The prodomain of Toxoplasma gondii GPI-anchored subtilase TgSUB1 mediates its targeting to micronemes

Abstract

Subtilisin-like proteases have been proposed to play an important role for parasite survival in Toxoplasma gondii (Tg) and Plasmodium falciparum. The T. gondii subtilase TgSUB1 is located in the microneme, an apical secretory organelle whose contents mediate adhesion to the host during invasion. TgSUB1 is predicted to contain a glycosyl-phosphatidylinositol (GPI) anchor. This is unusual as Toxoplasma GPI-anchored proteins are targeted to the parasite's surface. In this study, we report that the subtilase TgSUB1 is indeed a GPI-anchored protein but contains dominant microneme targeting signals. Accurate targeting of TgSUB1 to the micronemes is dependent upon several factors including promoter strength and timing, accurate processing and folding. We analyzed the targeting domains of TgSUB1 using TgSUB1 deletion constructs and chimeras made between TgSUB1 and reporter proteins. The TgSUB1 prodomain is responsible for trafficking to the micronemes and is sufficient for targeting a reporter protein to the micronemes. Trafficking is dependent upon correct folding or other context-dependent conformation as the prodomain expressed alone is unable to reach the micromenes. Therefore, TgSUB1 is a novel example of a GPI-anchored protein in T. gondii that bypasses the GPI-dependent surface trafficking pathway to traffic to micronemes, specialized regulated secretory organelles.

Figure 1. TgSUB1 behaves like a GPI-anchored protein

A) A schematic of TgSUB1 is shown indicating the signal sequence (ss), prodomain, catalytic domain, proline-rich domain and putative GPI anchor addition site. The amino acid residues encompassing the prodomain are indicated. The amino acids surrounding the proposed GPI addition site are illustrated. The GPI anchor addition site consists of a triad of amino acids (underlined) followed by a hydrophilic spacer region (lowercase) and a hydrophobic region (italics). The specific amino acids (aspartic acid, glycine and serine) of the GPI addition site and length of the subsequent domains are consistent with other known GPI-anchored proteins. B) TX-114 phase partitioning of TgSUB1. RH tachyzoite lysates were either treated or mock treated with PI-PLC, an enzyme that specifically cleaves GPI-anchored proteins inducing transition from a membrane-associated state (M) to a soluble state (S). SAG1, a known GPI-anchored protein, is shown for comparison. SAG1 is revealed with P30 antibody and TgSUB1 with AE653 antibody. Because of ongoing proteolysis of TgSUB1, AE653 antisera were used for all westerns. These antisera, which recognize a peptide adjacent to the GPI addition site, does not recognize TgSUB1 released into the media after secretion by micronemes (C). RH tachyzoite lysates treated or mock treated with PI-PLC were probed with the anti-CRD GPI-specific epitope antibody. Results are shown in duplicate. D) CRD antibody immunoprecipitates PI-PLC-cleaved TgSUB1. Immunoprecipitation was performed with CRD antibody. TgSUB1 was detected by western blot analysis with PfSUB1 antibody. Immunoprecipitations were performed (i) on untreated lysates with no primary CRD antibody, (ii) on untreated lysates with CRD antibody and (iii) with PI-PLC-treated tachyzoite lysates immunoprecipitated with CRD antibody. IP, precipitated protein fraction (precipitated with protein A beads); SN, supernatant containing non-precipitated proteins.

Figure 2. Biosynthetic labeling of the TgSUB1 GPI anchor

RH tachyzoites lysates were radiolabeled with [³H] ethanolamine and [³H] palmitate, markers of GPI anchors. Following radiolabeling, TgSUB1 was immunoprecipitated with the PfSUB1 antibody (arrow). Controls for immunoprecipitation were lysate incubated without antibody and antibody without lysate. The right panel represents palmitate-labeled proteins washed with hydroxylamine. Palmitate incorporated into GPI anchors is resistant to hydroxylamine treatment. Labeling of TgSUB1 with palmitate was sustained in the presence of hydroxylamine.

