Discovery of parasite virulence genes reveals a unique regulator of chromosome condensation 1 ortholog critical for efficient nuclear trafficking

Frankel et al. 10.1073/pnas.0701893104.

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SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10
SI Materials and Method

SI Figure 6

Fig. 6. Schematic of T. gondii STM screen. Pools of 60 uniquely tagged insertional mutants were combined and used to infect either fibroblasts in tissue culture or two mice at an inoculum of 2 ´ 10⁴. Parasites grown in tissue culture were serially passaged onto a fresh monolayer after lysis (typically 3 days). At 22 days, DNA was extracted from parasites in both conditions and the tag radiolabeled by PCR to probe identical filters containing all 60 tags. Shown is the filter from plate 73 from which the 73F9 mutant originated (circled in red). Clones with a decreased hybridization signal in mice were examined in a chronic infection model. Total cysts per brain were compared to wild-type Pru (WT). Clones that were »10-fold reduced in brain cysts are listed in Table 1. The number of cysts per brain for the 73F9 mutant is shown compared with the parental F9 strain (three separate experiments of four mice each; results were statistically significant using an unpaired Student's t test with a value of P < 0.0001). Examination of acute virulence in four mice each at 5 ´10⁵ and 1 ´ 10⁶ showed that the parental F9 strain was lethal, whereas 73F9 was not (this pilot experiment was performed once).

SI Figure 7

Fig. 7. TgRCC1 transcript is alternatively spliced. Primers P1 and P2 were designed from the TwinScan1173 gene prediction on ToxoDB and used in a PCR to amplify the ORF from cDNA. Analysis of the PCR revealed multiple products, which were cloned into the pCR2.1-TOPO cloning vector for sequence analysis. M13F and M13R primers were used for initial sequencing of the products followed by gene-specific primers. Four unique products were identified. A-D illustrate the differences in splicing. A represents the complete ORF containing eight exons. B-D are smaller splice forms of the TgRCC1 transcript that are missing the RCC1 domains.

SI Figure 8

Fig. 8. TgRCC1 has similarity to human RCC1. Amino acids 385-794 of TgRCC1 were aligned with residues 24-373 of the human RCC1 by using ClustalW and the Swissport database [Schwede T, Kopp J, Guex N, Peitsch MC (2003) Nucleic Acids Res 31:3381-3385; Guex N, Peitsch MC (1997) Electrophoresis 18:2714-2723]. These alignment models predict 27% sequence identity with an Evalue of 2.71e⁻²⁷. Identical residues are highlighted in red and shown on the known crystal structure of human RCC1. Similar residues are shaded in blue. Shown by arrows are regions of predicted TgRCC1 and known human RCC1 (hsRCC1) b-sheet folding. The sequence conservation of TgRCC1 and its predicted protein folding suggest that the structure of TgRCC1 is similar to other RCC1 proteins.

SI Figure 9

Fig. 9. Localization of TgRCC1. 73F9 parasites were transfected with either TgRCC1 containing an N-terminal FLAG-tag expressed from the a-tubulin promoter or a C- terminal HA-tag expressed from its native promoter. Stable transformants were selected and allowed to infect a monolayer of HFF cells for 24 h. Coverslips were mounted on slides with VectorShield containing DAPI to stain the nucleus. From left to right, differential interference contrast (DIC)/phase image of parasites, DAPI staining in red, TgRCC1 staining in green (labeled as FLAG or HA depending on construct), and the merge of the two fluorescent images. Because of low expression levels from the native promoter, visualization was difficult and we have shown what appears to be two dividing parasites, which yields a stronger fluorescent signal.

SI Figure 10

Fig. 10. 73F9 parasites are lethal to mice at high doses. A total of 8 ´ 10⁵ tachyzoites of the F9 parental, mutant 73F9, and complemented strains C1H and C2 were i.p. injected into mice. Morbidity and mortality were monitored for 23 days. Four mice were injected in each experiment, and the experiment was repeated twice for a total of eight mice per strain. At this high dose, 73F9 parasites are lethal to mice (63% survival) but still defective compared with the parental and complemented strains. C1H and C2 represent two independent clones from separate electroporations and are unique as confirmed by Southern blot (data not shown).

SI Materials and Methods

Cell Culture and Creation of the STM Library. All parasites were maintained in monolayers of HFF cells in standard T. gondii culture conditions (1). The Prugniaud strain of T. gondii deleted in hypoxanthine-xanthine-guanosine phosphoribosyl transferase (DHPT) and containing a signature-tag (2) was used for insertional mutagenesis. The library creation was described in ref. 3. After selection, parasites were cloned by limiting dilution and arrayed into 96-well plates of HFFs, not using the outer wells.

