Tagging of Weakly Expressed Toxoplasma gondii Calcium-Related Genes with High-Affinity Tags

Abstract

Calcium ions regulate a diversity of cellular functions in all eukaryotes. The cytosolic Ca²⁺ concentration is tightly regulated at the physiological cytosolic concentration of 50-100 nm. The Toxoplasma gondii genome predicts the presence of several genes encoding potential Ca²⁺ channels, pumps, and transporters. Many of these genes are weakly expressed and likely tightly regulated due to their potential impact to the physiology of the cell. Endogenous tagging has been widely used to localize proteins in T. gondii but low level of expression of many of them makes visualization of tags difficult and sometimes impossible. The use of high-performance tags for labeling proteins expressed at low level is ideal for investigating the localization of these gene products. We designed a Carboxy-terminus tagging plasmid containing the previously characterized "spaghetti monster-HA" (smHA) or "spaghetti monster-MYC" (smMYC) tags. These tags consist of 10 copies of a single epitope (HA or MYC) inserted into a darkened green fluorescence protein scaffold. We localized six proteins of various levels of expression. Clonal lines were isolated and validated by PCR, western blot, and immunofluorescence analyses. Some gene products were only visible when tagged with smHA and in one case the smHA revealed a novel localization previously undetected.

Keywords: C-terminus tagging; Calcineurin; hedgehog; phospholipase C; spaghetti monster.

Figure 1

Spaghetti monster high affinity tag. A. Strategy used for the smHA plasmid construction. A fragment between the NdeI and AvrII restriction sites corresponding to the 3HA tag in the p3HA.LIC.CAT plasmid was removed and replaced with the smHA gene tag. B. Linearized plasmids containing the smHA and 3HA tags showed a size of ~5.9 kb and ~4.9 kb, respectively (red arrows). (C) Scheme showing the strategy used for gene tagging via a single homology recombination event. F_GOI and R_pVAL indicate the position of the primers used for validation of tag insertion for each gene locus. Primers are shown in Table S1. HR, homology region, TUB, tubulin promoter, GOI, gene of interest, CAT, chloramphenicol acetyl transferase, resistance conferring gene U, uncut, C, cut.

Figure 2

The spaghetti monster tag does not alter protein localization. A. PCR analysis showing the correct insertion of the 3HA or smHA tag at the 3’ end of the TgCnA-3HA (3HA) (1.6 kbp) and TgCnA-smHA (smHA) (2.6 kbp) parasites. The PCR primers used are shown in Table S1. B. Cytosolic localization of TgCnA as observed with αHA antibodies (green). Extracellular and intracellular tachyzoites expressing TgCnA-HA and TgCnA-smHA are shown. The nucleus was stained with DAPI (blue). Scale bars = 5 μm. C. Western blot of lysates obtained from tachyzoites expressing TgCnA-3HA and TgCnA-smHA. Anti-HA antibodies detected bands of ~59 (3HA) or ~ 96 (smHA) kDa. The parental (P) line TatiΔku80 was used as negative control. α-tubulin antibody was used for loading control. D. Super-resolution microscopy shows cytosolic localization of TgCnA in both tagged lines. E. PCR analysis showing the correct insertion of the 3HA (3HA) (1.5 kbp) or smHA (smHA) (2.5 kbp) tags at the 3’ region of the TgELC2 gene. PCR products were absent in DNA samples obtained from WT tachyzoites. The PCR primers used are shown in Table S1. F. Immunofluorescence microscopy using αHA antibodies (green) confirmed plasma membrane localization in both extracellular and intracellular parasites expressing both tagged versions of TgELC2. Scale bars = 5 μm G. Western blot analysis using αHA antibodies detected bands of the expected size (~18 (3HA) and ~55 (smHA) kDa). Tubulin was used as loading control. H. Super-resolution microscopy images of TgELC2-3HA (3HA) and TgELC2-smHA (smHA) expressing cell lines, showing plasma membrane localization as detected by is-HA antibodies (green). Scale bars = 5 μm.

