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ATF6beta is a host cellular target of the Toxoplasma gondii virulence factor ROP18
YAMAMOTO, Masahiro, et al.
Abstract
The ROP18 kinase has been identified as a key virulence determinant conferring a high mortality phenotype characteristic of type I Toxoplasma gondii strains. This major effector molecule is secreted by the rhoptries into the host cells during invasion; however, the molecular mechanisms by which this kinase exerts its pathogenic action remain poorly understood. In this study, we show that ROP18 targets the host endoplasmic reticulum-bound transcription factor ATF6β. Disruption of the ROP18 gene severely impairs acute toxoplasmosis by the type I RH strain. Because another virulence factor ROP16 kinase modulates immune responses through its N-terminal portion, we focus on the role of the N terminus of ROP18 in the subversion of host cellular functions. The N-terminal extension of ROP18 contributes to ATF6β-dependent pathogenicity by interacting with ATF6β and destabilizing it. The kinase activity of ROP18 is essential for proteasome-dependent degradation of ATF6β and for parasite virulence. Consistent with a key role for ATF6β in resistance against this intracellular pathogen, ATF6β-deficient mice exhibit a high [...]
YAMAMOTO, Masahiro, et al . ATF6beta is a host cellular target of the Toxoplasma gondii virulence factor ROP18. Journal of Experimental Medicine , 2011, vol. 208, no. 7, p. 1533-46
DOI : 10.1084/jem.20101660
PMID : 21670204
The J our nal of Exper imental Medicine
SUPPLEMENTAL MATERIAL
Yamamoto et al., http://www.jem.org/cgi/content/full/jem.20101660/DC1
[ID]FIGS1[/ID][ID]FIGS2[/ID][ID]FIGS3[/ID][ID]FIGS4[/ID][ID]FIGS5[/ID][ID]FIGS6[/ID][ID]TBLS1[/ID]
Figure S1. Parasitological analysis of rop18-KO T. gondii . (A) Plaque assays. HFF monolayers were infected with WT or two independent rop18-KO parasites (#1 and #2) and then fi xed after 8–9 d and stained with Giemsa. (B) Intracellular growth assays. Replication was analyzed 24 h after infection.
Data are represented as means ± SEM for three independent experiments. P-values were calculated using a Student’s t test (equal variance, unpaired samples, two-tailed distribution). Generally, p-values were found to be not signifi cant (P > 0.1) when comparing equal numbers of parasites/vacuole be- tween the two strains. (A and B) Data are representative of three independent experiments.
Figure S2. Complementation of rop18-KO parasites by ROP18 mutants. (A and C) rop18-KO parental parasites were transfected with the indi- cated expression vectors. After pyrimethamine selection and cloning by limiting dilution, 10 7 parasites were lysed and subjected to Western blot. Expres- sion of each ROP18 mutant was also determined by Western blot on extracts of total cell lysates using anti-Flag antibodies. Expression levels of SAG1 were analyzed and are shown as loading controls (top). IB, immunoblot; WCL, whole cell lysate. (bottom) ROP18 mutants. The underlined regions corre-
Figure S3. Similar splenic cellularity in mice infected with WT or rop18-KO parasites. (A–C) Surface markers of splenocytes from mice unin- fected or infected with WT or rop18-KO parasites were analyzed by fl ow cytometry. Composition of B220/CD3 (A), CD8/CD4 (B), and Gr1/CD11b (C) popu- lations. Data are representative of two independent experiments.
Figure S4. Specifi c interaction of Nt-ROP18 with ATF6  in yeasts and similar infection levels between WT and rop18-KO parasites. (A) Plasmids expressing Nt-ROP18 or p53 fused to the GAL4 DNA-binding domain or an empty vector were cotransfected with a plasmid expressing ATF6  or SV40 large T antigen (SV40LT) fused to the GAL4 transactivation domain or an empty vector. Interactions were detected by the ability of cells to grow on medium lacking Ade, Trp, Leu, and His (-LWAH). Growth of cells on plates lacking Trp and Leu (-LW) was indicative of the effi ciency of the transfection. (B) 293T cells were transfected with 0.1 μg of plasmids to express HA-tagged ATF6  and T2A-CFP in tandem. 24 h after the transfection, cells were nonin- fected or infected with the indicated parasites expressing RFP at an MOI of 10. 24 h after infection, the infections were analyzed by FACS (x axis, CFP; y axis, RFP), and the regions “a” or “b” indicate CFP (HA-ATF6  )-expressing cells uninfected (top left) or infected with WT (top middle) or rop18-KO (top right) parasites. Histogram of cells uninfected (gray) or infected with WT or rop18-KO parasites to monitor the levels of RFP, indicating similar levels of infection by both parasites and achievement of almost 80% infection (top right). (A and B) Data are representative of two independent experiments.
