Journal: Molecular Biology of the Cell

Article Title: An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasite Toxoplasma gondii

doi: 10.1091/mbc.E15-11-0754

Figure Lengend Snippet: TLAP2 is conserved between T. gondii, Plasmodium spp., and C. velia and is dispensable for parasite growth. (A) Multiple sequence alignments of the C-terminal domains of TLAP2 homologues from T. gondii (TGGT1_232130), P. falciparum (-a, PF3D7_1034300 and -b, PF3D7_0517200), and C. velia (Cvel_2610). Tryptophan residues are highlighted in green. The consensus of the conserved residues is shown in the bottom row in red. (B) Deconvolved wide-field images of T. gondii transiently expressing mEmeraldFP-PfTLAP2-a from a T. gondii tubulin promoter. (C) Scheme for generating Δtlap2 parasites and Southern blotting strategy. RH Δhx parasites (parental; top) were used to generate mEmeraldFP-TLAP2 knock-in parasites (knock-in; middle) via double-crossover homologous recombination. The knock-in parasites were then transiently transfected with a plasmid expressing Cre recombinase to excise the genomic fragment between the two LoxP sites. mEmeraldFP(-) parasites were sorted by FACS to facilitate the cloning of Δtlap2 parasites (knockout; bottom). The positions of restriction sites and probe (red bar) used in Southern blotting (D) and the corresponding DNA fragment sizes are indicated. (D) Southern blotting analyses of the tlap2 locus in parental RH Δhx (P), mEmeraldFP-TLAP2 knock-in (KI), and Δtlap2 (KO) parasites generated as described in A. Genomic DNA of the parasites was digested with either Mfe I (left) or Kpn I- Sca I (right). A probe (red bar in C) hybridized to the upstream region of tlap2 gene was used for the Southern blotting. The predicted Mfe I- Mfe I fragment size recognized by the probe is 5575 base pairs for RH Δhx , 7511 base pairs for mEmeraldFP-TLAP2 knock-in, and 2967 base pairs for Δtlap2 parasites. The predicted Kpn I- Sca I fragment size recognized by the probe is 5850 base pairs for RH Δhx , 7786 base pairs for mEmeraldFP-TLAP2 knock-in, and 3242 base pairs for Δtlap2 parasites. (E) Analyses of the virulence of Δtlap2 parasites in mice. Each survival curve represents data from a group of four mice infected with an equal number of RH Δhx (parental) , mEmeraldFP-TLAP2 knock-in (mE-TLAP2 KI), or Δtlap2 parasites. Mice infected with the parental RH Δhx parasites died between days 7 and 8 postinfection, whereas mice infected with mEmeraldFP-TLAP2 knock-in or Δtlap2 parasites died between days 8 and 9 postinfection. (F) Projections of deconvolved wide-field images showing proper targeting of several coating proteins in Δtlap2 and Δspm1 parasites. Top, Δtlap2 parasites transiently expressing fluorescently tagged SPM1 or TLAP3. Bottom, Δspm1 parasites transiently expressing fluorescently tagged TLAP2 or TLAP3. The fluorescently tagged proteins were driven by the 2-kb genomic region immediately upstream of the respective genes in the pTKO2_II vector backbone. Scale bars, 2 μm. (G) Plaque assay of parental RH Δhx , Δtlap2 , Δtlap2Δspm1 , and Δtlap2Δspm1Δtlap3 parasites. HFF cultures were infected with an equal number of each line of parasites, grown for 7 d at 37°C, and then fixed and stained with crystal violet. Host cells remaining attached absorbed the crystal violet staining, whereas regions of host cells lysed by the parasites (“plaques”; arrows) were clear.

Article Snippet: Subsequently a DNA fragment containing the coding sequences for mEmeraldFP, SGLGS linker, and the N-terminus region of SPM1 was synthesized (GenScript; Supplemental Table S1), digested with Bgl II and Xma I, and subcloned into the Bgl II- Xma I sites on pTKO2-II-SPM1 to produce pTKO2-II-mEmeraldFP-SPM1 in which the coding sequence for mEmeraldFP was placed N-terminal to the SPM1 CDS. pmin-Cre-eGFP_Gra-mCherry: A Cre-eGFP expression cassette including a dhfr promoter and the dhfr 3′ untranslated region was released from the plasmid pmin-Cre-eGFP ( Heaslip et al. , 2010 ) by Apa I- Not I digestion and subcloned into the Apa I- Not I site on pTKO2-II-mCherryFP to replace the DHFR expression cassette. pmin-mEmeraldFP-TgCentrin1: The CDS of TgCentrin1 (TGGT1_247230; EuPathDB.org) was amplified by PCR using a cDNA library isolated from RH parasites as the template.

Techniques: Sequencing, Expressing, Southern Blot, Knock-In, Homologous Recombination, Transfection, Plasmid Preparation, Cloning, Knock-Out, Generated, Infection, Plaque Assay, Staining