JCS022350 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Fig. S1. MIC8 is not involved in replication, egress and microneme secretion. (A) Replication assay of parasites grown in the presence and absence of ATc. Parasites grown in the presence or absence of ATc were inoculated on HFF cells and allowed to grow for 24 hours. After fixation, the number of parasites inside the parasitophorous vacuole was determined in immunofluorescence assays. The depicted graph is representative for one out of three independent experiments. (B) Induced egress is not altered in the absence of MIC8. Parasites were grown in the presence or absence of ATc, before egress was stimulated with the Ca-Ionophore A23817. After 5 minutes, parasites and host cells were fixed and the number of lysed parasitophorous vacuoles vs intact parasitophorous vacuoles was determined. Mean values of three independent experiments (± s.d.) are shown. (C) Secretion of micronemal proteins is not affected by the depletion of MIC8, as shown for secretion of MIC2. Both stimulated (+EtOH) and constitutive (−EtOH) secretion appears to be not affected in the absence of MIC8 expression. SN, supernatant; P, pellet.

• Supplemental Table S2 -

  Fig. S2. Identification of two MIC8 homologues in the genome of T. gondii. (A) Alignment of MIC8 with MIC8-like 1 (76.m01679) and MIC8-like 2 (49.m03396). The overall domain organisation is identical, consisting of one Lectin-like (grey bar) and ten EGF-like (black bars) domains. The sequence of the transmembrane domain (thick bar) and cytoplasmic tail domain is relatively conserved for all three members. (B) Members of the MIC8 family are expressed in tachyzoites of T. gondii. RT-PCR analysis was performed on the CTD of MIC8 (upper panel), MIC8-like 1 (middle panel) and MIC8-like 2 (lower panel) using sequence-specific oligonucleotides. Different concentrations of total RNA have been used as template as indicated (in µg). To ensure that no genomic DNA contamination was present in the preparation, multiplex PCR was performed using oligonucleotides specific for alpha-tubulin exons. In the case of genomic DNA (gDNA) as template the intron of alpha-tubulin is amplified, resulting in a larger PCR fragment. (C). Alignment of the TMD and CTD of different micronemal proteins. Although the C-terminal W (box) appears to be conserved in case of MIC8, MIC8-like 1 (MIC8-1) and MIC8-like 2 (MIC8-2), in comparison to the members of the TRAP family there is no acidic amino acid stretch close to the C-terminal Trp (shaded aa).