A spatially localized rhomboid cleaves surface adhesins essential for invasion by Toxoplasma

Fabien Brossier†, Travis J. Jewett†, L. David Sibley†, and Sinisa Urban‡§

†Department of Molecular Microbiology, Center for Infectious Diseases, Washington University School of Medicine, St. Louis, MO 63110-1093; and ‡Center for Neurologic Diseases, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA 02115

Edited by Louis H. Miller, National Institutes of Health, Rockville, MD, and approved February 1, 2005 (received for review October 25, 2004) Apicomplexan parasites cause serious human and animal diseases, called protease 1, has not been identified. the treatment of which requires identification of new therapeutic Recently, the cleavage of MIC2 and MIC6 has been found to occur targets. Host-cell invasion culminates in the essential cleavage of within the first few residues of their TMDs (9, 10), providing an parasite adhesins, and although the cleavage site for several important clue regarding the identity of microneme protein pro- adhesins maps within their transmembrane domains, the protease tease 1. responsible for this processing has not been discovered. We have Rhomboids are that are unique in being able to identified, cloned, and characterized the five nonmitochondrial cleave within the first few residues of their TMDs to rhomboid intramembrane proteases encoded in the recently com- release domains to the outside of the cell, unlike other examples of pleted genome of . Four T. gondii rhomboids intramembranous processing. These proteases typically have seven (TgROMs) were active proteases with similar substrate specificity. TMDs and are proposed to work by forming a within TgROM1, TgROM4, and TgROM5 were expressed in the tachyzoite the membrane bilayer involving an asparagine, a histidine, and a stage responsible for the disease, whereas TgROM2 and TgROM3 serine residue contributed by different TMDs (11). Rhomboid-1 were expressed in the oocyst stage involved in transmission. from melanogaster (Rho-1) was the first member of this Although both TgROM5 and TgROM4 localized to the cell surface widespread protease family to be identified (11) and is responsible in tachyzoites, TgROM5 was primarily at the posterior of the for cleaving Spitz, the primary of the EGF receptor in parasite, whereas adhesins were sequestered in internal mi- cronemes. Upon microneme secretion, as occurs during invasion, Drosophila. Spitz is synthesized as an inert transmembrane precur- the MIC2 adhesin was secreted to the apical end and translocated sor; its cleavage by Rho-1 leads to secretion of the active EGF to the posterior, the site of cleavage, where it colocalized only with receptor ligand. TgROM5. Moreover, only TgROM5 was able to cleave MIC adhesins Importantly, Spitz contains a motif within its TMD that is in a cell-based assay, indicating that it likely provides the key necessary and sufficient for cleavage by many rhomboid proteases protease activity necessary for invasion. T. gondii rhomboids have (12). A similar motif exists around the cleavage site of MIC clear homologues in other apicomplexans including ; thus, adhesins, and this motif is necessary and sufficient for their cleavage our findings provide a model for studying invasion by this deadly (9, 12). Taken together, these observations suggest that a T. gondii pathogen and offer a target for therapeutic intervention. may be the microneme protein protease 1 activity for invasion of host cells. To address this hypothesis directly, microneme ͉ microneme protein protease 1 ͉ regulated intramembrane we have cloned rhomboid proteases expressed by T. gondii and proteolysis ͉ MIC2 ͉ protease investigated their role in the cleavage of adhesins.

