthening the physiological importance of PtdThr for T. gondii.


Phosphatidylthreonine and Lipid-Mediated Control of Parasite Virulence.

Arroyo-Olarte RD, Brouwers JF, Kuchipudi A, Helms JB, Biswas A, Dunay IR, Lucius R, Gupta N - PLoS Biol. (2015)

Knockdown of PSS in the Δtgpts/TgPSS-2HA-DD strain does not rescue growth defect caused by the loss of PTS alone.(A) Autoradiography of TLC-resolved lipids after labeling of the indicated strains with radioactive serine. Parasites were cultured during the intracellular phase without or with Shield1 (0.5 μM, 24 hr) followed by labeling of extracellular parasites (5 x 107) with 14C-serine (2 μCi, 100 μM, 2 hr, 37°C). Lipids were resolved in chloroform/ethanol/water/triethylamine (30:35:7:35). For corresponding quantitative radiolabeling and phospholipid analysis, refer to S12 Fig. (B–C) Relative growth fitness of the parasite strains incubated with or without Shield1. Plaque assays were executed and quantified, as described in Materials and Methods. Note that growth of the Δtgpts/TgPSS-2HA-DD and Δtgpts strains are equivalent irrespective of Shield1 in cultures. Statistics was performed with respect to the untreated parental strain (mean ± SEM, n = 3 assays; *p < 0.05, **p < 0.01, ***p < 0.001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643901&req=5

pbio.1002288.g007: Knockdown of PSS in the Δtgpts/TgPSS-2HA-DD strain does not rescue growth defect caused by the loss of PTS alone.(A) Autoradiography of TLC-resolved lipids after labeling of the indicated strains with radioactive serine. Parasites were cultured during the intracellular phase without or with Shield1 (0.5 μM, 24 hr) followed by labeling of extracellular parasites (5 x 107) with 14C-serine (2 μCi, 100 μM, 2 hr, 37°C). Lipids were resolved in chloroform/ethanol/water/triethylamine (30:35:7:35). For corresponding quantitative radiolabeling and phospholipid analysis, refer to S12 Fig. (B–C) Relative growth fitness of the parasite strains incubated with or without Shield1. Plaque assays were executed and quantified, as described in Materials and Methods. Note that growth of the Δtgpts/TgPSS-2HA-DD and Δtgpts strains are equivalent irrespective of Shield1 in cultures. Statistics was performed with respect to the untreated parental strain (mean ± SEM, n = 3 assays; *p < 0.05, **p < 0.01, ***p < 0.001).
Mentions: To examine whether an elevated level of PtdSer underlies the observed growth phenotype in the PTS mutant, we created a double mutant (Δtgpts/TgPSS-2HA-DD; S10A Fig). The TgPSS gene was fused with a Shield1-regulated degradation domain (DD) and 2HA epitopes at 3’-end [18] to achieve a conditional expression of PSS protein. The PSS-2HA-DD fusion protein showed a predominant fluorescent signal in the ER (S10B Fig). We also observed apparent staining of PSS with the markers of mitochondrion (F1B) [19] and acidocalcisomes/plant-like vacuole (vacuolar proton pyrophosphatase 1; VP1) [20], whereas other organelles, micronemes, rhoptries, dense granules, and apicoplast did not show evident PSS staining (S11 Fig). Expression of PSS-2HA-DD could be regulated by exposure to Shield1 (S10B and S10C Fig). Metabolic labeling of parasite lipids with serine (PtdSer and nascent decarboxylated product PtdEtn) also confirmed that PSS activity was restored to the parental level in the absence of Shield1 (Fig 7A, S12A Fig). A knockdown of PSS activity reinstated PtdSer content in the Δtgpts strain (S12B Fig). Even a normal PtdSer pool, however, was unable to rectify the growth defect in the Δtgpts/TgPSS-2HA-DD double mutant, which mirrored plaques formed by the Δtgpts strain (Fig 7B and 7C). These results exclude the impact of amplified PtdSer in disrupting the lytic cycle, while strengthening the physiological importance of PtdThr for T. gondii.

Bottom Line: The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum.The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion.Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Parasitology, Humboldt University, Berlin, Germany.

ABSTRACT
The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.

Show MeSH
Related in: MedlinePlus