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The interplay of self-assembly and target binding in centrin 1 from Toxoplasma gondii.

ABSTRACT: Centrins are conserved calcium (Ca2+)-binding proteins typically associated with centrosomes that have been implicated in several biological processes. In Toxoplasma gondii, a parasite that causes toxoplasmosis, three centrin isoforms have been recognized. We have recently characterized the metal binding and structural features of isoform 1 (TgCEN1), demonstrating that it possesses properties consistent with a role as a Ca2+ sensor and displays a Ca2+-dependent tendency to self-assemble. Herein, we expanded our studies, focusing on the self-association and target binding properties of TgCEN1 by combining biophysical techniques including dynamic light scattering, isothermal titration calorimetry, nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy. We found that the self-assembly process of TgCEN1 depends on different physicochemical factors, including Ca2+ concentration, temperature, and protein concentration, and is mediated by both electrostatic and hydrophobic interactions. The process is completely abolished upon removal of the first 21-residues of the protein and is significantly reduced in the presence of a binding target peptide derived from the human XPC protein (P17-XPC). Titration of P17-XPC to the intact protein and isolated domains showed that TgCEN1 possesses two binding sites with distinct affinities and Ca2+ sensitivity; a high-affinity site in the C-lobe which may be constitutively bound to the peptide and a low-affinity site in the N-lobe which is active only upon Ca2+ stimulus. Overall, our results suggest a specific mechanism of TgCEN1 for Ca2+-modulated target binding and support a N-to-C self-assembly mode, in which the first 21-residues of one molecule likely interact with the C-lobe of the other.

SUBMITTER: Conter C

PROVIDER: S-EPMC8286830 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Toxoplasmosis is a cosmopolitan zoonotic infection, caused by a unicellular protozoan parasite known as Toxoplasma gondii that belongs to the phylum Apicomplexa. It is estimated that over one-third of the world's population has been exposed and are latently infected with the parasite. In humans, toxoplasmosis is predominantly asymptomatic in immunocompetent persons, while among immunocompromised individuals may be cause severe and progressive complications with poor prognosis. Moreover, seronegative pregnant mothers are other risk groups for acquiring the infection. The life cycle of T. gondii is very complex, indicating the presence of a plurality of antigenic epitopes. Despite of great advances, recognize and construct novel vaccines for prevent and control of toxoplasmosis in both humans and animals is still remains a great challenge for researchers to select potential protein sequences as the ideal antigens. Notably, in several past years, constant efforts of researchers have made considerable advances to elucidate the different aspects of the cell and molecular biology of T. gondii mainly on microneme antigens, dense granule antigens, surface antigens, and rhoptry proteins (ROP). These attempts thereby provided great impetus to the present focus on vaccine development, according to the defined subcellular components of the parasite. Although, currently there is no commercial vaccine for use in humans. Among the main identified T. gondii antigens, ROPs appear as a putative vaccine candidate that are vital for invasion procedure as well as survival within host cells. Overall, it is estimated that they occupy about 1%-30% of the total parasite cell volume. In this review, we have summarized the recent progress of ROP-based vaccine development through various strategies from DNA vaccines, epitope or multi epitope-based vaccines, recombinant protein vaccines to vaccines based on live-attenuated vectors and prime-boost strategies in different mouse models.

Project description:Cyst formation is a key feature of the T. gondii life cycle but the genetic networks that drive this process are not yet fully characterized. To identify new components of this network, we compared T. gondii to its nearest extant relative Hammondia hammondi given the critical differences between these species in the timing and efficiency of cyst formation. Using transcriptional data from critical developmental and pH exposure time points from both species, we identified the gene TGVEG_311100, which we named Regulator of Cystogenesis 1 (ROCY1), as being both necessary and sufficient for cyst formation in T. gondii. Compared to WT parasites, TGVEG?ROCY1 parasites formed significantly fewer tissue cysts in response to alkaline pH stress in vitro and cysts were nearly undetectable in mouse brains for up to 9 weeks post-infection. Overexpression of tagged ROCY1 in WT parasites was sufficient to induce cyst formation in vitro in both WT and ROCY1-deficient parasites, demonstrating that ROCY1 is both necessary and sufficient for cyst formation. Moreover this induction of cyst formation required at least 1 of 3 predicted CCCH Zinc finger domains. Mice chronically infected with ?ROCY1 parasites had detectable tachyzoites in the brain for up to 37 days post-infection (while mice infected with WT parasites did not), and CNS transcriptional analyses at day 30 post-infection throughout the chronic phase of infection revealed inflammatory signatures consistent with acute infection in ?ROCY1 parasites compared to WT. Despite our inability to detect brain cysts in infected mice, both WT and ?ROCY1 knockout parasites reactivated after dexamethasone treatment with similar timing and magnitude for up to 5 months post infection, challenging the paradigm that long term parasite persistence in the CNS requires cyst formation. These data identify a new regulator of cyst formation in T. gondii that is both necessary and sufficient for cyst formation, and whose function relies on its conserved nucleic acid binding motif.

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The interplay of self-assembly and target binding in centrin 1 from Toxoplasma gondii.

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