Dataset Information

The GRA17 Parasitophorous Vacuole Membrane Permeability Pore Contributes to Bradyzoite Viability.

ABSTRACT: The Toxoplasma gondii parasitophorous vacuole membrane (PVM) offers protection from the host immune system but is also a barrier for uptake of nutrients from the host. Previously, we showed that GRA17 mediates the tachyzoite PVM permeability to small molecules. During the conversion from tachyzoites to encysted bradyzoites, the PVM become the cyst membrane that is the outer layer of the cyst wall. Little is known about how small molecules, such as nutrients, enter cysts. To characterize GRA17's role in cysts, we deleted GRA17 in the type II ME49 cyst-forming strain. ME49?gra17 parasites have reduced growth and formed grossly enlarged "bubble vacuoles," which have reduced PVM small molecule permeability. ME49?gra17 parasites formed cysts in vitro at rates comparable to the wild-type, but the viability of the bradyzoites inside these cysts was significantly reduced compared to wild-type bradyzoites. Genetic complementation of ME49?gra17 with GRA17 expressed from the endogenous or tachyzoite-specific SAG1 promoter recovered the viability of bradyzoites. Complementation with the bradyzoite-specific SRS9 promoter drastically increased the viability of bradyzoites, demonstrating the importance of GRA17 in regulating bradyzoite viability inside cysts. Mice infected with a high dose of ME49?gra17 parasites did not contain parasites in their brain nor did mice infected with ME49?gra17 complemented with GRA17 expressed from a bradyzoite-specific promoter. Our results suggest that the ME49?gra17 strain is avirulent and is cleared before it can reach the brain and that GRA17 not only plays an important role during acute infections but is also needed for viability of bradyzoites inside cysts.

SUBMITTER: Paredes-Santos T

PROVIDER: S-EPMC6751312 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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The GRA17 Parasitophorous Vacuole Membrane Permeability Pore Contributes to Bradyzoite Viability.

Paredes-Santos Tatiana T Wang Yifan Y Waldman Benjamin B Lourido Sebastian S Saeij Jeroen P JP

Frontiers in cellular and infection microbiology 20190912

The <i>Toxoplasma gondii</i> parasitophorous vacuole membrane (PVM) offers protection from the host immune system but is also a barrier for uptake of nutrients from the host. Previously, we showed that GRA17 mediates the tachyzoite PVM permeability to small molecules. During the conversion from tachyzoites to encysted bradyzoites, the PVM become the cyst membrane that is the outer layer of the cyst wall. Little is known about how small molecules, such as nutrients, enter cysts. To characterize G ...[more]

PMID: 31572690

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Project description:During their development within the vertebrate host, Plasmodium parasites infect hepatocytes and red blood cells. Within these cells, parasites are surrounded by a parasitophorous vacuole membrane (PVM). The PVM plays an essential role for the interaction of parasites with their host cells; however, only a limited number of proteins of this membrane have been identified so far. This is partially because systematic proteomic analysis of the protein content of the PVM has been difficult in the past, due to difficulties encountered in attempts to separate the PVM from other membranes such as the parasite plasma membrane. In this study, we adapted the BioID technique to in vitro-cultivated Plasmodium berghei blood stage parasites and utilized the promiscuous biotin ligase BirA* fused to PVM-resident exported protein 1 to biotinylate proteins of the PVM. These we further processed by affinity purification, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and label-free quantitation, leading to a list of 61 known and candidate PVM proteins. Seven proteins were analyzed further during blood and liver stage development. This resulted in the identification of three novel PVM proteins, which were the serine/threonine protein phosphatase UIS2 (PlasmoDB accession no. PBANKA_1328000) and two conserved Plasmodium proteins with unknown functions (PBANKA_0519300 and PBANKA_0509000). In conclusion, our report expands the number of known PVM proteins and experimentally validates BioID as a powerful method to screen for novel constituents of specific cellular compartments in P. berghei. IMPORTANCE Intracellular pathogens are often surrounded by a host-cell derived membrane. This membrane is modified by the pathogens to their own needs and is crucial for their intracellular lifestyle. In Plasmodium parasites, this membrane is referred to as the PVM and only a limited number of its proteins are known so far. Here, we applied in rodent P. berghei parasites a method called BioID, which is based on biotinylation of proximal and interacting proteins by the promiscuous biotin ligase BirA*, and demonstrated its usefulness in identification of novel PVM proteins.

Dataset Information

The GRA17 Parasitophorous Vacuole Membrane Permeability Pore Contributes to Bradyzoite Viability.

Publications

The GRA17 Parasitophorous Vacuole Membrane Permeability Pore Contributes to Bradyzoite Viability.

Similar Datasets

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