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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.

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Related in: MedlinePlus

The Δtgpts mutant is defective in egress and invasion but not in replication.(A) Representative images showing the in vitro growth fitness of the parental, Δtgpts, and PTS-complemented strains by plaque assays, which recapitulate successive lytic cycles of tachyzoites in host cells (see schematics). The mutant was generated as shown in Fig 3. Complemented strain expressed wild-type TgPTS-HA under the control of the TgGRA1 promoter at the TgUPRT gene locus. (B) Quantification of plaque area (left Y-axis) and numbers (right Y-axis). 120–300 plaques of each strain from 7 assays were scored. (C) Intracellular replication of the specified strains, as deduced by the mean number of parasites/vacuoles at different periods. A total of 100–200 vacuoles were analyzed for each strain (n = 4 assays). (D) The natural egress of the indicated parasite strains at 60 and 72 hr postinfection (MOI, 1). In total, 200–300 vacuoles were counted for each strain (n = 4 assays). (E) Invasion rates of the parasite strains (700–1,000 parasites of each strain from 5 assays). The number of egressed vacuoles and invaded parasites were estimated by dual-color staining, as described in Materials and Methods. Graphs in panels B–E indicate the mean ± standard error of the mean (SEM) (*p < 0.05, **p < 0.01, ***p < 0.001). Note that a partial rescue of plaque growth (panel B) in the complemented strain as opposed to near complete recovery of invasion and egress defects in panels D–E is caused by a mild replication defect due to overexpression of PTS in the Δtgpts mutant (panel C).
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pbio.1002288.g005: The Δtgpts mutant is defective in egress and invasion but not in replication.(A) Representative images showing the in vitro growth fitness of the parental, Δtgpts, and PTS-complemented strains by plaque assays, which recapitulate successive lytic cycles of tachyzoites in host cells (see schematics). The mutant was generated as shown in Fig 3. Complemented strain expressed wild-type TgPTS-HA under the control of the TgGRA1 promoter at the TgUPRT gene locus. (B) Quantification of plaque area (left Y-axis) and numbers (right Y-axis). 120–300 plaques of each strain from 7 assays were scored. (C) Intracellular replication of the specified strains, as deduced by the mean number of parasites/vacuoles at different periods. A total of 100–200 vacuoles were analyzed for each strain (n = 4 assays). (D) The natural egress of the indicated parasite strains at 60 and 72 hr postinfection (MOI, 1). In total, 200–300 vacuoles were counted for each strain (n = 4 assays). (E) Invasion rates of the parasite strains (700–1,000 parasites of each strain from 5 assays). The number of egressed vacuoles and invaded parasites were estimated by dual-color staining, as described in Materials and Methods. Graphs in panels B–E indicate the mean ± standard error of the mean (SEM) (*p < 0.05, **p < 0.01, ***p < 0.001). Note that a partial rescue of plaque growth (panel B) in the complemented strain as opposed to near complete recovery of invasion and egress defects in panels D–E is caused by a mild replication defect due to overexpression of PTS in the Δtgpts mutant (panel C).

Mentions: We next assessed the physiological impact of TgPTS ablation on the parasite growth by plaque assays. Compared to the parental strain, the Δtgpts strain formed noticeably smaller (−70%) and considerably fewer (−80%) plaques (Fig 5A and 5B). Ectopic expression of wild-type TgPTS largely rescued the parasite growth. In contrast, the catalytically-dead isoform of TgPTS(ΔECWWD), which was incapable of restoring PtdThr level in the Δtgpts strain (Fig 3D), could not amend the growth defect (S9 Fig), confirming the physiological need of the PTS activity for the parasite. It should be mentioned that the Δtgpts strain expressing TgPTS(ΔECWWD)-myc showed an accentuated growth defect when compared to the mutant (S9A Fig), which prevented its prolonged culture and detailed biochemical analyses.


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)

The Δtgpts mutant is defective in egress and invasion but not in replication.(A) Representative images showing the in vitro growth fitness of the parental, Δtgpts, and PTS-complemented strains by plaque assays, which recapitulate successive lytic cycles of tachyzoites in host cells (see schematics). The mutant was generated as shown in Fig 3. Complemented strain expressed wild-type TgPTS-HA under the control of the TgGRA1 promoter at the TgUPRT gene locus. (B) Quantification of plaque area (left Y-axis) and numbers (right Y-axis). 120–300 plaques of each strain from 7 assays were scored. (C) Intracellular replication of the specified strains, as deduced by the mean number of parasites/vacuoles at different periods. A total of 100–200 vacuoles were analyzed for each strain (n = 4 assays). (D) The natural egress of the indicated parasite strains at 60 and 72 hr postinfection (MOI, 1). In total, 200–300 vacuoles were counted for each strain (n = 4 assays). (E) Invasion rates of the parasite strains (700–1,000 parasites of each strain from 5 assays). The number of egressed vacuoles and invaded parasites were estimated by dual-color staining, as described in Materials and Methods. Graphs in panels B–E indicate the mean ± standard error of the mean (SEM) (*p < 0.05, **p < 0.01, ***p < 0.001). Note that a partial rescue of plaque growth (panel B) in the complemented strain as opposed to near complete recovery of invasion and egress defects in panels D–E is caused by a mild replication defect due to overexpression of PTS in the Δtgpts mutant (panel C).
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Related In: Results  -  Collection

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pbio.1002288.g005: The Δtgpts mutant is defective in egress and invasion but not in replication.(A) Representative images showing the in vitro growth fitness of the parental, Δtgpts, and PTS-complemented strains by plaque assays, which recapitulate successive lytic cycles of tachyzoites in host cells (see schematics). The mutant was generated as shown in Fig 3. Complemented strain expressed wild-type TgPTS-HA under the control of the TgGRA1 promoter at the TgUPRT gene locus. (B) Quantification of plaque area (left Y-axis) and numbers (right Y-axis). 120–300 plaques of each strain from 7 assays were scored. (C) Intracellular replication of the specified strains, as deduced by the mean number of parasites/vacuoles at different periods. A total of 100–200 vacuoles were analyzed for each strain (n = 4 assays). (D) The natural egress of the indicated parasite strains at 60 and 72 hr postinfection (MOI, 1). In total, 200–300 vacuoles were counted for each strain (n = 4 assays). (E) Invasion rates of the parasite strains (700–1,000 parasites of each strain from 5 assays). The number of egressed vacuoles and invaded parasites were estimated by dual-color staining, as described in Materials and Methods. Graphs in panels B–E indicate the mean ± standard error of the mean (SEM) (*p < 0.05, **p < 0.01, ***p < 0.001). Note that a partial rescue of plaque growth (panel B) in the complemented strain as opposed to near complete recovery of invasion and egress defects in panels D–E is caused by a mild replication defect due to overexpression of PTS in the Δtgpts mutant (panel C).
Mentions: We next assessed the physiological impact of TgPTS ablation on the parasite growth by plaque assays. Compared to the parental strain, the Δtgpts strain formed noticeably smaller (−70%) and considerably fewer (−80%) plaques (Fig 5A and 5B). Ectopic expression of wild-type TgPTS largely rescued the parasite growth. In contrast, the catalytically-dead isoform of TgPTS(ΔECWWD), which was incapable of restoring PtdThr level in the Δtgpts strain (Fig 3D), could not amend the growth defect (S9 Fig), confirming the physiological need of the PTS activity for the parasite. It should be mentioned that the Δtgpts strain expressing TgPTS(ΔECWWD)-myc showed an accentuated growth defect when compared to the mutant (S9A Fig), which prevented its prolonged culture and detailed biochemical analyses.

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