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Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma

ABSTRACT: Toxoplasma gondii is an intracellular parasite that causes the disease toxoplasmosis in humans. It is a member of the phylum Apicomplexa that also includes significant human pathogens such as Plasmodium spp. and Cryptosporidium spp. causing malaria and diarrheal disease, respectively. These parasites use a programmed sequence of secretory events to find, invade, and then reengineer their host cells to enable parasite growth and proliferation. After invasion, Toxoplasma secretes proteins for host cell remodelling and manipulation from dense granules. The site(s) of dense granule exocytosis, however, has been unknown. In Toxoplasma gondii, small subapical annular structures that are embedded in the inner membrane complex have been observed, but the role or significance of these apical annuli to plasma membrane function has also been unknown. We have identified four integral membrane proteins of the plasma membrane that occur specifically at these apical annular sites. These proteins are an LMBR-domain containing protein TgLMBD3, and three SNARE proteins TgStxPM, TgNPSN, and TgSyp7. Using widefield-microscopy, we discovered that depletion of each of these proteins results in reduced secretion of dense granule proteins, implicating the apical annuli as a site of exocytosis in Toxoplasma. To further understand the role of these proteins and the impact of their depletion on the parasite, we performed whole-cell quantitative proteomics in control and knockdown cell lines. Several hundred proteins have been identified and quantified in three biological replicates for every knockdown cell line and the untreated control line. The majority of enriched proteins in the knockdown cell lines are those packaged in dense granules, indicating their failure to be secreted outside the cell. The apical annuli are therefore sites of SNARE-mediated exocytosis of dense granules in Toxoplasma.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Toxoplasma Gondii

TISSUE(S): Hff-1 Cell

SUBMITTER: Thomas Krueger

LAB HEAD: Ross Waller

PROVIDER: PXD044588 | Pride | 2024-01-25

REPOSITORIES: Pride

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Project description:Toxoplasma gondii is a widely prevalent parasite of animals and humans that causes a broad spectrum of conditions ranging from asymptomatic or mild cold-like infections to abortion, severe ocular disease, encephalitis, and sometimes death. It is a member of the phylum Apicomplexa that also includes such significant human pathogens as Plasmodium spp. and Cryptosporidium spp. causing malaria and diarrheal disease, respectively. The toxoplasma cell has a multilayered pellicle: under the plasma membrane lies the inner membrane complex (IMC) comprised of flat membrane sacks supported by cortical microtubules and a meshwork of cytoskeletal filaments. The IMC acts as a mechanical barrier for membrane vesicles trafficking to and from the plasma membrane, necessitating the existence of dedicated cites for endo- and exocytosis where the IMC is interrupted. One such site is located at the apical end of the cell where the contents of the apical secretory organelles is exocytosed upon invasion. However, endocytosis in Toxoplasma and other apicomplexans is poorly understood, although conspicuous invaginations of the plasma membrane observed microscopically have been dubbed the micropore and postulated to be involved in endocytosis. We found that the endocytic markers including the subunits α and μ of the AP2 adaptor complex, dynamin-related protein C (DrpC), an Eps15 homology domain-containing protein, and the kelch-domain protein K13 localise to distinct persistent foci in the pellicle of Toxoplasma. Another type of persistent punctate structures found in the IMC are the enigmatic apical annuli whose function is completely unknown. We have discovered a new component of the annuli which is a putative LMBR1 family region protein embedded into the plasma membrane. Our experiments indicate that this protein – and annuli by extension – has a role in exocytosis. To better understand the molecular composition of these endocytic and exocytic structures and gain an insight into their function, we genetically fused them with a promiscuous bacterial biotin-ligase BirA* and used them as baits in proximity-dependent biotin identification experiments (BioID). We employed TMT10plex labelling and MS3-level tandem mass spectrometry with synchronous precursor selection (SPS-MS3) to achieve accurate quantification of multiple samples. Several hundred proteins have been identified and quantified in three biological replicates for every bait and the parental cell line control. Proteins significantly enriched in the BirA*-tagged cells relative to the control have been identified using linear modelling with LIMMA.

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Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma

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