Materials and Methods oxoplasma gondii is a member of the phylum , Twhich contains obligate intracellular parasites including Plas- Identification and Cloning of TgROMs. Bacterial, fly, mouse, human, modium, the agent of malaria, and Cryptosporidium, an agent of and plant rhomboids were used to search the EST (www.cbil. diarrheal disease. During its complex life cycle, T. gondii alternates upenn.edu͞apidots) database and the complete draft sequence of between three different invasive stages: sporozoites, bradyzoites, the genome (http:͞͞ToxoDB.org) of T. gondii by using TBLASTX. and tachyzoites. The first two of these stages are involved in Five putative nonmitochondrial T. gondii rhomboid proteases were transmission: sporozoites are contained within oocysts that are shed identified. TgROM1 (scaffold no. TGG࿝995345, chromosome into the environment by cats, whereas bradyzoites are persistent VIII), TgROM2 (scaffold no. TGG࿝995366, chromosome VI), tissue forms that are responsible for chronic infection. Tachyzoites TgROM3 (scaffold no. TGG࿝995283, chromosome V), TgROM4 are the replicating form responsible for dissemination within the (scaffold no. TGG࿝995368, chromosome VIII), and TgROM5 (scaf- host during acute infection and, thus, are most often associated with fold no. TGG࿝994723, chromosome Ia). Complete ORFs for each symptoms of toxoplasmosis. gene were amplified from full-length cDNAs generated by using a Apicomplexans contain a set of apically localized organelles SMART cDNA library construction kit (Becton Dickinson) from whose sequential secretion is required for invasion of host cells (1). the RH strain, the TgRH* tachyzoite cDNA or VEG sporozoites Polarized attachment of T. gondii to the surface of host cells is cDNA, by using the high fidelity polymerase Klentaq LA. The mediated by the secretion of adhesins stored in organelles called sequences of completed cDNAs were confirmed by cycle sequenc- (2). Microneme containing a transmembrane ing with BIGDYE 3.1. Cloning and sequencing primers are available domain (TMD), namely MIC2, MIC6, MIC8, and MIC12, form on request. heterologous–homologous complexes that expose a variety of adhesive domains at the surface of the parasite (3–6). After binding host-cell receptors, adhesins are rapidly redistributed toward the This paper was submitted directly (Track II) to the PNAS office. posterior through an actin-myosin-dependent process and, ulti- Abbreviations: HA, hemagglutinin; TgROM, Toxoplasma gondii rhomboid; TMD, trans- mately, released into the medium by proteolysis (2, 7). membrane domain; TGF␣, type ␣ TGF. Cleavage of MIC2 is required for efficient invasion to occur; in Data deposition: The sequence reported in this paper has been deposited in the GenBank its absence, parasites remain attached to cells in a nonpolarized database (accession no. AY587208–AY587210, AY704175, and AY704176). manner and fail to enter (8). However, the parasite protease §To whom correspondence should be addressed. E-mail: [email protected]. responsible for C-terminal processing of adhesins including MIC2, © 2005 by The National Academy of Sciences of the USA

4146–4151 ͉ PNAS ͉ March 15, 2005 ͉ vol. 102 ͉ no. 11 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407918102 Downloaded by guest on September 25, 2021 Phylogenetic Analysis. The protein sequences for 24 rhomboids was removed after 2 h and concentrated. Cleavage of Spitz and representing the major branches of rhomboids were aligned by MIC2 were tested in the presence of Batimastat and BB1101 (20 using CLUSTALX 1.81 (13) with a gap opening of 10, a gap extension ␮M), respectively, which are potent metalloprotease inhibitors. of 0.1, and using protein weight matrix Gonnet 250. Regions of the Proteins in supernatants or in cells were detected by Western blot alignment, where Ͻ75% of taxa contained data, were excluded analysis. from the phylogenetic analysis. Dendrograms were generated in PAUP* 4.0 (14) by neighbor-joining and parsimony methods and Electron Microscopy. Parasites were fixed in 4% paraformaldehyde͞ bootstrapped for 1,000 replicates. Identical trees were obtained 0.5% glutaraldehyde in 100 mM Pipes for1hat4°C. Samples were from both analyses. then embedded in 10% gelatin and infiltrated overnight with 2.3 M sucrose͞20% polyvinyl pyrrolidone in Pipes at 4°C. Samples were Total RNA Extraction and RT-PCR. Total RNA from the oocysts was frozen in liquid nitrogen and sectioned with a cryoultramicrotome. extracted, ethanol precipitated twice, and resuspended in RNase- Sections were probed with rabbit anti-⌯〈 (1513) followed by a free water (15). Total RNA from Me49 tachyzoites and bradyzoites 18-nm colloidal gold-conjugated anti-rabbit antibody, stained with were obtained after incubation in TRIzol for 5 min at room ͞ temperature, extraction with 20% chloroform, and precipitation uranyl acetate methyl cellulose, and analyzed by transmission with 50% isopropanol. RNAs were resuspended in water to a final electron microscopy. Parallel controls omitting the primary anti- concentration of 1 mg͞ml. mRNAs of genes of interest were reverse body were consistently negative at the concentration of colloidal transcribed with SuperScript II, and the cDNAs obtained were then gold conjugated secondary antibodies used in these studies. amplified for 30 cycles by using Klentaq LA polymerase.

