The Hsp90 co-chaperone p23 of Toxoplasma gondii: Identification, functional analysis and dynamic interactome determination

Abstract

Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. Recently a link between the T. gondii Hsp90 chaperone machinery and parasite development was observed. Here, the T. gondii Hsp90 co-chaperones p23 and Hip were identified mining the Toxoplasma- database (www.toxodb.org). Their identity was confirmed by domain structure and blast analysis. Additionally, analysis of the secondary structure and studies on the chaperone function of the purified protein verified the p23 identity. Studies of co-immunoprecipitation (co-IP) identified two different types of complexes, one comprising at least Hip-Hsp70-Hsp90 and another containing at least p23-Hsp90. Indirect immunofluorescence assays showed that Hip is localized in the cytoplasm in tachyzoites and as well in bradyzoites. For p23 in contrast, a solely cytoplasmic localization was only observed in the tachyzoite stage whereas nuclear and cytosolic distribution and co-localization with Hsp90 was observed in bradyzoites. These results indicate that the T. gondii Hsp90-heterocomplex cycle is similar to the one proposed for higher eukaryotes, further highlighting the implication of the Hsp90/p23 in parasite development. Furthermore, co-IP experiments of tachyzoite/bradyzoite lysates with anti-p23 antiserum and identification of the complexed proteins together with the use of the curated interaction data available from different source (orthologs and Plasmodium databases) allowed us to construct an interaction network (interactome) covering the dynamics of the Hsp90 chaperone machinery.

Figure 1. Characterization of T. gondii Hip co-chaperone

A. Sequence alignment generated by clustal W (Bioedit program). Letters in black indicate identical residues; letters in grey indicate conserved residues. Gaps are indicated by dashes and were introduced to improve the alignment. TgHip, T. gondii Hip (Accession number [AN]: DQ680015), PbHip, P. berghei Hip (AN: Q08168), RnHip, R. norvegicus Hip (AN: P50503). TgHip and RnHip sequences showed 36% of identity and 55% of positives, whereas with PbHip identity was of 45% and positives were 58% (Blast2 program at www.ncbi.nlm.nih.gov/Blast). B. Schematic representation of T. gondii and R norvegicus Hip proteins with the putative protein motifs. Numbers denote the amino acid positions of motifs of the protein. C. Sequence alignment of the T. gondii and R. novergicus´s tetratricopeptide units from the TPR domains of Hip proteins. Consensus primary structure of the TPR domains: “b”: hydrophobic aminoacids, “l”: hydrophilic aminoacids. Above, the schematic representation of α helices determined by prediction of secondary structure is indicated. Conserved amino acids are marked in black. Stars indicate the most important positions in this structure (residues 4, 8, 11, 20, 24 and 27). The amino acid in position 32 is normally a hydrophobic residue (e.g. proline).

Figure 2. Characterization of T. gondii p23

A. Sequence alignment was generated by clustal W (Bioedit program). Letters in black indicate identical residues; letters in grey indicate conserved residues. Gaps are indicated by dashes and were introduced to improve the alignment. Tgp23, T. gondii p23 (Accesion number [AN]: DQ680014), Pbp23, P. berghei p23 (AN: XP_680236), Hup23, human p23 (AN: L24804). TgHip and Pbp23 sequences showed 40% of identity and 57% of positives, whereas with Hup23 it was of 28% of identity and 45% of positives (Blast2 program at www.ncbi.nlm.nih.gov/Blast). Domains, β-sheet regions and conserved residues are indicated in figure. β-sheet was deduced by using the http://protevo.eb.tuebingen.mpg.de/toolkit. BT. gondii p23 shows the characteristic, (β-sheet rich secondary structure. The far-UV-CD spectrum of the native T. gondii p23 was recorded at a protein concentration of 0.3 mg/ml in 50 mM Tris/HCl, pH 8.0 at 20°C. Inset: Calculated percentage of secondary structure elements.

