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A family of transmembrane microneme proteins of Toxoplasma gondii contain EGF-like domains and function as escorters

Markus Meissner^1,*, Matthias Reiss^1,*, Nicola Viebig², Vern B. Carruthers³, Catherine Toursel⁴, Stanislas Tomavo⁴, James W. Ajioka⁵ and Dominique Soldati^1,

¹ Imperial College of Science, Technology and Medicine, Department of Biology, Sir Alexander Fleming Building, Imperial College Road, London, SW7 2AZ, UK
² ZMBH Im Neuheimer Feld 28, 69120 Heidelberg, Germany
³ W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA
⁴ UMR CNRS 8576 Université des Sciences et Technologies de Lille, France
⁵ Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK

View larger version (56K): [in a new window]   Fig. 1. Comparison of EGF-like proteins from apicomplexan parasites. (A) Apicomplexan proteins containing EGF-like domains. P. falciparum merozoite surface antigen PfMSP1, P. falciparum circumsporozoite protein PfCSP, Plasmodium ookinete surface antigens PfP21/P25, P. falciparum 125 kDa protein, Pf125 (PFC1045C, ACO97302), E. tenella microneme protein EtMIC4, and T. gondii microneme proteins TgMIC3, TgMIC6, TgMIC7, TgMIC8 and TgMIC9. (B) Amino acid sequences alignment of the cytoplasmic tails of transmembrane microneme proteins across the Apicomplexa. The amino acids colored in green correspond to the membrane-spanning domain and terminate with an almost strictly conserved tyrosine residue in blue. The conserved tryptophan residue is colored in red. (C) Alignment of the lectin-like domains present on TgMIC3 (TGO132530), TgMIC8 (AAK19757), NcMIC3 and E. tenella ESTs.

View larger version (37K): [in a new window]   Fig. 2. Nucleotide and predicted amino acid sequences of TgMIC6, TgMIC7, TgMIC8 and TgMIC9. The predicted signal peptides and transmembrane spanning domains are underlined. The accession numbers are: TgMIC6, AAD28185; TgMIC7, AF357911; TgMIC8, AAK19757; and TgMIC9, AAK19758.

View larger version (58K): [in a new window]   Fig. 3. Developmental regulation of the transcripts coding for EGF-like domains containing proteins. Semi-quantitative RT-PCR analysis of transcripts corresponding to TgMIC2, TgMIC4, TgMIC6, TgMIC7, TgMIC8 and TgMIC9 genes, performed with tenfold serial dilutions of in vitro tachyzoite and in vivo bradyzoite cDNAs. To ensure that equal amounts of cDNA from each parasitic stage were being compared, the housekeeping -tubulin primers were used as controls.

View larger version (47K): [in a new window]   Fig. 4. TgMIC6 and TgMIC8 are microneme proteins, processed at their C-terminus upon secretion. Western blot analysis of T. gondii tachyzoites probed with antisera raised against NterMIC6 or the CterMIC6 (A) and with antisera raised against NterMIC8 and CterMIC8 (B). Lysates were prepared from freshly lysed parasites from RH, mic6ko in RH or Prugniaud (Pru) strains and from vero cells. (C) Western blot analysis of lysates from parasite pellets and supernatants of secretion assays (ESA). TgMIC6 is processed from a 53 kDa precursor into a 45 kDa product in the parasites during its transport to the micronemes. In the ESA, the 35 kDa form is detectable with -N-terMIC6 but failed to be recognized by the -CterMIC6 antibodies. (D) A similar analysis was performed on TgMIC8 and showed that the 65 kDa secreted form of TgMIC8 is not detectable with -CterMIC8, establishing that the C-terminal domain has been removed. (E) Immunofluorescent labeling of permeabilized HFF cells infected with tachyzoites from RH strain. Double labeling with mouse -MIC3 and rabbit -MIC8 confirmed the microneme localization of TgMIC8. The antibodies were visualized with AlexaTM 488 goat -mouse IgG and AlexaTM 594 goat -rabbit IgG antibodies. Bar, 1 µm.