Figure S5. ER localization of ROP18 and PVM–ER fusion. (A) Correct protein expression in cells transfected with vectors harboring T2A signal. Ly- sates of 293T cells transiently cotransfected with 2 μg of the indicated expression vectors were immunoprecipitated with antibodies and detected by Western blot. The data indicated independent and reliable expression of both HA_hATF6  _YFP and CFP_KDROP18_Flag proteins after cleavage between the two. IB, immunoblot; IP, immunoprecipitation. (B) 293T cells tandemly expressing HA_ATF6  _YFP and T2ACFP-tagged KDROP18_F were transfected with ER-localizing RFP expression vectors. Arrows indicate cells showing the colocalization of RFP and CFP. (C) 4 h after infection of parasites, HFFs were analyzed by electron microscopy. The bottom image includes indication of color lines morphologically determined by the top raw image. P, parasite. Bars:
(B) 30 μm; (C) 2 μm. (A–C) Data are representative of two independent experiments.
Figure S6. Generation of ATF6  -defi cient mice and comparable susceptibility in WT or ATF6  -defi cient mice infected with WT T. gondii . (A) The structure of the Atf6  gene, the targeting vector, and the predicted disrupted gene. The closed box denotes the coding exon. (B) Southern blot analysis of offspring from the heterozygote intercrosses. Genomic DNA was extracted from mouse tails, digested with BamHI, electrophoresed, and hy- bridized with the radiolabeled probe indicated in A. Southern blotting resulted in a single 8.0-kb band for WT (+/+), a 4.0-kb band for homozygotes ( ⫺ / ⫺ ), and both bands for heterozygous mice (+/ ⫺ ). (C) Northern blot analysis of splenocytes. 10 μg total RNA extracted from cells was electrophoresed, transferred to a nylon membrane, and hybridized using cDNA fragments for Atf6  and  -actin. (D) WT or ATF6  -defi cient ( n = 5) mice were infected with 10 3 WT parasites, and the survival rates were monitored for 10 d. (E) ATF6  -defi cient (each n = 5 for each group) mice were infected with 10 3 of the indi- cated rop18-KO complemented parasite lines, and the survival rates were monitored for 10 d. (F) WT or ATF6  -defi cient ( n = 5) mice were infected with 10 3 type III CTG parasites, and the survival rates were monitored for 20 d. (B–F) Data are representative of two (B and C) or the cumulative percentages of two (D–F) independent experiments.
Table S1. Primers used in this study
Primer name Enzyme Sequence Resulting plasmid
OVA_F EcoRI 5 ⬘ -GAATTCCCTTTTTCGACAAAATGGAAGACGCCAAAAACATAAAG-3 ⬘ pOVRFPluc
OVA_R XhoI 5 ⬘ -CTCGAGCACGGCGATCTTTCCGCCCTTCTT-3 ⬘ pOVRFPluc
RFP_F XhoI 5 ⬘ -CTCGAGGCCTCCTCCGAGGACGTCAT-3 ⬘ pOVRFPluc
RFP_R PacI 5 ⬘ -TTAATTAACTACAGGAACAGGTGGTGGC-3 ⬘ pOVRFPluc
R18KOLA_F NotI 5 ⬘ -GCGGCCGCGATCCTGCGTCTGTTGAGTTC-3 ⬘ pKO-ROP18 R18KOLA_R BamHI 5 ⬘ -GGATCCTCGGACGACGGTACGCGTAAGA-3 ⬘ pKO-ROP18 R18KOSA_F HindIII 5 ⬘ -AAGCTTACTCAAAATGAAAAGGGGAACGTGGC-3 ⬘ pKO-ROP18 R18KOSA_R HindIII 5 ⬘ -AAGCTTGCCGGGGACTTAGTTCAAGAGGGT-3 ⬘ pKO-ROP18