Growth of Host Cells and Parasites. T. gondii tachyzoites of the RH hxgprtϪ (obtained from David Roos, University of Pennsylvania, Philadelphia), the Me49, and the VEG strains were maintained by growth in monolayers of human foreskin fibroblasts as described in ref. 8. Bradyzoites of the Me49 strain were obtained by in vitro differentiation by cultivation in RPMI medium 1640͞1% FBS͞50 mM Hepes, pH 8.1 at 37°C for 5–7 days in presence of air (16). Fully developed cysts (50–60% of parasites) were harvested by scraping the monolayer and the bradyzoites released from the cysts by digestion in 170 mM NaCl-pepsin (0.1 mg⅐mlϪ1)͞60 mM HCl for 1 min at 37°C and neutralization with 94 mM Na2CO3. Oocysts of the vascular endothelial growth strain (partially sporulated for 48 h and representing a mixture of VEG oocysts at different stages of sporulation) were obtained as described in ref. 17.

Expression of Tagged TgROMs in T. gondii. Full-length ORFs and an N-terminal hemagglutinin (HA) 9 tag were inserted into the plasmid pGRA1͞LacZ (18) replacing the LacZ gene. To express TgROMs under their endogenous promoters, Ϸ1 kb genomic regions upstream of the start codon were used to replace the GRA1 promoter in the above vector. The promoters and tagged genes were finally cloned into the pHLEM vector and transfected into the RH hxgprtϪ strain with a plasmid harboring the selectable marker HXGPRT (19). Transfected parasites were passaged under drug selection and analyzed between passages 6 and 11. At this point, only a portion of parasites expressed the tagged protein, ranging from 10% to 30% of the total cells.

Indirect Immunofluorescence Microscopy. TgROMs were detected in transiently or semistably transfected parasites grown in human foreskin fibroblast cells. In some cases, extracellular parasites were incubated with 1% ethanol for 1 min at 37°C to induce the secretion of microneme proteins. Cells were fixed in 3.7% paraformaldehyde for 30 min at 4°C, blocked in PBS͞Ca2ϩ and 10% FBS and permeabilized by using 0.05% saponin. Cells were incubated with ͞ Fig. 1. T. gondii contains five nonmitochondrial rhomboids, all of which are rabbit anti-HA9 (Zymed), anti-SAG1 (monoclonal DG52) and or closely related to the Rho members of the family. (A) Schematic representation of anti-MIC2 (monoclonal 6D10) (20) antibodies and were examined the predicted T. gondii rhomboids. The seven TMDs (gray bars) are numbered. by using a Zeiss Axioplan microscope equipped with phase contrast The three conserved catalytic triad residues (N in TMD2, the catalytic S in TMD4, and epifluorescence optics. and H in TMD6) and the GAST or GSSG motifs surrounding the putative active are indicated. (B) Unrooted phylogram based on neighbor-joining anal- Rhomboid Cleavage Assay. COS cell transfection experiments were ysis of selected rhomboid proteases. Toxoplasma rhomboids grouped with or- performed as described in refs. 11, 12, and 21 by using FuGENE 6 thologues in spp. were not closely aligned with other major groups and 250 ng of GFP-Spitz (11) and Star (11), GFP-Spi-type ␣ TGF of rhomboids found in , plants, or animals, thus forming several separate ␣ parasite groups. Bootstrap values are shown with colored circles on the tree (TGF ) chimeras (12), or GFP-MIC2, 100 ng of Rho-1 (11), or 100 corresponding the values given in the lower right corner. Dm, D. melanogaster; or 250 ng of TgROMs. TgROMs were tagged with a triple HA tag Hs, Homo sapiens; Mm, Mus musculus; Tg, T. gondii; A. thaliana, Arabidopsis immediately after the initiating methionine. Medium was removed thaliana; O. sativa, Oryza sativa; P. syringae, Pseudomonas syringae; AarA, Provi-

24 h after transfection, and cells were incubated in fresh serum-free dencia stuartii; R. sphaerodies, Rhodobacter sphaerodies (GenBank accession MICROBIOLOGY DMEM for 24 h, except for inhibitor experiments in which media numbers are beneath each entry).