Figure 3

High affinity tags improved detection of proteins expressed at low levels. A. Predicted topology of TgGT1_222060 protein based on its predicted amino acid sequence using the TMHMM server 2.0 and Protter for visualization. Transmembrane domains predicted to be part of a voltage-gated channel are shown in purple. B. PCR analysis showing the correct insertion of the 3HA (3HA) (1.8 kbp) or smHA (smHA) (2.8 kbp) tags at the 3’ region of the TgGT1_222060 gene. The PCR primers used are shown in Table S1. C. Western blot analysis of TgGT1_222060-3HA (3HA) and TgGT1_222060-smHA (smHA) expressing cell lines. Anti-HA antibodies detected a band of ~140 kDa in smHA tachyzoite lysates (black arrow). No band was detected in lysates from 3HA cells. Lysates of the TatiΔku80 parental line were used as negative controls. α-tubulin was used as loading control. D. Immunofluorescence microscopy using α-HA antibodies (green) show cytosolic vesicular localization in extracellular and intracellular parasites expressing TgGT1_222060-smHA but no signals in parasites expressing TgGT1_222060-3HA. Nuclei are stained with DAPI (blue). Scale bars are 5 μm. E. Predicted Topology of the TgGT1_290870 protein from its predicted amino acid sequence using the TMHMM server 2.0 and Protter. Domains of the full patch family were colored in yellow and TM domains part of the SSD are gray while the TM domains part of RNA family are shown in pink. F. PCR analysis showing the correct insertion of the 3HA (3HA) (1.9 kbp) or smHA (smHA) (2.9 kbp) tags at the 3’ region of the TgGT1_29080 gene. The PCR primers used are shown in Table S1. G. Western blot analysis using α-HA antibodies detected a band of the expected size (~170 kDa) in lysates of the smHA-tagged tachyzoites but no band was observed in lysates of the 3HA-tagged cells. Tubulin was used as loading control. H. Immunofluorescence microscopy using α-HA antibodies (green) detected plasma membrane localization in both extracellular and intracellular parasites expressing TgGT1_290870-smHA. Very weak signals were observed in the 3HA-tagged strain. Nuclei were stained with DAPI (blue). Scale bars = 5 μm.

Figure 4

Spaghetti monster high affinity tag can be used for immunoprecipitation assays. A. Predicted topology of the TgGT1_247370 protein amino acid sequence using the TMHMM server 2.0 and Protter. The PKD_channel Polycystin cation domain is shown in orange. B. PCR analysis showing the correct insertion of the 3HA (3HA) (1.6 kbp) or smHA (smHA) (2.6 kbp) tags at the 3’ region of the TgGT1_247370 gene. The PCR primers used are shown in Table S1. C. Western blot analysis using α-HA antibodies detected a band of ~130 kDa in smHA-tagged tachyzoites (black arrowhead) and no band in 3HA-tagged cells. Tubulin was used as loading control. D. Western blot analysis of the immunoprecipitation using α-HA antibodies showed a band of the same size in the elution sample (E) as indicated by the black arrowhead. No band was observed in the flow-through (FT) and elution (E) of the 3HA-tagged cells. The parental line TatiΔku80 was used as a negative control and no band was detected. E. IFAs with α-HA antibodies (green) shows punctate staining around the ER that co-localizes with the reaction of α-SERCA (ER marker) (red) in extracellular parasites expressing TgGT1_247370-smHA. Parasite nuclei were labeled with DAPI (blue). Scale bars = 5 μm.

Figure 5

Spaghetti monster tag reveals a novel localization for TgPI-PLC. A. PCR analysis showing the correct insertion of the 3HA or smHA tags at the 3’ region of the TgPI-PLC gene. A DNA fragment of 1.5 kbp was amplified. B. Western blot analysis of lysates obtained from parasites expressing TgPI-PLC-smHA. Anti-HA antibodies detected a band of ~160 kDa (SM). The parental (P) line TatiΔku80 was used as negative control. The α-tubulin antibody was used as loading control. C. IFA with α-HA antibodies of extracellular and intracellular tachyzoites expressing TgPI-PLC-smHA showing peripheral localization and an additional dotted localization at the apical end (arrow). D. Super-resolution microscopy image reveal six dots at the apical end using α-HA antibodies (green) in smHA tagged parasites. Nuclei are labeled with DAPI (blue). E. IFA using α-Ty antibodies (green) to stain 3Ty tagged parasites. A weak signal at the apical end is observed (white arrow). DAPI (blue) labels the nuclei. F. Super resolution microscopy images of smHA-tagged parasites double labeled with α-HA antibodies (green) and α-GFP antibodies (red) showing co-localization with centrin2 (top) but not with cytoskeleton marker tubulin (red). Scale bars on C, D, E and F = 5 μm.

Figure 6

CRISPR-Cas9 dependent C-terminal endogenous tagging with the high affinity tag smMyc. A. PCR analysis showing the correct insertion of the smMyc (smMyc) tag at the 3’ region of the TgCnA gene. A DNA fragment of 770 bp was amplified from smMyc-tagged tachyzoites. B. Western blot analysis of TgCnA-smMyc expressing cell lines. Anti-Myc antibodies detected a band of ~110 kDa (smMyc). The parental (P) line TatiΔku80 was used as negative control. α-tubulin antibody was used as loading control. C. IFA using anti-Myc antibodies (green) to label the cytosol of smMyc-tagged extracellular and intracellular parasites. DAPI (blue) labels the nuclei. Scale bars = 5 μm.