R18frg2common_F EcoRI 5 ⬘ -CGACTGGATAGAATTCTGACTGTCG-3 ⬘ ⌬ 27-ROP18_F, ⌬ 27 ⌬ N2-ROP18_F, ⌬ 240- ROP18_F
R18frg2common_R NotI 5 ⬘ -GCGGCCGCTACTTGTCATCGTCGTCCTTGTAGTCTTCTGTGTGGAGATG-
3 ⬘ ⌬ 27-ROP18_F, ⌬ 27 ⌬ N2-ROP18_F, ⌬ 240- ROP18_F
R18D27frg1_F BamHI 5 ⬘ -GGATCCACCACCATGCAGGATGGGACCGGA-3 ⬘ ⌬ 27-ROP18_F R18D240frg1_F BamHI 5 ⬘ -GGATCCACCACCATGACGCTGGTGAGAGGT-3 ⬘ ⌬ 240-ROP18_F
R18frg1_R EcoRI 5 ⬘ -CGACAGTCAGAATTCTATCCAGTCG-3 ⬘ ⌬ 27-ROP18_F, ⌬ 27 ⌬ N2-ROP18_F, ⌬ 240- ROP18_F
ParasiteR18frg1_F BamHI 5 ⬘ -GGATCCACCACCATGTTTTCGGTACAGCGGCCACCTCTT ACGCGTACCGTCGTCCGAATGGGTTTAGCGACTCTTCTCCC-3 ⬘
ROP18_F, KD-ROP18_F, ⌬ N2-ROP18_F ATF6  _common_F EcoRI 5 ⬘ -GAATTCATGGCGGAGCTGATGCTGCTCAGCGA-3 ⬘ HA-ATF6  (Full), HA-ATF6  ( ⌬ C), HA-
ATF6  ( ⌬ C ⌬ TM)
ATF6  _full_R NotI 5 ⬘ -GCGGCCGCTCAGGGATGATTGAGGTAGAGGGG-3 ⬘ HA-ATF6  (Full) ATF6  _DC_R NotI 5 ⬘ -GCGGCCGCTATAACTTGAGCTCGCTGTTTTCAGCC-3 ⬘ HA-ATF6  ( ⌬ C) ATF6  _DCDTM_R NotI 5 ⬘ -GCGGCCGCTAAACTCCCTGAACTGGCTCTTGCTCT-3 ⬘ HA-ATF6  ( ⌬ C ⌬ TM) ATF6  _full_XhoI_R XhoI 5 ⬘ -CTCGAGGGGATGATTGAGGTAGAGGGGCTGG-3 ⬘ HA_hATF6  _T2ACFP, HA_hATF6  _YFP-
T2ACFP_KDROP18_Flag T2ACFP_F XhoI, SacI 5 ⬘ -CTCGAGCTCGGTGAGGGCAGAGGAAGTCTGCTAACATGCGGTG
ACGTCGAGGAGAATCCTGGCCCAATGGTGAGCAAGGGCGAGGAGC-3 ⬘
HA_hATF6  _T2ACFP, HA_hATF6  _YFP- T2ACFP_KDROP18_Flag T2ACFP_R NotI 5 ⬘ -GCGGCCGCTATGCTGAAACGAATTCGGA
TCCCAGCTCGTCCATGCCGAGAGTGATCCC-3 ⬘
HA_hATF6  _T2ACFP, HA_hATF6  _YFP- T2ACFP_KDROP18_Flag YFP_F SalI 5 ⬘ -GTCGACTCTAGAACCACCATGGTGAGCAAGGGCGAGGAGC-3 ⬘ HA_hATF6  _YFP-T2ACFP_KDROP18_Flag,
HA_hATF6  _YFP
YFP_R SacI, EcoRI 5 ⬘ -GAATTCTAGAGCTCGTCCATGCCGAGAGT-3 ⬘ HA_hATF6  _YFP-T2ACFP_KDROP18_Flag, HA_hATF6  _YFP
ATF6  KO_LA_F NotI 5 ⬘ -GCGGCCGCACCTGCTCAGGTCTGAACTACACTTG-3 ⬘ pKOATF6 
ATF6  KO_LA_R SalI 5 ⬘ -GTCGACTCTGTCTTCACTTCCAGAACCTCC-3 ⬘ pKOATF6 
ATF6  KO_SA_F BamHI 5 ⬘ -GGATCCATGGAGCTGAGCCTCACAGATGAGCTG-3 ⬘ pKOATF6  ATF6  KO_SA_R ClaI 5 ⬘ -ATCGATTCAGGTTTCTGAAGTGAAGCAGAGGAG-3 ⬘ pKOATF6  SB_F 5 ⬘ -AATGAGTGTCTGCGGAGTCT-3 ⬘ Used in Southern blot analysis in Fig. 1 B SB_R 5 ⬘ -CCACTGTTGGTCGGTTATAC-3 ⬘ Used in Southern blot analysis in Fig. 1 B NB_F 5 ⬘ -GTGACTACCGGTGAAACCCGG-3 ⬘ Used in Northern blot analysis in Fig. 1 C NB_R 5 ⬘ -AAAATAGTTTGGTACCCACCG-3 ⬘ Used in Northern blot analysis in Fig. 1 C R18ex01 5 ⬘ -AGCTACACGCTGGTTCGCTTGAGATCT-3 ⬘ Used for screening of rop18-KO parasites DHFRrc01 5 ⬘ -CGGGTTTGAATGCAAGGTTTCGTGCTG-3 ⬘ Used for screening of rop18-KO parasites
⬘ ⬘
Table S1. Primers used in this study (Continued)
Primer name Enzyme Sequence Resulting plasmid
ROP18Y2H_R SalI 5 ⬘ -GTCGACCCGGGTTTCACCGGTAGTCA-3 ⬘ Nt-ROP18
mIrgb6_F EcoRI 5 ⬘ -GAATTCATGGCTTGGGCCTCCAGCTTTGATG-3 ⬘ Myc-Irgb6-YFP mIrgb6_R XhoI 5 ⬘ -CTCGAGAGCTTCCCAGTACTCGGGGGGCTCAGA-3 ⬘ Myc-Irgb6-YFP Empty cells indicate that the information is not applicable.
