Characterization of Two EF-hand Domain-containing Proteins from Toxoplasma gondii

Abstract

The universal role of calcium (Ca²⁺ ) as a second messenger in cells depends on a large number of Ca²⁺ -binding proteins (CBP), which are able to bind Ca²⁺ through specific domains. Many CBPs share a type of Ca²⁺ -binding domain known as the EF-hand. The EF-hand motif has been well studied and consists of a helix-loop-helix structural domain with specific amino acids in the loop region that interact with Ca²⁺ . In Toxoplasma gondii a large number of genes (approximately 68) are predicted to have at least one EF-hand motif. The majority of these genes have not been characterized. We report the characterization of two EF-hand motif-containing proteins, TgGT1_216620 and TgGT1_280480, which localize to the plasma membrane and to the rhoptry bulb, respectively. Genetic disruption of these genes by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) resulted in mutant parasite clones (Δtg216620 and Δtg280480) that grew at a slower rate than control cells. Ca²⁺ measurements showed that Δtg216620 cells did not respond to extracellular Ca²⁺ as the parental controls while Δtg280480 cells appeared to respond as the parental cells. Our hypothesis is that TgGT1_216620 is important for Ca²⁺ influx while TgGT1_280480 may be playing a different role in the rhoptries.

Keywords: Calcium entry; plasma membrane; rhoptry.

Figure 1

Tagging and localization of TgGT1_216620. (A) Schematic model showing the predicted topology of TgGT1_216620. The EF-hand-motifs are marked in blue. The model was generated using the Protter web application (Omasits et al. 2014). (B) Scheme depicting the strategy used for the C-terminal tagging of TgGT1_216620 in the parental strain TatiΔku80. The genomic region and recombination fragment are shown in green, the C-terminal HA tag in yellow and the selection marker, chloramphenicol acetyl transferase (CAT), in blue. (C) Top panel, PCR analysis showing the correct insertion of 3HA tag (3HA) at the 3’ region of the TgGT1_216620gene. Bottom panel, PCR positive control of parental and 3HA tagged parasites. (D) Western blot analysis of total lysates from TgGT1_216620-3HA and TatIΔku80 parental cells, respectively. Top panel, two bands above ~250 kDa were detected in lysates of the tagged strain, but not in lysates of the parental strain. Bottom panel, loading controls developed with anti-tubulin. (E) Immunofluorescence analysis of intracellular and extracellular tachyzoites showing a punctuated localization of α-HA in both intracellular and extracellular parasites with a higher concentration at the plasma membrane. Scale bars = 5 μm (F) Top panel, IFAs showing co-localization of α-HA and α-GAP45 in intracellular parasites at the periphery. Bottom panel, co-localization of α-HA and α-SAG1, a plasma membrane marker, in extracellular tachyzoites (bottom). Scale bars = 5 μm

Figure 2

Characterization of TgGT1_216620. (A) Scheme showing the strategy used for generation of TgGT1_216620 gene knockouts in the Toxoplasma gondii RH strain. The cartoon shows the genomic region and recombination fragment, protospacer (blue), and selection marker (orange; dihydrofolate reductase, DHFR). (B) Top panel, disruption of the TgGT1_216620 gene by insertion of a DHFR cassette. PCR reaction with primers upstream and downstream of insertion site produced a fragment of 4.1 kbp including the DHFR (Δtg216620]. PCR product obtained with template DNA from the parental strain produces a fragment of ~1 kbp corresponding to the original TgGT1_216620 gene. Bottom panel, PCR reaction of positive controls for Δtg216620 and parental parasites. (C) qPCR analysis of total RNA harvested from the Δtg216620 mutant strain and parental strain RH. Left panel, transcript levels obtained with primers downstream of the insertion site. Right panel, mRNA levels using primers upstream and downstream of the drug cassette insertion site. (D) Plaque assays showing growth of the Δtg216620 mutant strain compared with the RH strain. Plaque size measurements and statistic analysis n = 3, P<0.05. (E) Changes in cytosolic Ca²⁺ levels of Fura-2 AM loaded tachyzoites [Δtg216620 mutants and RH). Parasites were in suspension and Ca²⁺ (2 mM) was added at the time indicated. The bar graph to the right shows the quantification and statistical analysis from three biological experiments, each in duplicate. (F) Changes in cytosolic Ca²⁺ levels of Fura-2 AM loaded tachyzoites [Δtg216620 mutants and RH). Parasites were in suspension and 1 μM Thapsigargin (TG) and 2 mM Calcium (Ca²⁺) were added at the times indicated. The bar graph to the right shows the quantification and statistical analysis of three biological experiments, each in duplicate. P<0.05.