Identification of DNA Flanking the Insertion Site. Genomic DNA of the mutant strains was digested with a restriction enzyme (primarily HindIII, NcoI, or SacI) that cuts once within the insertion plasmid. Digested DNA was ligated by using T4 DNA Ligase (NEB), ethanol precipitated, and electroporated into GC10 Thunderbolt Escherichia coli (Gene Choice, Frederick, MD) according to the manufacturer's instructions. Plasmid DNA was sequenced by using a primer specific to the insertional plasmid. Sequenced DNA was then compared with the genome database at www.toxodb.org by using the BLASTN and BLASTX search engines. Gene predictions and ESTs were used to predict the disrupted gene.

RNA Isolation, cDNA Synthesis, 5′-RACE, and Northern Blot Analyses. Total RNA was isolated by using ULTRASPEC (BIOTECX) according to the manufacturer's instructions. Parasites were force-lysed from the host cell by syringing through a 27-gauge needle twice. For cDNA synthesis, RNA was treated with amplification grade DNase (Invitrogen). DNase-treated RNA was then reverse transcribed by using either Thermoscript or Superscript III RT kits (Invitrogen) according to the manufacturer's instructions. 5′-RACE was performed to identify the 5′ end of the 73F9 transcript by using the First Choice RLM-RACE kit (Ambion). Nested primers used were 5′-AAGTCTGTCGCTTCTCCAGGAC-3′ and 5′-ATGGAAGGCGGAAAAACGGAGC-3′. For Northern blot analysis, RNA was run on a 0.8% formaldehyde gel. The gel was transferred to a Zeta-probe blotting membrane (Bio-Rad) followed by prehybridization (50% formamide, 0.12 M Na₂HPO₄, 0.25 M NaCl, 7% wt/vol SDS, 1 mM EDTA) for 4-6 h. Radiolabeled DNA was hybridized overnight at 42°C. The blot was then washed in 3´ SSC, 0.1% SDS; 2´ SSC, 0.1% SDS; 1´ SSC, 0.1% SDS; 0.1´ SSC, 0.1% SDS; all washes were performed at 42°C for 30 min each.

Polyclonal Antibody Production. Codons 992-1,101 of TgRCC1 were PCR amplified by using primers 5′-GAATTCTCGCGCGCGAAAGCCGCTC-3′ and 5′-AAGCTTTTTCTTCCCCGTCGCCTTC-3′. The underlined regions denote an EcoRI and HindIII site, respectively, for subsequent subcloning into the pET28a (Novagen) vector at the same restriction sites. This construct produced a »17-kDa protein with both N- and C-terminal 6x-HIS tags. The protein was induced with 1 mM IPTG for 4 h and the soluble fraction was purified by using the His•Bind Quick 900 Cartridges (Novagen) according to the manufacturer's protocol. The purified protein was then used to immunize two rats according to protocol by Harlan Bioproducts (Madison, WI).

Western Blot Analysis. Lysates from 5 ´ 10⁶ parasites each was loaded per well and separated in an 8% SDS/PAGE gel and transferred to Immobilon-P membrane (Millipore). The membranes were blocked in 5% nonfat dry milk in PBS/1% Tween 20, incubated in the anti-TgRCC1 antisera at a 1:1,000 dilution, and then goat anti-rat secondary antibody conjugated to horseradish peroxidase at a 1:5,000 dilution (Jackson ImmunoResearch). Protein was visualized by using ECL Plus Western Blotting Detection Reagents (Amersham Pharmacia Biosciences). The blot was stripped by placing it in 25 mM glycine•HCl, pH 2, with 1% SDS for 30 min, and then reprobed with rabbit b-tubulin antiserum (4) diluted 1:2,000 and antirabbit conjugated to horseradish peroxidase diluted 1:10,000.

Mapping of TgRCC1. We used TwinScan1173 as a basis for amplifying the predicted disrupted gene. Primers 5′-CGGCTGTCTCTGCGTTTCTTTCTA-3′ and 5′-CGGCGTCGTTGCTTTTCTTCCTGT-3′ with a nested reaction by using primers 5′-GATGCCCTATTTGCCTCGTCGTCA-3′ and 5′-CCGCCTTCTTCTCTGCTTTGCTCT-3′ were used to amplify the predicted ORF from cDNA and the product was cloned into the pCR2.1-TOPO cloning vector. All PCRs were performed with Accuprime Pfx DNA polymerase (Invitrogen) according to manufacturer's instructions. Primers were then designed from predicted exons and used to sequence the PCR product to determine the intro/exon junctions. The 3′ transcript end was determined by using the EST library available at ToxoDB in which an EST (TgESTzyg79c08.y1) contained a poly-adenosine tail.

