Conditional Knockdown of Proteins Using Auxin-inducible  Degron (AID) Fusions in Toxoplasma gondii

Brown, Kevin M.; Long, Shaojun; Sibley, L. David

doi:10.21769/bioprotoc.2728

Cited by 99 publications

(122 citation statements)

References 15 publications

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“…To investigate TgGC function, we utilized the auxin-inducible degron (AID) system for conditional knockdown of proteins in T. gondii (Brown et al, 2017, 2018; Long et al,2017). Starting with the auxin receptor (TIR1) expressing line RH TIR1-3FLAG, we used CRISPR/Cas9 genome editing to create epitope-tagged TgGC lines with 6Ty near the N-terminus and/or an AID-3HA (AID or mini-AID) at the C-terminus (Figure 2A, Figure S2A).…”

Section: Resultsmentioning

confidence: 99%

“…Since TgGC resisted several knockout attempts with CRISPR/Cas9, we utilized traditional epitope tagging and an auxin-inducible degron (AID) system (Brown et al, 2017, 2018; Long et al, 2017) to detect and regulate TgGC expression. TgGC localizes to the apical cap region of the plasma membrane in both intracellular and extracellular parasites.…”

Section: Discussionmentioning

confidence: 99%

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Essential cGMP Signaling in Toxoplasma Is Initiated by a Hybrid P-Type ATPase-Guanylate Cyclase

Brown

Sibley

2018

Cell Host & Microbe

101

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Summary (150) Apicomplexan parasites rely on cyclic nucleotide-dependent kinases for host cell infection, yet the mechanisms that control their activation remain unknown. Here we show that an apically-localized guanylate cyclase (GC) controls microneme secretion and lytic growth in the model apicomplexan Toxoplasma gondii. Cell permeable cGMP reversed the block in microneme secretion seen in a knockdown of TgGC, linking its function to production of cGMP. TgGC possesses an N-terminal P-type-ATPase domain fused to a C-terminal heterodimeric guanylate cyclase domain, an architecture found only in Apicomplexa and related protists. Complementation with a panel of mutants revealed a critical requirement for the P-type ATPase domain for maximum GC function. We further demonstrate that knockdown of TgGC in vivo protects mice from lethal infection by blocking parasite expansion and dissemination. Collectively this work demonstrates that cGMP-mediated signaling in Toxoplasma relies on a multi-domain architecture, which may serve a conserved role in related parasites.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Essential cGMP Signaling in Toxoplasma Is Initiated by a Hybrid P-Type ATPase-Guanylate Cyclase

Brown

Sibley

2018

Cell Host & Microbe

101

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“…A detailed protocol for using the auxin-inducible degron system is also available at Bio-protocol (Brown et al , 2018). …”

Section: Methodsmentioning

confidence: 99%

CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii

2018

Self Cite

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Defining protein interaction networks can provide key insights into how protein complexes govern complex biological problems. Here we define a method for proximity based labeling using permissive biotin ligase to define protein networks in the intracellular parasite Toxoplasma gondii. When combined with CRISPR/Cas9 based tagging, this method provides a robust approach to defining protein networks. This approach detects interaction within intact cells, it is applicable to both soluble and insoluble components, including large proteins complexes that interact with the cytoskeleton and unique microtubule organizing center that comprises the apical complex in apicomplexan parasites.

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“…To investigate the role of myristoylation in CDPK1 function, we generated an inducible knock-down (iKD) line using the mini auxin inducible degron (mAID) strategy (Brown et al, 2018), which allows for proteasomal degradation of mAID-tagged proteins in an indole acetic acid (IAA) dependent manner. To generate the CDPK1 iKD line, we C-terminally tagged endogenous CDPK1 in RHΔku80 TIR1 parasites with a mAID-Myc cassette ( Figure 3C).…”

Section: Myristoylation Is Important For Cdpk1 Functionmentioning

confidence: 99%

“…To generate the inducible CDPK1 knock-down strain (RH TIR-1-3FLAG_CDPK1_mAID_Myc, referred to here as iKD CDPK1), the sequence encoding mAID_Myc_HXGPRT was PCR amplified from pTUB1_YFP_mAID_Myc using primer pair 35/36, and co-transfected into the RH TIR1-3FLAG (Brown et al, 2018) strain with pSag1_Cas9-U6_sg3'UTRCDPK1. Recombinant parasites were selected 24 h post transfection by addition of mycophenolic acid (MPA; 25µg/mL) and xanthine (XAN; 50 µg/mL) to culture medium.…”

Section: Parasite Strain Generationmentioning

confidence: 99%

Global profiling of myristoylation inToxoplasma gondiireveals key roles for lipidation in CDPK1 and MIC7 function

Broncel

Dominicus

Hunt

et al. 2019

Preprint

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N-myristoylation is a ubiquitous class of protein lipidation across eukaryotes and N-myristoyl transferase has been proposed as an attractive drug target in several pathogens. Functionally the myristate often primes for subsequent palmitoylation and stable membrane attachment, however, growing evidence also suggests additional regulatory roles for myristoylation on proteins. Here we describe the first global chemoproteomic screening of protein myristoylation in Toxoplasma gondii.Through quantitative mass spectrometry coupled with validated chemoproteomic tools, we identify 65 myristoylated proteins. We report functionally important myristoylation on the key signalling protein CDPK1 and, surprisingly, myristoylation of the microneme protein 7 (MIC7), a predicted type-Itransmembrane protein. We demonstrate that myristoylation of MIC7 is not important for the trafficking to micronemes, but appears to play a role in host cell invasion. This dataset represents a large fraction of the parasite's myristoylated proteome and a prerequisite to investigate this modification in Toxoplasma.

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Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii

Cited by 99 publications

References 15 publications

Essential cGMP Signaling in Toxoplasma Is Initiated by a Hybrid P-Type ATPase-Guanylate Cyclase

Essential cGMP Signaling in Toxoplasma Is Initiated by a Hybrid P-Type ATPase-Guanylate Cyclase

CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii

Global profiling of myristoylation inToxoplasma gondiireveals key roles for lipidation in CDPK1 and MIC7 function

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