Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins

Mineo, Tiago Wilson Patriarca; Chern, Jessica H; Thind, Amara C; Mota, Caroline Martins; Nadipuram, Santhosh M.; Torres, Juan A; Bradley, Peter J.

doi:10.1128/msphere.00896-21

Cited by 7 publications

(6 citation statements)

References 56 publications

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“…We established a method to generate NcGRA7 -complemented parasite insertion of the NcGRA7 gene into the Neospora uracil phosphoribosyl transferase (NcUPRT) gene ( Nishikawa et al, 2018 ), but we failed to obtain NcSAG1 -complemented parasites. Although we tried other methods, such as insertion of the NcSAG1 gene into the Neospora hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) gene ( Mineo et al, 2022 ) or random insertion of the NcSAG1 gene into the N. caninum genome ( Abe et al, 2015 ), we could not obtain the NcSAG1 -complemented parasite. Thus, we confirmed the results using two different clones of NcSAG1KO parasites in this study.…”

Section: Discussionmentioning

confidence: 99%

Neospora caninum surface antigen 1 is a major determinant of the pathogenesis of neosporosis in nonpregnant and pregnant mice

Abdelbaky,

Rahman,

Shimoda

et al. 2024

Front. Microbiol.

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IntroductionNcSAG1 is one of most widely investigated antigens of Neospora caninum in various research fields. Such studies demonstrated the proficiency of NcSAG1 in the regulatory process of parasite adhesion and invasion of host cells. Accordingly, the contribution of NcSAG1 to the pathogenesis of neosporosis can undoubtedly be extrapolated, but direct evidence is lacking. Herein, we provide the first successful attempt at the gene disruption of NcSAG1 and novel data on the invasion and virulence potentials of N. caninum in vitro and in vivo.MethodsThe disruption of the NcSAG1 gene was applied using the CRISPR/Cas9 system and confirmed by PCR, western blot and indirect fluorescent antibody tests as NcSAG1 knockout parasites (NcSAG1KO). Then, we investigated the role of NcSAG1 in the growth kinetics of the parasite in vitro.Results and discussionThe deletion of the NcSAG1 gene significantly decreased the infection rate and reduced the egress rate of the parasite. An in vivo study using nonpregnant female and male BALB/c mice revealed a significantly higher survival rate and lower body weight change in the group infected with the NcSAG1KO parasite than in the parental strain (Nc-1)-infected group. Regarding the vertical transmission model of BALB/c mice, the absence of the NcSAG1 gene significantly enhanced the survival of pups and greatly lowered the parasite burden in the brains of pups. In conclusion, our study suggested NcSAG1 as a key molecule in the pathogenesis of N. caninum.

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Section: Discussionmentioning

confidence: 99%

Neospora caninum surface antigen 1 is a major determinant of the pathogenesis of neosporosis in nonpregnant and pregnant mice

Abdelbaky,

Rahman,

Shimoda

et al. 2024

Front. Microbiol.

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“…N. caninum also does not significantly activate NF-κB ( 65 ), suggesting that GRA83 may play a role in enabling GRA15 to bind to TRAF2/6 and consequently translocate NF-κB to the nucleus, or that a second function for GRA83 in both organisms has yet to be discovered. Recently developed tools for genetic manipulation of N. caninum will aid in the already proven approach of comparing these pathogens using heterologous expression or knockouts of various effectors to assess how individual or groups or effectors are able to mediate infection of these pathogens in their various mammalian hosts ( 26 , 66 ).…”

Section: Discussionmentioning

confidence: 99%

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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“…Another interactor with the TgBDP1 core complex was TGGT1_235420, a protein of unknown function that localizes to the nucleus. Although the function of this protein is unknown, it is essential for tachyzoite survival in both Toxoplasma and the related coccidian Neospora caninum ( 39 ).…”

Section: Discussionmentioning

confidence: 99%

An apicomplexan bromodomain protein, TgBDP1, associates with diverse epigenetic factors to regulate essential transcriptional processes in Toxoplasma gondii

Fleck

McNutt

Chu

et al. 2023

mBio

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The protozoan pathogen Toxoplasma gondii relies on tight regulation of gene expression to invade and establish infection in its host. The divergent gene regulatory mechanisms of Toxoplasma and related apicomplexan pathogens rely heavily on regulators of chromatin structure and histone modifications. The important contribution of histone acetylation for Toxoplasma in both acute and chronic infection has been demonstrated, where histone acetylation increases at active gene loci. However, the direct consequences of specific histone acetylation marks and the chromatin pathway that influences transcriptional regulation in response to the modification are unclear. As a reader of lysine acetylation, the bromodomain serves as a mediator between the acetylated histone and transcriptional regulators. Here we show that the bromodomain protein, TgBDP1, which is conserved among Apicomplexa and within the Alveolata superphylum, is essential for Toxoplasma asexual proliferation. Using cleavage under targets and tagmentation, we demonstrate that TgBDP1 is recruited to transcriptional start sites of a large proportion of parasite genes. Transcriptional profiling during TgBDP1 knockdown revealed that loss of TgBDP1 leads to major dysregulation of gene expression, implying multiple roles for TgBDP1 in both gene activation and repression. This is supported by interactome analysis of TgBDP1 demonstrating that TgBDP1 forms a core complex with two other bromodomain proteins and an ApiAP2 factor. This core complex appears to interact with other epigenetic factors such as nucleosome remodeling complexes. We conclude that TgBDP1 interacts with diverse epigenetic regulators to exert opposing influences on gene expression in the Toxoplasma tachyzoite. IMPORTANCE Histone acetylation is critical for proper regulation of gene expression in the single-celled eukaryotic pathogen Toxoplasma gondii . Bromodomain proteins are “readers” of histone acetylation and may link the modified chromatin to transcription factors. Here, we show that the bromodomain protein TgBDP1 is essential for parasite survival and that loss of TgBDP1 results in global dysregulation of gene expression. TgBDP1 is recruited to the promoter region of a large proportion of parasite genes, forms a core complex with two other bromodomain proteins, and interacts with different transcriptional regulatory complexes. We conclude that TgBDP1 is a key factor for sensing specific histone modifications to influence multiple facets of transcriptional regulation in Toxoplasma gondii .

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Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins

Cited by 7 publications

References 56 publications

Neospora caninum surface antigen 1 is a major determinant of the pathogenesis of neosporosis in nonpregnant and pregnant mice

Neospora caninum surface antigen 1 is a major determinant of the pathogenesis of neosporosis in nonpregnant and pregnant mice

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

An apicomplexan bromodomain protein, TgBDP1, associates with diverse epigenetic factors to regulate essential transcriptional processes in Toxoplasma gondii

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