Proximity biotinylation reveals novel secreted dense granule proteins of Toxoplasma gondii bradyzoites

Nadipuram, Santhosh M.; Thind, Amara C; Rayatpisheh, Shima; Wohlschlegel, James A.; Bradley, Peter J.

doi:10.1371/journal.pone.0232552

Cited by 42 publications

(41 citation statements)

References 75 publications

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“…The mechanisms inducing cyst wall turnover in vivo are unclear, although host cell macrophages were reported to secrete chitinase to lyse cysts in vitro [65]. Additionally, proximity biotinylation has shown the presence of GRA56, which is predicated to belong to melibiase family of polysaccharide degrading enzymes, on the cyst wall [66]. Collectively, these findings suggest that synthesis and degradation of polysaccharides likely contribute both to cyst wall maturation and turnover in vivo.…”

Section: Discussionmentioning

confidence: 99%

Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress

Jones

et al. 2021

Preprint

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Toxoplasma gondii has evolved different developmental stages of tachyzoites for disseminating during acute infection and bradyzoites for establishing chronic infection. Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unknown. Here we show that Ca2+ signals and egress by bradyzoites in response to agonists are highly restricted. Development of dual-reporter parasites revealed dampened calcium responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with down-regulation of calcium ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisome. Once liberated from cysts by trypsin digestion, bradyzoites displayed weaker gliding motility associated with Ca2+ oscillations compared with tachyzoites, although gliding motility of bradyzoites was enhanced by uptake of exogenous Ca2+. Collectively, our findings indicate that bradyzoites exhibit dampened Ca2+ signaling due to a decreased amount of stored Ca2+, limiting microneme secretion and egress, likely constituting an adaptation to their long-term intracellular niche.

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Section: Discussionmentioning

confidence: 99%

Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress

Jones

et al. 2021

Preprint

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“…This interactome analysis identified MAG1 as a hub protein that is probably critical for cyst wall composition. Other proximity labeling studies also identified additional GRA proteins (GRA1 [22], GRA13, GRA17, GRA25 [23], and GRA55 through GRA59 [24]) as interacting partners, further illustrating the role of MAG1 as a hub in cyst wall composition. A large-scale in vivo CRISPR knockout screen identified MAG1 as a key protein necessary for either dissemination to the brain or establishment of brain cysts (25).…”

mentioning

confidence: 84%

Toxoplasma gondii Matrix Antigen 1 Is a Secreted Immunomodulatory Effector

Tomita

Mukhopadhyay

Han

et al. 2021

mBio

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Our studies on novel cyst wall proteins serendipitously led us to the discovery that cyst wall and vacuolar matrix protein MAG1, first identified a quarter of a century ago, functions as a secreted immunomodulatory effector. MAG1 is a dense granular protein that is found in the parasitophorous vacuolar matrix in tachyzoite vacuoles and the cyst wall and matrix in bradyzoite vacuoles. In the current study, we demonstrated that MAG1 is secreted beyond the parasitophorous vacuole into the host cytosol in both tachyzoites and bradyzoites. Secretion of MAG1 gradually decreases as the parasitophorous vacuole matures, but prominent MAG1 puncta are present inside host cells even at 4 and 6 days following infection. During acute murine infection, Δmag1 parasites displayed significantly reduced virulence and dissemination. In the chronic stage of infection, Δmag1 parasites generated almost no brain cysts. To identify the mechanism behind the attenuated pathology seen with Δmag1 parasites, various immune responses were screened in vitro using bone marrow-derived macrophages (BMDM). Infection of BMDM with Δmag1 parasites induced a significant increase in interleukin 1β (IL-1β) secretion, which is a hallmark of inflammasome activation. Heterologous complementation of MAG1 in BMDM cells prevented this Δmag1 parasite-induced IL-1β release, indicating that secreted MAG1 in host cytosol dampens inflammasome activation. Furthermore, knocking out GRA15 (an inducer of IL-1β release) in Δmag1 parasites completely inhibited all IL-1β release by host cells following infection. These data suggest that MAG1 has a role as an immunomodulatory molecule and that by suppressing inflammasome activation, it would favor survival of the parasite and the establishment of latent infection. IMPORTANCE Toxoplasma gondii is an Apicomplexan that infects a third of humans, causing encephalitis in AIDS patients and intellectual disabilities in congenitally infected patients. We determined that one of the cyst matrix proteins, MAG1, which had been thought to be an innate structural protein, can be secreted into the host cell and suppress the host immune reaction. This particular immune reaction is initiated by another parasite-secreted protein, GRA15. The intricate balance of inflammasome activation by GRA15 and suppression by MAG1 protects mice from acute death while enabling parasites to disseminate and establish chronic cysts. Our finding contributes to our understanding of how parasites persist in the host and how T. gondii modulates the host immune system.

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“…For plaque assays, intracellular parasites were collected by scraping and passaging through a 27-gauge needle, and then equivalent parasite numbers were allowed to infect HFF monolayers. 7 days after infection, cells were fixed with 100% ice-cold methanol and stained with crystal violet (44). The area of 50 plaques per condition was measured using ZEN software (Zeiss).…”

Section: Gene Knockout and Phenotypic Analysesmentioning

confidence: 99%

“…CRISPR/Cas9 and homologous recombination was used to knockout the ISAP1 gene, as previously described using primers p5-p10 (44). Knockout clones were verified by IFA and PCR using primers p11-p14.…”

Section: Gene Knockout and Phenotypic Analysesmentioning

confidence: 99%

A novel Toxoplasma IMC sutures-associated protein regulates suture protein targeting and colocalizes with membrane trafficking machinery

et al. 2021

Preprint

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The cytoskeleton of Toxoplasma gondii is composed of the inner membrane complex (IMC) and an array of underlying microtubules that provide support at the periphery of the parasite. Specific subregions of the IMC carry out distinct roles in replication, motility, and host cell invasion. Building on our previous in vivo biotinylation (BioID) experiments of the IMC, we identify here a novel protein that localizes to discrete punctae that are embedded in the parasite's cytoskeleton along the IMC sutures. Gene knockout analysis shows that loss of the protein results in defects in cytoskeletal suture protein targeting, cytoskeletal integrity, parasite morphology, and host cell invasion. We then use deletion analyses to identify a domain in the N-terminus of the protein that is critical for both localization and function. Finally, we use the protein as bait for in vivo biotinylation which identifies several other proteins that colocalize in similar spot-like patterns. These putative interactors include several proteins that are implicated in membrane trafficking and are also associated with the cytoskeleton. Together, this data reveals an unexpected link between the IMC sutures and membrane trafficking elements of the parasite and suggests that the sutures punctae are likely a portal for trafficking cargo across the IMC.

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Proximity biotinylation reveals novel secreted dense granule proteins of Toxoplasma gondii bradyzoites

Cited by 42 publications

References 75 publications

Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress

Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress

Toxoplasma gondii Matrix Antigen 1 Is a Secreted Immunomodulatory Effector

A novel Toxoplasma IMC sutures-associated protein regulates suture protein targeting and colocalizes with membrane trafficking machinery

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