Functional Characterization of 15 Novel Dense Granule Proteins in Toxoplasma gondii Using the CRISPR-Cas9 System

Zheng, Xiao-Nan; Wang, Jin‐Lei; Elsheikha, Hany M.; Wang, Meng; Zhang, Zhiwei; Sun, Li-Xiu; Wang, Xincheng; Zhu, Xing‐Quan; Li, Tingting

doi:10.1128/spectrum.03078-22

Cited by 8 publications

(4 citation statements)

References 68 publications

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“…We show that GRA83 is secreted into the vacuole in both tachyzoites and bradyzoites, suggesting that it functions in both lifecycle stages. While this manuscript was in preparation, another group also demonstrated that the epitope-tagged protein is secreted into the vacuole in tachyzoites ( 54 ). Together with the hyperLOPIT prediction ( 34 ), which tends to be reliable for dense granule proteins, these results confirm that GRA83 is indeed a secreted GRA protein.…”

Section: Discussionmentioning

confidence: 98%

The Toxoplasma gondii effector GRA83 modulates the host’s innate immune response to regulate parasite infection

Thind,

Mota,

Gonçalves

et al. 2023

mSphere

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Toxoplasma gondii ’s propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole (PV) in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both acute and chronic infections. Murine macrophages infected with ∆ gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro , which was confirmed with reduced IL-12 and interferon-gamma in vivo . This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the nuclear factor-κB (NF-κB) complex. While GRA15 similarly regulates NF-κB, infection with ∆ gra83/ ∆ gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labeling experiments to reveal candidate GRA83 interacting T. gondii -derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit the parasite burden. IMPORTANCE Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite’s ability to efficiently invade and regulate components of the host’s infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma’ s ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite’s diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection is important for understanding the complexity of the pathogen’s tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell’s response to limit infection.

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Section: Discussionmentioning

confidence: 98%

The Toxoplasma gondii effector GRA83 modulates the host’s innate immune response to regulate parasite infection

Thind,

Mota,

Gonçalves

et al. 2023

mSphere

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“…The CRISPR-Cas9 mediated homologous gene recombination was used to delete the had2a gene in the wild-type RH strain [ 23 ]. The CRISPR plasmid pSAG1::CAS9-U6::Sg HAD2a and homologous dihydrofolate reductase (DHFR) drug-selective plasmid were constructed.…”

Section: Methodsmentioning

confidence: 99%

Immunization with Live-Attenuated RHΔhad2a Strain Confers Partial Protective Immunity against Acute and Chronic Infection of Toxoplasma gondii in Mice

Zhang,

Cao,

et al. 2024

Pathogens

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Toxoplasmosis caused by Toxoplasma gondii is an important zoonosis of human and animal health significance. Current chemical therapeutics have side effects, and no commercially available vaccine is licensed for the prevention of toxoplasmosis in humans and most animals. Developing a safe and effective vaccine with long-term protection against T. gondii infection is necessary to control toxoplasmosis. HAD2a is a key member of the haloacid dehalogenase (HAD) phosphatase family, which is essential for T. gondii daughter budding. However, the role of HAD2a in T. gondii virulence remains unknown. In this study, we successfully constructed the had2a gene knockout strain in the T. gondii-type I RH strain (RHΔhad2a) and determined its role in virulence and vaccination. These results demonstrate that HAD2a played an important role in parasite daughter budding and in vitro replication. Knockout of the had2a gene attenuated the virulence of the T. gondii-type I RH strain. Vaccination with RHΔhad2a tachyzoites induced a Th1-biased immune response, provided partial protection against acute T. gondii infection in mice by highly virulent tachyzoites of RH and PYS (ToxoDB#9, Chinese I) strains, and conferred strong protection against challenge infection by cysts and oocysts of the less virulent type II Pru strain. These results demonstrate that T. gondii had2a is important for its in vitro proliferation and virulence in mice and that RHΔhad2a may be used as a candidate strain to generate a multiple gene knockout live-attenuated strain or be collaboratively applied with other live-attenuated strains to confer more effective protection against T. gondii infection.

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“…To investigate the expression and localization of ZFPs in the tachyzoite stage of the RH strain, we performed C-terminal endogenous tagging for eight ZFPs. CRISPR plasmid targeting the 3 -untranslated region (3 -UTR) of the zfp gene was obtained as previously described [17]. A short homology fragment containing 6 × HA tag products and DHFR fragments was amplified from the pLIC-6×HA-DHFR plasmid using specific primers located near the SgRNA at the 3 end of the corresponding zfp gene.…”

Section: Construction Of Epitope Tagging Strainsmentioning

confidence: 99%

Functional Characterization of Eight Zinc Finger Motif-Containing Proteins in Toxoplasma gondii Type I RH Strain Using the CRISPR-Cas9 System

Gao,

Wu,

Zheng

et al. 2023

Pathogens

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The Zinc finger protein (ZFP) family is widely distributed in eukaryotes and interacts with DNA, RNA, and various proteins to participate in many molecular processes. In the present study, the biological functions of eight ZFP genes in the lytic cycle and the pathogenicity of Toxoplasma gondii were examined using the CRISPR-Cas9 system. Immunofluorescence showed that four ZFPs (RH248270-HA, RH255310-HA, RH309200-HA, and RH236640-HA) were localized in the cytoplasm, and one ZFP (RH273150-HA) was located in the nucleus, while the expression level of RH285190-HA, RH260870-HA, and RH248450-HA was undetectable. No significant differences were detected between seven RHΔzfp strains (RHΔ285190, RHΔ248270, RHΔ260870, RHΔ255310, RHΔ309200, RHΔ248450, and RHΔ236640) and the wild-type (WT) strain in the T. gondii lytic cycle, including plaque formation, invasion, intracellular replication, and egress, as well as in vitro virulence (p > 0.05). However, the RHΔ273150 strain exhibited significantly lower replication efficiency compared to the other seven RHΔzfp strains and the WT strain, while in vivo virulence in mice was not significantly affected. Comparative expression analysis of the eight zfp genes indicates that certain genes may have essential functions in the sexual reproductive stage of T. gondii. Taken together, these findings expand our current understanding of the roles of ZFPs in T. gondii.

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Functional Characterization of 15 Novel Dense Granule Proteins in Toxoplasma gondii Using the CRISPR-Cas9 System

Abstract: Dense granule proteins (GRAs) play important roles in Toxoplasma gondii pathogenicity. However, the functions of many putative GRAs have not been elucidated.

Cited by 8 publications

References 68 publications

The Toxoplasma gondii effector GRA83 modulates the host’s innate immune response to regulate parasite infection

The Toxoplasma gondii effector GRA83 modulates the host’s innate immune response to regulate parasite infection

Immunization with Live-Attenuated RHΔhad2a Strain Confers Partial Protective Immunity against Acute and Chronic Infection of Toxoplasma gondii in Mice

Functional Characterization of Eight Zinc Finger Motif-Containing Proteins in Toxoplasma gondii Type I RH Strain Using the CRISPR-Cas9 System

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