Figure 3. Determination of T. gondii p23 chaperone activity

A: Influence of p23 on the thermal aggregation of CS. CS (final concentration: 1.0 µM) was diluted into a thermostated solution (43°C) containing 1.0 µM (□), 2.0 µM (◊), 4.0 µM (∇) or 8.0 µM (Δ) p23. Circles (◦) represent the spontaneous aggregation of CS at 43°C. The kinetics of aggregation was determined by measuring the light scattering of the samples at 400 nm. B: Influence of p23on the thermal inactivation of CS. Inactivation of CS (0.15 µM) at 43°C in the absence (◦) and in the presence of increasing concentrations of p23; 0.075 µM (+), 0.15 µM (□), 0.3 µM (◊) or 0.6 µM (∇).

Figure 4. Detection of native Hip and p23 proteins

Western-blot with murine anti-T. gondii Hip (α-TgHip), murine anti-T. gondii p23 (α-Tgp23) and murine anti-Histidines (α-His) antibodies. Tg: T. gondii PK strain lysate. Protein extract from uninfected HFF cells were also assayed. Preimmune serum samples did not show reactivity (data not shown). Migration of size marker is indicated (in kilo Daltons [kDa]).

Figure 5. Co-IP analysis of Hsp90, Hip and p23 proteins

Toxoplasma gondii PK strain lysates (Tg) were used for immunoprecipitation (IP) using either preimmune (Pre) or specific αHsp90, αHip and αp23 antisera (vertical columns). Pulled down materials were analyzed by western blot with specific sera antibodies and a commercial anti-Hsp70 antibody (horizontal columns).

Figure 6. Indirect immune-fluorescence assay (IFA) and subcellular localization of Hip and p23 in intracellular PK tachyzoites (Tz) and bradyzoites (Bz)

PK Tz were grown in vitro in human foreskin fibroblasts (HFF) and induced to Bz.[2]. To induce Bz formation in PK strain, parasites were grown 4 days at pH 8.1 and low CO₂). IFA was performed on fixed intracellular parasites with antibodies against T. gondii Hip (murine serum), -p23 (murine serum) and -Hsp90 (rabbit serum) antibodies [2]. Propidium iodide (PI) staining reveals the location of the nucleus (red color). Mouse anti-P30 (α-SAG1) or anti-P34 serum (α-p34, mAb T82C2)[59] were used as marker of Tz and Bz respectively (data not shown). Rabbit anti-BAG1 polyclonal serum was used as cytoplasmic bradyzoite marker. The efficiency of in vitro conversion to Bz was 82–88%. White arrows heads are signaling the localization of the nucleus in both panels.

Figure 7. Differential interactome of Hsp90/p23 complex in tachyzoites and bradyzoites

Interactors are visualized in a scheme of T. gondii tachyzoite and bradyzoite. Combined interactome of Hsp90 and p23 proteins was built using the Cytoscape platform based on the data obtained from proteomic analysis and verified by: co-IP assays from the present study (grey interactors), mining Hsp90 and p23 interactomes present in the public PPI database POINT (http://point.bioinformatics.tw) and the specific database for P. falciparum (http://apidb.org/apidb/showQuestion.do?questionFullName=GeneQuestions) (rectangle and dashed contour, respectively). Cellular localization is inferred from Gene Ontology data. ACT, actin; ADT,ADP/ADT carrier; BCKDH, 2-oxoisovalerate dehydorgenase; CLH, clatrin heavy chain; CRMP, Cysteine repeat modular protein; EF1-A, elongation factor 1 alpha; EF-2, elongation factor 2; ENO2, enolase 2; FBP/aldo, Fructose 1,6 biphosphatase aldolase; GAPDH, glyceraldehyde-3-phosphate dehydorgenase; LDH, lactate dehydrogense 1, MyoA, myosin A; NTP1, nucleoside triphosphatase I; PDIA, protein disulfide isomerase; PKM, pyruvate kinase I; Trp and Asn-tRNAs, tryptophanyl and asparaginyl tRNA synthase.