Brossier et al. PNAS ͉ March 15, 2005 ͉ vol. 102 ͉ no. 11 ͉ 4147 Downloaded by guest on September 25, 2021 Fig. 2. Activity analysis of TgROMs. (A) Plasmids encoding GFP-Spitz and Star, Fig. 3. Substrate specificity of TgROM proteases. (A) Substrates in schematic the transport factor for Spitz (26), were cotransfected into COS cells alone (Ϫ), form are depicted above each panel (membrane is indicated by two horizontal with 100 ng or 250 ng of a plasmid encoding one of the TgROMs, or 100 ng of lines, with extracellular up). Mutating the first seven residues of the Spitz TMD Drosophila Rho-1 (Dm) as a positive control. When Spitz is cleaved, it is detected strongly reduced its cleavage by Drosophila Rho-1 (DmR1) but completely abro- by anti-GFP Western blot analysis as a smaller band in cells (arrowhead in A and gated its cleavage by TgROMs. Failure of Spitz cleavage was detected in both B) and as a secreted product in cell culture media. All TgROMs were tagged at media and cells (bottom arrowhead for TgROM2). The mutations changed the their extreme N termini with a triple HA tag to verify their expression levels in conserved Spitz substrate motif ASIASGA (WT) (12) to VALVIGV (Mut) (9). Note mammalian cells [tagged forms retained wild-type (WT) levels of activity com- that the mutant form of Spitz was expressed well in cells and was trafficked pared with untagged forms]. Standards in kDa are shown to the right of each efficiently by Star, as evidenced by the presence of the higher, glycosylated band panel. High amounts of TgROM5 (250 ng) resulted in cytotoxicity, which was also (top arrowhead). (B) The requirement for the GA motif within a substrate TMD apparent as a reduction of Spitz in cells. Note that when cleavage occurs in the was assessed with TgROMs. The substrates were chimeras that contained the ␣ endoplasmic reticulum [Dm Rho-1-KDEL (R1K)], the cleaved product accumulates TMD and cytosolic tail of human TGF , which is not a substrate for rhomboids (12), in cells and is poorly secreted (21) This observation might explain why the cleaved and the extracellular ectodomain of Spitz (to reduce the background cellular ␣ product was poorly secreted for TgROMs 1 and 2. (B) Although TgROM2 had cleavage that occurs with the TGF ectodomain). The second molecule was ␣ weak activity against Spitz, a cleaved product was detected in cells, and its level identical, except that it contained a GA within its TGF TMD (GA). Note that a depended on the amount of TgROM2. (C) Mutating the putative serine reduction in the full-length glycosylated substrate band in cells accompanied of TgROM5 to alanine (SA) completely abolished Spitz cleavage and resulted in efficient cleavage by both DmR1 and TgROM5 (arrowhead). the accumulation of a glycosylated full-length band in cells (arrow), similar to when no rhomboid was transfected (Ϫ). sequence and 6 TMDs, typical of the PARL of mitochondrial rhomboids that are involved in mitochondrial mem- Results brane remodeling (not cell surface shedding) (22). Identification of Rhomboid-Like Genes in T. gondii. Five putative To better depict the relationship between nonmitochondrial T. gondii rhomboids, TgROM1 (AY587210; 249 aa), TgROM2 (AY704176; rhomboids and other members of this class of proteases, we performed a phylogenetic analysis of a select set of rhomboids from 283 aa), TgROM3 (AY587209; 264 aa), TgROM4 (AY704175, 634 flies, mice, humans, plants, bacteria, and parasites. Parasite rhom- aa) and TgROM5 (AY587208; 585 aa), were identified by searching boids formed three distinct clades that were distantly related to the EST database and in the recently completed genome of T. rhomboids in other taxa (Fig. 1B). Notably, several of the T. gondii gondii (Fig. 1A and Table 1, which is published as supporting ͞͞ ͞ rhomboids occur in closely related pairs (TgROM1 with TgROM2 information on the PNAS web site) (http: toxodb.org and TgROM4 with TgROM5) and, thus, appear to be paralogs, ToxoDB.shtml). Their predicted translation products contained the likely derived by gene duplication events. Each of the main groups conserved residues belonging to the catalytic triad, an invariant of T. gondii rhomboids has a single ortholog present in Plasmodium upstream glycine predicted to form the oxyanion-binding hole, and falciparum and Plasmodium yoellii (Fig. 1). In addition, there are at seven TMDs similar to other eukaryotic rhomboids (Fig. 1A; see least four distinct rhomboids in Plasmodium that have no apparent also Fig. 8, which is published as supporting information on the homologs in T. gondii (data not shown). Related genes are also PNAS web site). TgROMs 4 and 5 were unusual in having long found in the genomes of other apicomplexans, including Crypto- hydrophilic C termini and large loops connecting several TMDs. An sporidium, Eimeria, and Neospora (data not shown). additional putative rhomboid, TgROM6 (TgTwinScan࿝4036), was also encoded in the genome sequence (D. Soldati, personal com- TgROMs Are Active Proteases. To examine the capacity of putative munication), but it contained a predicted mitochondrial targeting TgROMs to act as proteases, we tested their ability to cleave

