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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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PtdThr and PtdSer are synthesized by PTS and PSS in the ER of T. gondii.(A) Immunostained images of the HA-tagged PtdThr synthase (TgPTS) and PtdSer synthase (TgPSS) targeted at the uracil phosphoribosyltransferase (UPRT) locus and expressed under the control of the regulatory elements of TgGRA1 or TgSAG1, respectively. Colocalization was done with TgDer1-GFP (ER marker). Yellow fluorescence in the merged panel indicates expression of TgPTS-HA and TgPSS-HA in the ER (bars, 5 μm). No crossfluorescence from green to red channel or vice versa was observed. For costaining with other organelle markers, refer to S5 and S11 Figs. (B) TLC-resolved lipid profiles of E. coli strains harboring the specified expression constructs. To assess the TgPTS and TgPSS activities, ORFs (open reading frames) were cloned into the M15/pREP4 strain of E. coli and expression was induced by IPTG (isopropyl β-D-1-thiogalactopyranoside) in cultures supplemented with 5 mM threonine or serine. Total lipids were resolved in chloroform/methanol/acetate (130:50:20) and visualized by ninhydrin spray.
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pbio.1002288.g002: PtdThr and PtdSer are synthesized by PTS and PSS in the ER of T. gondii.(A) Immunostained images of the HA-tagged PtdThr synthase (TgPTS) and PtdSer synthase (TgPSS) targeted at the uracil phosphoribosyltransferase (UPRT) locus and expressed under the control of the regulatory elements of TgGRA1 or TgSAG1, respectively. Colocalization was done with TgDer1-GFP (ER marker). Yellow fluorescence in the merged panel indicates expression of TgPTS-HA and TgPSS-HA in the ER (bars, 5 μm). No crossfluorescence from green to red channel or vice versa was observed. For costaining with other organelle markers, refer to S5 and S11 Figs. (B) TLC-resolved lipid profiles of E. coli strains harboring the specified expression constructs. To assess the TgPTS and TgPSS activities, ORFs (open reading frames) were cloned into the M15/pREP4 strain of E. coli and expression was induced by IPTG (isopropyl β-D-1-thiogalactopyranoside) in cultures supplemented with 5 mM threonine or serine. Total lipids were resolved in chloroform/methanol/acetate (130:50:20) and visualized by ninhydrin spray.

Mentions: Ectopic expression of epitope-tagged TgPTS-HA and TgPSS-HA showed a marked distribution in the endoplasmic reticulum (ER) of the parasite (Fig 2A). Because overexpression under the control of a foreign promoter may cause localization artifacts, we detected endogenous levels of PSS and PTS in transgenic parasite lines, in which the corresponding genes had been tagged with HA-epitope at the 3’-ends. As discussed below (S10B and S11 Figs), PSS fusion protein regulated by its promoter localized mainly in the parasite ER/mitochondrion intersecting with each other, and to some extent in acidocalcisomes/plant-like vacuole. The native expression of PTS was too low to be visualized (not shown). We nonetheless tested potential localization of PTS in other organelles using the parasites overexpressing TgPTS-HA; however, we found no apparent signal in micronemes, rhoptries, dense granules, mitochondrion, apicoplast, and acidocalcisomes/plant-like vacuole (S5 Fig). To evaluate the enzymatic function of both enzymes, we expressed them in Eschericia coli and assessed their catalytic activity in the presence of serine or threonine (Fig 2B). Lipid analyses of bacterial strains harboring empty vector (negative control), TgPTS, TgPSS, or Arabidopsis thaliana PSS (positive control [17]) showed synthesis of PtdSer by AtPSS and TgPSS as well as by TgPTS when using serine as substrate. Unlike AtPSS and TgPSS, however, TgPTS also produced PtdThr in presence of threonine, indicating that TgPSS is indeed a PtdSer synthase, whereas TgPTS can synthesize both PtdThr and PtdSer.


