Toxoplasma gondii, the causative agent of toxoplasmosis, chronically infects one-third of the world’s human population (Robert-Gangneux and Darde, 2012; Wang ZD, 2017; Elsheikha et al., 2021). Toxoplasma gondii encompasses three main lifecycle stages. Tachyzoites, the rapidly replicating form of T. gondii, are essential for acute infection (Robert-Gangneux and Darde, 2012, Elsheikha et al., 2021). Bradyzoites enclosed within tissue cysts are responsible for chronic infection (Elsheikha et al., 2021). Sporozoites-containing oocysts, the product of parasite sexual reproduction in the feline gut, are shed in cat faeces and infect many intermediate hosts (Robert-Gangneux and Darde, 2012; Wang ZD, 2017; Elsheikha et al., 2021). The ingestion of sporulated oocysts in water or food contaminated with feline feces, or ingestion of tissue cysts in raw infected meat, results in horizontal transmission of infection to humans (Robert-Gangneux and Darde, 2012). When a pregnant woman is infected, tachyzoites can colonize the placenta and infect the fetus, leading to a detrimental health impact on the offspring (Robert-Gangneux and Darde, 2012). Primary T. gondii infection in immunocompetent individuals is often asymptomatic. However, switching of bradyzoites within the cysts back to tachyzoites can lead to life-threatening brain pathologies in immunocompromised individuals (Luft and Remington, 1992; Wang ZD, 2017; Elsheikha et al., 2021). Because none of the current therapies can eliminate parasite cysts, there is a clear need for better understanding of the mechanisms of T. gondii pathogenesis and identification of the virulence factors controlling tachyzoite transformation into bradyzoite-containing cysts.

The ability of T. gondii to infect a broad range of host cell types is mediated by sequential secretion of effector proteins including microneme proteins (MICs), rhoptry neck proteins (RONs), rhoptry body proteins (ROPs) and dense granule proteins (GRAs) (Bradley et al., 2005, Clough and Frickel, 2017, Bullen et al., 2019, Wang et al., 2020d). Once inside the host cell, the parasite replicates inside a parasitophorous vacuole (PV) surrounded by a parasitophorous vacuole membrane (PVM) (Clough and Frickel, 2017, Wang et al., 2020d). After GRAs are released from the dense granules and secreted into the PV lumen, some of GRAs are associated with the PVM and intravacuolar network (IVN), which connects the parasites and the PVM inside the PV (Clough and Frickel, 2017, Wang et al., 2020d, Griffith et al., 2022). In addition to these GRAs in the PV, IVN or PVM, some GRAs such as GRA16, GRA18 and GRA24 are exported into the host cells and hijack cellular immune functions (Clough and Frickel, 2017, Hakimi et al., 2017).

GRAs promote T. gondii survival and replication by modifying the functions of the PV, IVN, PVM and host cell environment. For example, GRA17 and GRA23 facilitate diffusion of small molecules and nutrients across the PVM (Gold et al., 2015). GRA14 and GRA64 are indispensable for the internalization of host cytosolic proteins by interacting with components of the host endosomal sorting complex required for transport (ESCRT) (Rivera-Cuevas et al., 2021, Mayoral et al., 2022). In addition to their roles in vesicle trafficking and nutrient acquisition, some GRAs are involved in trafficking and export of secreted proteins into the host cell or the PVM. The Myc regulation 1 (MYR) complex on PVM, encompassing MYR1, MYR2 and MYR3, interacts with GRA44, GRA45 and TgPPM3C, and forms a putative PVM-localized translocon, which is necessary for trafficking of effectors such as GRA16, GRA18, T. gondii inhibitor of STAT transcription (TgIST) and Toxoplasma E2F4-asociated EIH2-inducing gene regulator (TEEGR) (Blakely et al., 2020, Cygan et al., 2020, Wang et al., 2020d). The effectors GRA16, GRA24, TEEGR and TgIST translocate to the host cell nucleus, and manipulate host signal pathways and gene expression to counter the host immune system (Clough and Frickel, 2017, Wang et al., 2020d). Several secreted GRAs, which localize in host cells or PVMs, can subvert the host immune response. GRA7 and GRA60, virulence factors of T. gondii, enable the parasites to evade the host innate immune mechanism by targeting the immunity regulated GTPases (Alaganan et al., 2014, Nyonda et al., 2021).

While functions of known GRAs have been characterized in the tachyzoite stage, some of them are also important in bradyzoites. In the bradyzoite stage, the IVN and PVM localized GRAs are located in the cyst wall and deletion of these gras, such as GRA2, GRA5, GRA7, GRA8 and GRA14, affect accumulation of cyst wall proteins at the cyst periphery (Guevara et al., 2019, Guevara et al., 2020). In addition, some bradyzoite-specific GRA proteins are involved in cyst formation and persistence, including CST1 (Tomita et al., 2013) and GRA55 (Nadipuram et al., 2020). CST2/GRA50 and GRA55 are important virulence factors, affecting the establishment and maintenance of cysts in chronic infection (Tu et al., 2019, Nadipuram et al., 2020). Although significant progress has been made to uncover the mechanism of cyst formation, only a few cyst wall proteins have been identified and their involvement in parasite differentiation and cyst formation remains unknown.

In recent years, multiple novel T. gondii GRAs have been reported (Barylyuk et al., 2020), including a novel gene TGME49_299780 which was predicted as a putative GRA using hyperplexed Localisation of Organelle Proteins by Isotopic Tagging (hyperLOPIT) (Barylyuk et al., 2020) and identified as a contributing factor to T. gondii fitness (Young et al., 2019, Butterworth et al., 2022). However, the function of this putative GRA (termed GRA76) remains unknown. In this study, we characterized the role of GRA76 in pathogenicity of T. gondii infection using CRISPR-Cas9 technology, immunofluorescence assay (IFA), plaque assay, intracellular replication assay, invasion assay, and egress assay. We also analyzed the effect of deletion of gra76 on the expression of bradyzoites-associated genes in PruΔgra76 using RNA sequencing (RNA-Seq). Our findings suggest that the newly characterized GRA76 plays a role in parasite growth, virulence, and bradyzoite differentiation.