Figure 3

Tagging and localization of TgGT1_280480. (A) Model showing the predicted transmembrane topology of TgGT1_280480. The EF-hand Ca²⁺ binding domains are marked in blue, and the COPI associate domain marked in yellow. (B) Scheme showing the strategy used for C-terminal tagging of TgGT1_280480 through homologous recombination in RHTatIΔku80 strain. The genomic region and recombination fragment are shown in green, the C-terminal HA tag in yellow and the selection marker, chloramphenicol acetyl transferase (CAT), in blue. (C) Top panel, PCR validation of TgGT1_280480-3HA tagged strain (Table S2). A 2.4 kbp band confirmed correct HA integration as indicated by the bar in B. Bottom panel, PCR positive control of parental and 3HA tagged parasites. (D) Top panel, Western blot analysis of total lysates from TgGT1_280480-3HA and parental cells, showing a band at 37 kDa in the tagged line. Bottom panel, tubulin was used as loading control (α-Tubulin antibodies) (E) IFA analysis of intracellular and extracellular tachyzoites showing that TgGT1_280480-3HA localizes to the rhoptry bulb region. Scale bars = 5 μm (F) Top panel, IFA of extracellular tachyzoites showing co-localization of α-HA and α-ROP7, a rhoptry bulb marker. Bottom panel, IFA showing co-localizations of rhoptry marker α-TgCA_RP (carbonic anhydrase related protein) and α-HA antibodies in intracellular parasites. Scale bars = 5 μm.

Figure 4

Characterization of TgGT1_280480. (A) Scheme showing the strategy used for TgGT1_280480 gene knockout. CRISPR/Cas9 gene knockout strategy was done in the RH strain. Genomic region (green) and recombination fragment, protospacer (blue), and selection marker (orange; DHFR) are shown. (B) Top panel, insertion of the drug cassette disrupts the TgGT1_280480 gene. PCR product using the primers indicated in the cartoon in A produce a product of 5 kbp validating the insertion in Δtg280480 (fragment is marked in the scheme in A) while a fragment of 1.9 kbp is obtained from the PCR reaction of the parental strain. Bottom panel, PCR positive control from parental and Δtg280480. (C) qPCR analysis of total RNA harvested from Δtg280480 mutant strain and parental strain RH. Left, expression level obtained with primers downstream of the drug cassette insertion site. Right, mRNA levels using primers upstream and downstream of the drug cassette insertion site. (D) Plaque assays showing growth of plaques formed by Δtg280480 mutant and RH parasites. Plaque size measurements and statistic analysis n = 3, P<0.05. (E) Changes in cytosolic Ca²⁺ levels of Fura-2 AM loaded tachyzoites [Δtg280480 mutants and RH). Parasites were in suspension as described in Material and Methods and Ca²⁺ (2 mM) was added at the time indicated. The bar graph to the right shows the quantification and statistical analysis from three biological experiments, each in duplicate. (F) Changes in cytosolic Ca²⁺ levels of Fura-2 AM loaded tachyzoites [Δtg280480 mutants and RH). 1 μM Thapsigargin and 2 mM Calcium (Ca²⁺) were added at the times indicated. The bar graph to the right shows the quantification and statistical analysis of three biological experiments, each in duplicate. P<0.05.