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)

PtdThr and PtdSer are synthesized by PTS and PSS in the ER of T. gondii.(A) Immunostained images of the HA-tagged PtdThr synthase (TgPTS) and PtdSer synthase (TgPSS) targeted at the uracil phosphoribosyltransferase (UPRT) locus and expressed under the control of the regulatory elements of TgGRA1 or TgSAG1, respectively. Colocalization was done with TgDer1-GFP (ER marker). Yellow fluorescence in the merged panel indicates expression of TgPTS-HA and TgPSS-HA in the ER (bars, 5 μm). No crossfluorescence from green to red channel or vice versa was observed. For costaining with other organelle markers, refer to S5 and S11 Figs. (B) TLC-resolved lipid profiles of E. coli strains harboring the specified expression constructs. To assess the TgPTS and TgPSS activities, ORFs (open reading frames) were cloned into the M15/pREP4 strain of E. coli and expression was induced by IPTG (isopropyl β-D-1-thiogalactopyranoside) in cultures supplemented with 5 mM threonine or serine. Total lipids were resolved in chloroform/methanol/acetate (130:50:20) and visualized by ninhydrin spray.
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Related In: Results  -  Collection

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pbio.1002288.g002: PtdThr and PtdSer are synthesized by PTS and PSS in the ER of T. gondii.(A) Immunostained images of the HA-tagged PtdThr synthase (TgPTS) and PtdSer synthase (TgPSS) targeted at the uracil phosphoribosyltransferase (UPRT) locus and expressed under the control of the regulatory elements of TgGRA1 or TgSAG1, respectively. Colocalization was done with TgDer1-GFP (ER marker). Yellow fluorescence in the merged panel indicates expression of TgPTS-HA and TgPSS-HA in the ER (bars, 5 μm). No crossfluorescence from green to red channel or vice versa was observed. For costaining with other organelle markers, refer to S5 and S11 Figs. (B) TLC-resolved lipid profiles of E. coli strains harboring the specified expression constructs. To assess the TgPTS and TgPSS activities, ORFs (open reading frames) were cloned into the M15/pREP4 strain of E. coli and expression was induced by IPTG (isopropyl β-D-1-thiogalactopyranoside) in cultures supplemented with 5 mM threonine or serine. Total lipids were resolved in chloroform/methanol/acetate (130:50:20) and visualized by ninhydrin spray.
Mentions: Ectopic expression of epitope-tagged TgPTS-HA and TgPSS-HA showed a marked distribution in the endoplasmic reticulum (ER) of the parasite (Fig 2A). Because overexpression under the control of a foreign promoter may cause localization artifacts, we detected endogenous levels of PSS and PTS in transgenic parasite lines, in which the corresponding genes had been tagged with HA-epitope at the 3’-ends. As discussed below (S10B and S11 Figs), PSS fusion protein regulated by its promoter localized mainly in the parasite ER/mitochondrion intersecting with each other, and to some extent in acidocalcisomes/plant-like vacuole. The native expression of PTS was too low to be visualized (not shown). We nonetheless tested potential localization of PTS in other organelles using the parasites overexpressing TgPTS-HA; however, we found no apparent signal in micronemes, rhoptries, dense granules, mitochondrion, apicoplast, and acidocalcisomes/plant-like vacuole (S5 Fig). To evaluate the enzymatic function of both enzymes, we expressed them in Eschericia coli and assessed their catalytic activity in the presence of serine or threonine (Fig 2B). Lipid analyses of bacterial strains harboring empty vector (negative control), TgPTS, TgPSS, or Arabidopsis thaliana PSS (positive control [17]) showed synthesis of PtdSer by AtPSS and TgPSS as well as by TgPTS when using serine as substrate. Unlike AtPSS and TgPSS, however, TgPTS also produced PtdThr in presence of threonine, indicating that TgPSS is indeed a PtdSer synthase, whereas TgPTS can synthesize both PtdThr and PtdSer.

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