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

The Δtgpts strain is avirulent in mice and provides protection against subsequent acute and chronic toxoplasmosis.(A) Infection of mice with the parental, Δtgpts, and Δtgpts/TgPTS-HA strains to test the virulence and vaccination potential of the mutant against acute infection. Naïve (C57BL/6J) animals were infected either with the RHΔku80-hxgprt- (50), Δtgpts (500, 5,000), or Δtgpts/TgPTS-HA (50) strains by intraperitoneal route and monitored for 28 d (n = 2–3 experiments, each with 3–5 mice). Animals surviving the Δtgpts infection were reinfected with a lethal dose of the parental strain and examined for additional 4 wk. A control group of naïve mice (n = 4) was also infected with the same inoculum. (B–C) Infection of the Δtgpts-immunized animals with a cyst-forming strain of T. gondii. C57BL/6J mice were initially infected with the Δtgpts parasites (500) for 4 wk and subsequently challenged with 3 cysts of the type II (ME49) strain. A control group of naïve mice was also infected. Parasite burden in the brain tissue was evaluated either by real-time PCR (panel B) or by microscopic counting of the parasite cysts (panel C). Panel B shows normalized levels of TgB1 (cyst-specific T. gondii marker) with respect to the mouse reference (MmASL). The PCR results show the mean ± SEM of 3 assays, each with 3 mice (***p < 0.001). (D) Cerebral histopathological alterations in the control naïve and Δtgpts-vaccinated animals infected with the ME49 strain. Arrows indicate inflammatory foci and meningeal thickening in the brain tissue stained by hematoxylin-eosin (n = 2 assays, each with 4 mice).
© Copyright Policy
Related In: Results  -  Collection

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

pbio.1002288.g008: The Δtgpts strain is avirulent in mice and provides protection against subsequent acute and chronic toxoplasmosis.(A) Infection of mice with the parental, Δtgpts, and Δtgpts/TgPTS-HA strains to test the virulence and vaccination potential of the mutant against acute infection. Naïve (C57BL/6J) animals were infected either with the RHΔku80-hxgprt- (50), Δtgpts (500, 5,000), or Δtgpts/TgPTS-HA (50) strains by intraperitoneal route and monitored for 28 d (n = 2–3 experiments, each with 3–5 mice). Animals surviving the Δtgpts infection were reinfected with a lethal dose of the parental strain and examined for additional 4 wk. A control group of naïve mice (n = 4) was also infected with the same inoculum. (B–C) Infection of the Δtgpts-immunized animals with a cyst-forming strain of T. gondii. C57BL/6J mice were initially infected with the Δtgpts parasites (500) for 4 wk and subsequently challenged with 3 cysts of the type II (ME49) strain. A control group of naïve mice was also infected. Parasite burden in the brain tissue was evaluated either by real-time PCR (panel B) or by microscopic counting of the parasite cysts (panel C). Panel B shows normalized levels of TgB1 (cyst-specific T. gondii marker) with respect to the mouse reference (MmASL). The PCR results show the mean ± SEM of 3 assays, each with 3 mice (***p < 0.001). (D) Cerebral histopathological alterations in the control naïve and Δtgpts-vaccinated animals infected with the ME49 strain. Arrows indicate inflammatory foci and meningeal thickening in the brain tissue stained by hematoxylin-eosin (n = 2 assays, each with 4 mice).

Mentions: We also explored the prophylactic potential of the Δtgpts mutant. Examination of virulence in a mouse model demonstrated that nearly all animals infected with the Δtgpts mutant survived as opposed to the parental and PTS-complemented strains, both of which were explicitly lethal (Fig 8A). Importantly, all mice enduring the mutant infection became categorically resistant to a subsequent lethal challenge by a hypervirulent type I strain of T. gondii causing acute toxoplasmosis (Fig 8A). To further expand the therapeutic utility of our strain as a potential vaccine against chronic infection, we challenged the Δtgpts-infected animals with the cyst-forming type II strain. Remarkably, in contrast to naïve animals, the mutant-vaccinated mice showed no signs of chronic stage cysts in their brain tissue (Fig 8B and 8C). In accord, unlike the naïve control mice, we did not observe any inflammatory lesions in the cortex or meninges of the Δtgpts-immunized animals infected with the type II strain (Fig 8D). In brief, these results demonstrate a requirement of PtdThr for the parasite virulence and illustrate the prophylactic potential of a metabolically attenuated whole-cell “vaccine” against acute and chronic toxoplasmosis.