TgRCC1 Complementation Constructs. Plasmid description. pMF106 a-tubulin promoter, TgRCC1 cDNA ORF; pMF107, pMF106 with C-terminal HA-tag; pMF108, pMF107 with N-terminal FLAG-tag; pMF110 native promoter, TgRCC1 cDNA ORF.

For plasmid pMF106, the TgRCC1 ORF was PCR amplified from cDNA by using primers 5′-ATGCATGCAAACGCATCCGTCGAGGCA-3′ and 5′-CCTTAATTAAATTCGACTGTTTGGGCGCCGCTTGGC-3′. The underlined regions denote NsiI and PacI sites, respectively. The PCR product and vector pT230-TUB/5CAT (5) were partially digested with NsiI and completely with PacI. The TgRCC1 ORF replaced the CAT gene. DHFR was digested from pDHFR-TSc3 (6) with SpeI and HindIII, blunted with Klenow DNA polymerase, and then subcloned into the blunted NotI site of tub-TgRCC1. For plasmid pMF107, the C-terminal end of the ORF was amplified by using primers 5′-GTCCGCAACCTCTCCATCAG-3′ and 5′-CCTTAATTAAATCGCGTAGTCTGGGACGTCGTATGGGTATCGACTGTTTGGGCGCCGCTTGGC-3′. The underlined portion indicates the PacI site at the stop codon, and the bold represents the HA epitope tag. The product was digested with NcoI and PacI and subcloned into pMF107 cut with the same restriction enzymes. For plasmid pMF108, primers 5′-CCATGCATGACTACAAGGACGACGACGACAAGGACGCAAACGCATCCGTCGAGGA-3′ and 5′-GTCGGTCGCAGCTAGGCAGTGGTC-3′ were used to amplify the N-terminal region of TgRCC1. Underlined region denotes an NsiI site and bold represents the FLAG epitope tag. This product was partially digested with NsiI and completely with NcoI and cloned into pMF107 digested identically to create pMF108. For plasmid pMF110, the a-tubulin promoter of pMF107 was replaced with the native promoter from a PCR product by using primers 5′-GGTACCACTGCGGGGAGAGGCGGTAGGAGA-3′ and 5′-TGCCGGCGACCAGACCTACG-3′ (underlined region denotes a KpnI site) at the KpnI and EcoRI sites. To replace the a-tubulin 5′-UTR, primers 5′-GTGGATCCGAGAAGAAGCGTTGGGGAATGGTC-3′ and 5′-CGTCTGTGTCTGGTCGCTGTCTCT-3′ amplified the 73F9 5′-UTR and coding sequence from cDNA and cloned into the EcoRI site of pMF107. Between 10 and 50 mg of each plasmid was linearized before electroporation into the 73F9 strain. Stable clones were selected with 1 mM pyrimethamine.

Immunofluoresence analysis. Parasites containing the TgRCC1 ORF with either a C-terminal HA tag or an N-terminal FLAG tag were infected onto a monolayer of HFFs on glass coverslips and grown for 24 h. The samples were then fixed, blocked, and permeabilized as described in the main text. For localization using the HA epitope containing parasites, the rabbit anti-HA polyclonal antibody (Zymed Laboratories) was used at a 1:300 dilution and incubated for 1 h at room temperature. For the parasites expressing the FLAG tag epitope, a 1:1,000 dilution of the rabbit anti-FLAG polyclonal antibody (Sigma) was used. An Alexa Fluor 488 goat anti-rabbit secondary antibody (Molecular Probes) was used at either a 1:500 (HA tag) or 1:1,000 (FLAG) dilution. Samples were mounted by using VectaShield mounting media with DAPI (Vector Laboratories). Serial image stacks (0.2-mm Z increment) were collected at ´100 (PlanApo oil immersion, 1.4 na) on a motorized Zeiss Axioplan IIi equipped with a rear-mounted excitation filter wheel, a triple pass (DAPI/FITC/Texas Red) emission cube, differential interference contrast optics, and a Hamamatsu ORCA-AG CCD camera. Fluorescence images were captured by using Openlabs 4.0 software and deconvolved by a constrained iterative algorithm, pseudocolored, and merged by using Volocity 4.0.1 software (Improvision, Lexington, MA).

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