A Novel Secreted Protein, MYR1, Is Central to Toxoplasma's Manipulation of Host Cells.
2016; 7 (1)
Impact of Regulated Secretion on Antiparasitic CD8 T Cell Responses
2014; 7 (5): 1716-1728
The intracellular protozoan Toxoplasma gondii dramatically reprograms the transcriptome of host cells it infects, including substantially up-regulating the host oncogene c-myc. By applying a flow cytometry-based selection to infected mouse cells expressing green fluorescent protein fused to c-Myc (c-Myc-GFP), we isolated mutant tachyzoites defective in this host c-Myc up-regulation. Whole-genome sequencing of three such mutants led to the identification of MYR1 (Myc regulation 1; TGGT1_254470) as essential for c-Myc induction. MYR1 is a secreted protein that requires TgASP5 to be cleaved into two stable portions, both of which are ultimately found within the parasitophorous vacuole and at the parasitophorous vacuole membrane. Deletion of MYR1 revealed that in addition to its requirement for c-Myc up-regulation, the MYR1 protein is needed for the ability of Toxoplasma tachyzoites to modulate several other important host pathways, including those mediated by the dense granule effectors GRA16 and GRA24. This result, combined with its location at the parasitophorous vacuole membrane, suggested that MYR1 might be a component of the machinery that translocates Toxoplasma effectors from the parasitophorous vacuole into the host cytosol. Support for this possibility was obtained by showing that transit of GRA24 to the host nucleus is indeed MYR1-dependent. As predicted by this pleiotropic phenotype, parasites deficient in MYR1 were found to be severely attenuated in a mouse model of infection. We conclude, therefore, that MYR1 is a novel protein that plays a critical role in how Toxoplasma delivers effector proteins to the infected host cell and that this is crucial to virulence.Toxoplasma gondii is an important human pathogen and a model for the study of intracellular parasitism. Infection of the host cell with Toxoplasma tachyzoites involves the introduction of protein effectors, including many that are initially secreted into the parasitophorous vacuole but must ultimately translocate to the host cell cytosol to function. The work reported here identified a novel protein that is required for this translocation. These results give new insight into a very unusual cell biology process as well as providing a potential handle on a pathway that is necessary for virulence and, therefore, a new potential target for chemotherapy.
View details for DOI 10.1128/mBio.02231-15
View details for PubMedID 26838724
De Novo Growth Zone Formation from Fission Yeast Spheroplasts
2011; 6 (12)
CD8 T cells play a key role in defense against the intracellular parasite Toxoplasma, but why certain CD8 responses are more potent than others is not well understood. Here, we describe a parasite antigen, ROP5, that elicits a CD8 T cell response in genetically susceptible mice. ROP5 is secreted via parasite organelles termed rhoptries that are injected directly into host cells during invasion, whereas the protective, dense-granule antigen GRA6 is constitutively secreted into the parasitophorous vacuole. Transgenic parasites in which the ROP5 antigenic epitope was targeted for secretion through dense granules led to enhanced CD8 T cell responses, whereas targeting the GRA6 epitope to rhoptries led to reduced CD8 responses. CD8 T cell responses to the dense-granule-targeted ROP5 epitope resulted in reduced parasite load in the brain. These data suggest that the mode of secretion affects the efficacy of parasite-specific CD8 T cell responses.
View details for DOI 10.1016/j.celrep.2014.04.031
View details for Web of Science ID 000338324200034
Spatial control of Cdc42 activation determines cell width in fission yeast
MOLECULAR BIOLOGY OF THE CELL
2011; 22 (20): 3801-3811
Eukaryotic cells often form polarized growth zones in response to internal or external cues. To understand the establishment of growth zones with specific dimensions we used fission yeast, which grows as a rod-shaped cell of near-constant width from growth zones located at the cell tips. Removing the cell wall creates a round spheroplast with a disorganized cytoskeleton and depolarized growth proteins. As spheroplasts recover, new growth zones form that resemble normal growing cell tips in shape and width, and polarized growth resumes. Regulators of the GTPase Cdc42, which control width in exponentially growing cells, also control spheroplast growth zone width. During recovery the Cdc42 scaffold Scd2 forms a polarized patch in the rounded spheroplast, demonstrating that a growth zone protein can organize independent of cell shape. Rga4, a Cdc42 GTPase activating protein (GAP) that is excluded from cell tips, is initially distributed throughout the spheroplast membrane, but is excluded from the growth zone after a stable patch of Scd2 forms. These results provide evidence that growth zones with normal width and protein localization can form de novo through sequential organization of cellular domains, and that the size of these growth zones is genetically controlled, independent of preexisting cell shape.
View details for DOI 10.1371/journal.pone.0027977
View details for Web of Science ID 000298370400008
View details for PubMedID 22194800
The fission yeast Schizosaccharomyces pombe is a rod-shaped cell that grows by linear extension at the cell tips, with a nearly constant width throughout the cell cycle. This simple geometry makes it an ideal system for studying the control of cellular dimensions. In this study, we carried out a near-genome-wide screen for mutants wider than wild-type cells. We found 11 deletion mutants that were wider; seven of the deleted genes are implicated in the control of the small GTPase Cdc42, including the Cdc42 guanine nucleotide exchange factor (GEF) Scd1 and the Cdc42 GTPase-activating protein (GAP) Rga4. Deletions of rga4 and scd1 had additive effects on cell width, and the proteins localized independently of one another, with Rga4 located at the cell sides and Scd1 at the cell tips. Activated Cdc42 localization is altered in rga4?, scd1?, and scd2? mutants. Delocalization and ectopic retargeting experiments showed that the localizations of Rga4 and Scd1 are crucial for their roles in determining cell width. We propose that the GAP Rga4 and the GEF Scd1 establish a gradient of activated Cdc42 within the cellular tip plasma membrane, and it is this gradient that determines cell growth-zone size and normal cell width.
View details for DOI 10.1091/mbc.E11-01-0057
View details for Web of Science ID 000296346600006
View details for PubMedID 21849474