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 strain is avirulent in mice and provides protection against subsequent acute and chronic toxoplasmosis.(A) Infection of mice with the parental, Δtgpts, and Δtgpts/TgPTS-HA strains to test the virulence and vaccination potential of the mutant against acute infection. Naïve (C57BL/6J) animals were infected either with the RHΔku80-hxgprt- (50), Δtgpts (500, 5,000), or Δtgpts/TgPTS-HA (50) strains by intraperitoneal route and monitored for 28 d (n = 2–3 experiments, each with 3–5 mice). Animals surviving the Δtgpts infection were reinfected with a lethal dose of the parental strain and examined for additional 4 wk. A control group of naïve mice (n = 4) was also infected with the same inoculum. (B–C) Infection of the Δtgpts-immunized animals with a cyst-forming strain of T. gondii. C57BL/6J mice were initially infected with the Δtgpts parasites (500) for 4 wk and subsequently challenged with 3 cysts of the type II (ME49) strain. A control group of naïve mice was also infected. Parasite burden in the brain tissue was evaluated either by real-time PCR (panel B) or by microscopic counting of the parasite cysts (panel C). Panel B shows normalized levels of TgB1 (cyst-specific T. gondii marker) with respect to the mouse reference (MmASL). The PCR results show the mean ± SEM of 3 assays, each with 3 mice (***p < 0.001). (D) Cerebral histopathological alterations in the control naïve and Δtgpts-vaccinated animals infected with the ME49 strain. Arrows indicate inflammatory foci and meningeal thickening in the brain tissue stained by hematoxylin-eosin (n = 2 assays, each with 4 mice).
© Copyright Policy
Related In: Results  -  Collection

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

pbio.1002288.g008: The Δtgpts strain is avirulent in mice and provides protection against subsequent acute and chronic toxoplasmosis.(A) Infection of mice with the parental, Δtgpts, and Δtgpts/TgPTS-HA strains to test the virulence and vaccination potential of the mutant against acute infection. Naïve (C57BL/6J) animals were infected either with the RHΔku80-hxgprt- (50), Δtgpts (500, 5,000), or Δtgpts/TgPTS-HA (50) strains by intraperitoneal route and monitored for 28 d (n = 2–3 experiments, each with 3–5 mice). Animals surviving the Δtgpts infection were reinfected with a lethal dose of the parental strain and examined for additional 4 wk. A control group of naïve mice (n = 4) was also infected with the same inoculum. (B–C) Infection of the Δtgpts-immunized animals with a cyst-forming strain of T. gondii. C57BL/6J mice were initially infected with the Δtgpts parasites (500) for 4 wk and subsequently challenged with 3 cysts of the type II (ME49) strain. A control group of naïve mice was also infected. Parasite burden in the brain tissue was evaluated either by real-time PCR (panel B) or by microscopic counting of the parasite cysts (panel C). Panel B shows normalized levels of TgB1 (cyst-specific T. gondii marker) with respect to the mouse reference (MmASL). The PCR results show the mean ± SEM of 3 assays, each with 3 mice (***p < 0.001). (D) Cerebral histopathological alterations in the control naïve and Δtgpts-vaccinated animals infected with the ME49 strain. Arrows indicate inflammatory foci and meningeal thickening in the brain tissue stained by hematoxylin-eosin (n = 2 assays, each with 4 mice).
Mentions: We also explored the prophylactic potential of the Δtgpts mutant. Examination of virulence in a mouse model demonstrated that nearly all animals infected with the Δtgpts mutant survived as opposed to the parental and PTS-complemented strains, both of which were explicitly lethal (Fig 8A). Importantly, all mice enduring the mutant infection became categorically resistant to a subsequent lethal challenge by a hypervirulent type I strain of T. gondii causing acute toxoplasmosis (Fig 8A). To further expand the therapeutic utility of our strain as a potential vaccine against chronic infection, we challenged the Δtgpts-infected animals with the cyst-forming type II strain. Remarkably, in contrast to naïve animals, the mutant-vaccinated mice showed no signs of chronic stage cysts in their brain tissue (Fig 8B and 8C). In accord, unlike the naïve control mice, we did not observe any inflammatory lesions in the cortex or meninges of the Δtgpts-immunized animals infected with the type II strain (Fig 8D). In brief, these results demonstrate a requirement of PtdThr for the parasite virulence and illustrate the prophylactic potential of a metabolically attenuated whole-cell “vaccine” against acute and chronic toxoplasmosis.

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