View larger version (61K): [in a new window]   Fig. 5. TgMIC6 is N-terminally processed in the late Golgi. (A,B,C). Double IFA analyzed by confocal microscopy on intracellular parasites expressing mycMIC6. (A) The precursor of mycMIC6 (in red) is detectable only in less than 30% of the vacuoles, whereas all parasites are positive for MIC6 (green). (B) Overlays using -MIC6 (green) and -MIC2 (red). (C) Overlays using -myc (red) and -MIC4 (green). The compartments of the secretory pathway are indicated with arrows. (D) Western blot analysis of lysates from stable transgenic mic6ko parasites transformed with pTMIC6GPI or pTMIC6EGF1-2GPI (MIC6 lacking the first two EGF-like domains and the TMCD domains replaced by a GPI anchoring signal from SAG1). MIC6GPI was targeted to the plasma membrane but nevertheless underwent N-terminal processing. By contrast, the deletion of EGF-1-2 removes the processing site, and no processed form was detectable. (E) Mass spectrometry fragmentation peaks of the MIC6 specific peptide corresponding to the N-terminal cleavage site of the protein.

View larger version (52K): [in a new window]   Fig. 6. Determination of type I membrane topology of TgMIC6, both during its transport and during its storage in the micronemes. (A) The three forms of TgMIC6. The 53, 45 and 35 kDa forms are present in the ER/Golgi, in micronemes and secreted, respectively. The two processing sites are indicated by arrows. (B) Western blot analysis of lysate from wild-type parasites (left panel) after transient permeabilization and proteinase K (0.1 mg/ml and 0.05 mg/ml) treatment either in the presence or absence of detergent (0.2% Triton X-100). The same analysis was repeated using mic1ko mutant parasites (right panel) in which TgMIC6 was retained in the early compartments of the secretory pathway and consequently was not processed at the N-terminus. The western blots were probed with -NterMIC6 antibodies. The same material was analyzed in the lower panels with the -CterMIC6 antibodies, establishing that the tail of TgMIC6 was degraded by proteinase K treatment both in RH and mic1ko strains. (C) A similar experiment was repeated on the recombinant cell line expressing MIC6Ty in mic1ko. In this experiment, TgM2AP, a 43 kDa microneme protein associated with the lumenal domain of TgMIC2, was included as control to demonstrate the integrity of the micronemes.

View larger version (70K): [in a new window]   Fig. 7. TgMIC7 and TgMIC9 are microneme proteins poorly expressed in tachyzoites. (A) Western blot analysis of lysates from wild-type RH or Prugniaud strain and transgenic RH parasites stably transformed with MIC7Ty or MIC9Ty under the control of the constitutive promoter TUB1. (B) Double IFA by confocal microscopy of transgenic parasites expressing MIC7Ty. MIC7Ty colocalized (-Ty, red) with the microneme marker TgMIC3 (green). (C) Double IFA of transgenic parasites expressing MIC9Ty. MIC9Ty (red) colocalized with TgMIC3 (green). (D) In addition to the microneme localization, MIC9Ty (green) partially localized to the rhoptries, as documented by double IFA with -ROP2 (red). Bar, 1 µm.

View larger version (85K): [in a new window]   Fig. 8. MIC8 serves as escorter for the non-membrane adhesin TgMIC3. (A) Double IFA analysis by confocal microscopy of parasites transiently transfected with pTMIC8GPI. TgMIC8 covalently linked to a GPI anchor localized perfectly at the plasma membrane of the parasites. TgMIC3 redistributed to the plasma membrane in the transiently transfected parasites, while the protein is perfectly sorted to the micronemes in a vacuole containing non-transfected parasites. (B) Three examples of vacuoles containing parasites transfected with 30 µg of pTMIC8GPI vectors and analyzed 48 hours post-electroporation. MIC8GPI is stained with the -Ty1 (red) and accumulates at the plasma membrane specifically in the zones of contacts between parasites. The -MIC6 is used as microneme marker (green). Bar, 1 µm.