Biology subjects

Dey, V.

Publications and source records attributed to Dey, V..

4 recordsLinked to original sources

bioRxiv2025

GCN5a is a telomeric lysine acetyltransferase whose loss primes Toxoplasma gondii for latency

Toxoplasma gondii is a protozoan parasite that causes persistent infection in warm-blooded vertebrates by undergoing differentiation from a replicative stage (tachyzoites) to a latent encysted stage (bradyzoites). Stage differentiation is critical for transmission and pathogenesis and relies on gene regulation driven by a network of transcription and epigenetic factors. We previously found in non-cystogenic type I RH strain parasites that the lysine acetyltransferase (KAT), GCN5a, is dispensable in tachyzoites but required to upregulate stress-response genes, suggesting a link with bradyzoite conversion. To address this possibility, we generated endogenously tagged GCN5a parasites and a genetic knockout in cystogenic type II Pru strain. We show that GCN5a protein, but not mRNA, increases during differentiation and complexes with unique protein partners, most of which contain AP2 domains. Pru strain tachyzoites lacking GCN5a augment bradyzoite-specific gene expression in the absence of stress. Loss of GCN5a slowed tachyzoite replication and heightened sensitivity to bradyzoite conversion but resulted in smaller cyst sizes compared to wildtype. Using CUT&Tag, we delineated the chromosomal occupancy of GCN5a relative to the essential KAT, GCN5b. While GCN5b localizes to coding regions, GCN5a surprisingly localizes exclusively to telomeres. These findings suggest that the loss of GCN5a leads to telomere dysfunction, which slows replication and promotes the transition to latency. IMPORTANCEToxoplasma gondii is a single-celled parasite that persists in warm-blooded hosts, including humans, because it converts into latent tissue cysts. Switching from its replicating form into dormant cysts is a tightly regulated process that involves epigenetic factors such as lysine acetyltransferases GCN5a and GCN5b. This study is the first to examine the role of GCN5a in a cyst-forming Toxoplasma strain. We found that GCN5a protein, but not mRNA, increases during cyst development. Additionally, parasites lacking GCN5a replicate more slowly and are quicker to form cysts when stressed. We show that GCN5a and GCN5b work in different multi-protein complexes and localize to different areas of the genome; while GCN5b targets promoters of gene coding regions, GCN5a is exclusively found at telomeric regions. Our findings suggest a novel role for GCN5a in telomere biology that, when depleted, produces a fitness defect that favors development of latent stages.

microbiology↗

bioRxiv2024

Cap-independent translation directs stress-induced differentiation of the protozoan parasite Toxoplasma gondii

Translational control mechanisms modulate microbial latency of eukaryotic pathogens, enabling them to evade immunity and drug treatments. The protozoan parasite Toxoplasma gondii persists in hosts by differentiating from proliferative tachyzoites to latent bradyzoites, which are housed inside tissue cysts. Transcriptional changes facilitating bradyzoite conversion are mediated by a Myb domain transcription factor called BFD1, whose mRNA is present in tachyzoites but not translated into protein until stress is applied to induce differentiation. We addressed the mechanisms by which translational control drives BFD1 synthesis in response to stress-induced parasite differentiation. Using biochemical and molecular approaches, we show that the 5-leader of BFD1 mRNA is sufficient for preferential translation upon stress. The translational control of BFD1 mRNA is maintained when ribosome assembly near its 5-cap is impaired by insertion of a 5-proximal stem-loop and upon knockdown of the Toxoplasma cap-binding protein, eIF4E1. Moreover, we show that a trans-acting RNA-binding protein called BFD2/ROCY1 is necessary for cap-independent translation of BFD1 through its binding to the 5-leader. Translation of BFD2 mRNA is also suggested to be preferentially induced under stress, but by a cap-dependent mechanism. These results show that translational control and differentiation in Toxoplasma proceed through cap-independent mechanisms in addition to canonical cap-dependent translation. Our identification of cap-independent translation in protozoa underscores the antiquity of this mode of gene regulation in cellular evolution and its central role in stress-induced life-cycle events.

microbiology↗

bioRxiv2023

mRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency

The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and dormant cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2 accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogenous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence. SignificanceToxoplasma gondii is an opportunistic pathogen of importance to global human and animal health. There are currently no chemotherapies targeting the encysted form of the parasite. Consequently, a better understanding of the mechanisms controlling encystation are required. Here we show that the mRNA cap-binding protein, eIF4E1, is involved in regulating the encystation process. Encysted parasites reduce eIF4E1 levels and depletion of eIF4E1 decreases the translation of ribosome-associated machinery and drives Toxoplasma encystation. Together, these data reveal a new layer of mRNA translational control that regulates parasite encystation and latency.

microbiology↗

bioRxiv2022

Toxoplasma gondii importin α shows weak auto-inhibition

Importin is a nuclear transporter that binds to nuclear localization signals (NLSs), consisting of 7-20 positively charged amino acids found within cargo proteins. In addition to cargo binding, intramolecular interactions also occur within the importin protein due to binding between the importin {beta}-binding (IBB) domain and the NLS-binding sites, a phenomenon called auto-inhibition. The interactions causing auto-inhibition are driven by a stretch of basic residues, similar to an NLS, in the IBB domain. Consistent with this, importin proteins that do not have some of these basic residues lack auto-inhibition; a naturally occurring example of such a protein is found in the apicomplexan parasite Plasmodium falciparum. In this report, we show that importin from another apicomplexan parasite, Toxoplasma gondii, harbors basic residues (KKR) in the IBB domain and exhibits auto-inhibition. This protein has a long, unstructured hinge motif (between the IBB domain and the NLS-binding sites) that does not contribute to auto-inhibition. However, the IBB domain may have a higher propensity to form an -helical structure, positioning the wild-type KKR motif in an orientation that results in weaker interactions with the NLS-binding site than a KRR mutant. We conclude that the importin protein from T. gondii shows auto-inhibition, exhibiting a different phenotype from that of P. falciparum importin . However, our data indicate that T. gondii importin may have a low strength of auto-inhibition. We hypothesize that low levels of auto-inhibition may confer an advantage to these important human pathogens.

biochemistry↗