Figure 3. Generation of a TgSUB1 KO parasite

A) A diagram of the strategy used to disrupt the TgSUB1 gene by homologous recombination in the RH background is shown. The TgSUB1KO plasmid is seen on the top line showing 2.9 kb of 5′ and 3′ DNA flanking the coding region of TgSUB1. Dashed lines indicate the rest of the targeting plasmid sequence. TgSUB1 is replaced by the HXGPRT cassette, which includes the HXGPRT gene surrounded by the DHFR promoter and terminator sequences. Dots signify the rest of the genomic locus. B and C) IFA and western blot analysis of TgSUB1KO and clone 5 parasites (TgSUB1 was revealed with AE653 antibody). MIC5 is used as internal control for western blot loading. TgSUB1 immunofluorescence signal depicts the conventional apical microneme labeling. Scale bar: 5 μm.

Figure 4. TgSUB1 localization depends upon the promoter

A) Western blot analysis of tachyzoites transiently transfected with TgSUB1-HA (FL5SUB1-HA) driven by the M2AP, GRA1 and ROP1 promoters. Blots were probed with anti-TgSUB1 AE653 antibody or probed with antibodies against SAG1 to ensure that equivalent levels of parasite proteins were being compared. Transfection efficiencies are indicated. B) Immunofluorescence of tachyzoites transiently transfected with TgSUB1-HA driven by the M2AP, GRA1 and ROP1 promoters. Antibody AE653 was used to show localization of the different TgSUB1 constructs. Scale bar: 5 μm.

Figure 5. Effect of position of the HA tag in TgSUB1 upon microneme localization

A) A schematic of TgSUB1-HA is represented with different domains (ss, signal sequence; pp, prodomain; catalytic, catalytic site; proline, proline-rich domain and gpi, C-terminal part containing GPI anchor signal). The dark blue rectangle represents the HA tag in FL5SUB1-HA. The arrows indicate other positions where the HA tag has been inserted. B) Western blot analysis of M2AP-driven TgSUB1-HA tagged constructs after transient transfection of RH TgSUB1KO tachyzoites. Membranes were probed with a rat anti-HA antibody. C) IFA of tachyzoites transfected with different TgSUB1-HA constructs. TgSUB1 expression was detected with a rat anti-HA antibody or the AE653 anti-TgSUB1 antibody. MIC2 and M2AP were used as markers for micronemes and were visualized with proper antibodies. Scale bar: 5 μm.

Figure 6. Expression of TgSUB1-HA deletion constructs

Deletion constructs were transiently transfected into RH TgSUB1KO parasites. The dark blue rectangle indicates the HA tag (A, C–E). Immunofluorescence of intracellular tachyzoites containing the constructs was performed with a rat anti-HA antibody. In (B), the GPI domain corresponds to the C-terminal part of SAG1 containing the GPI anchor signal fused to the full length of TgSUB1 sequence. The construct was visualized with the AE653 anti-TgSUB1 antibody. M2AP and MIC2 were used as markers for micronemes. Scale bar: 5 μm.

Figure 7. Expression of TgSUB1 prodomain constructs

Chimeras containing GFP and SAG1 fused to the signal sequence and prodomain of TgSUB1 were transiently transfected into RH TgSUB1KO and RH SAG1KO parasites, respectively. The GFP reporter construct was detected by fluorescence of the GFP protein (A), whereas the SAG1 reporter construct was detected with a SAG1 antibody (B, C). The prodomain–HA and the prodomain–HA–GPI constructs were detected with a rat anti-HA antibody (D, E). Colocalizations performed with antibodies specific for microneme proteins M2AP and MIC5. All the TgSUB1 prodomain reporter constructs consist of the full length of the prodomain with the cleavage site except for the constructs consisting of the prodomain lacking the last 26 amino acids: pp(1–166) (C–E). Scale bar: 5 μm.