4148 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407918102 Brossier et al. Downloaded by guest on September 25, 2021 rely on helix-destabilizing residues within the top of their substrate TMD for cleavage (12). Accordingly, introduction of mutations into the first seven residues of the Spitz TMD reduced cleavage by Rho-1 but completely abrogated cleavage by all TgROMs, indicating that substrate specificity relies on the same residues within the Spitz TMD (Fig. 3A). Many rhomboid proteases rely, in particular, on a GA residue pair within the 7-aa substrate motif for cleavage (12). Similarly, we compared the ability of TgROMs to cleave two substrates that differed only in the presence or absence of this GA motif (Fig. 3B); inclusion of the GA facilitated cleavage only by TgROM5. This comparison served as an independent and more precise way of assessing substrate specificity and directly confirmed that TgROM5 conforms to the same specificity determinant previously charac- Fig. 4. Stage-specific expression of TgROMs. Schematic representation of terized for a large class of rhomboid proteases (12). the three main invasive stages of the T. gondii life cycle. RT-PCR analysis of TgROM expression in invasive stages of the parasite showed that TgROM1, Expression Patterns of TgROMs. TgROM4, and TgROM5 were expressed primarily in tachyzoites but also in To begin addressing the biological sporozoites. Conversely, TgROM2 and TgROM3 were expressed primarily in roles of these rhomboid proteases, we examined their expression sporozoites. SAG1 and BAG1 were used as positive control for tachyzoites and patterns by RT-PCR of RNA from the three invasion stages of the bradyzoites, respectively. ACT (actin) was used as an internal control. The parasite: tachyzoites, bradyzoites, and sporozoites (Fig. 4). smaller bands seen in TgROM5 are nonspecific amplification products. TgROM1, TgROM4, and TgROM5 were primarily expressed in tachyzoites, the proliferative stage of the life cycle (but were also detected weakly in sporozoites). Conversely, TgROM2 and GFP-Spitz expressed in COS cells, which is an efficient surrogate TgROM3 were expressed primarily in sporozoites. TgROM1 and substrate for many rhomboids from diverse organisms (23). The TgROM4 were also expressed in bradyzoites, although this result cleaved form of GFP-Spitz was detected in the media of cells may reflect, in part, their expression in tachyzoites because the in transfected with TgROM1, TgROM3, and TgROM5 (Fig. 2A), vitro tachyzoite-to-bradyzoite conversion was Ͻ100%. The dynamic whereas cleavage was only apparent in cell lysates with TgROM2 expression of TgROMs in different life cycle stages is consistent (Fig. 2B). No activity was detected with TgROM4. Increasing the with their regulation being primarily transcriptional, like other amounts of transfected DNA encoding each active rhomboid increased the amount of cleaved Spitz in cells and media, except for members of this family, and suggests roles in a variety of biological TgROM5 (because high levels of TgROM5 resulted in cytotoxic- processes, including invasion. ity). Cleavage of Spitz depends on the active-site serine and its mutation to alanine in TgROM5 completely abolished cleavage Intracellular Localization of TgROMs. Two of the five TgROMs were (Fig. 2C). All five TgROMs were expressed well in transfected unlikely to be responsible for cleavage of adhesins during tachyzoite mammalian cells (Fig. 2A), and their relative proteolytic activities invasion: TgROMs 2 and 3 were not expressed or minimally were not due to trivial differences in relative expression levels. expressed in tachyzoites. Thus, we analyzed the subcellular local- izations of the remaining TgROMs, TgROM1, TgROM4, and TgROMs Share Substrate Specificity with Drosophila Rho-1. Rhom- TgROM5. Parasite rhomboids were tagged with HA9 at their N boids from different organisms are highly specific proteases that termini (because C-terminal tags abolished proteolytic activity;

Fig. 5. Subcellular localization of HA9-tagged TgROM1, TgROM4, and TgROM5 in tachyzoites by using immunofluorescence and electron microscopy. (A) TgROM1 (␣HA9) was localized at the apical part of intracellular (Upper) and extracellular parasites (Lower) where it colocalized with MIC2 in micronemes. (B) TgROM4 (␣-HA9) was uniformly distributed at the surface of intracellular (Upper) and on extracellular parasites (Lower). (C) TgROM5 (␣-HA9) was localized mostly at the posterior end of intracellular parasites (Top). In extracellular parasites, TgROM5 appeared to be distributed along the surface of the cell in patches (Middle and Bottom). After

treatment with ethanol to induce microneme secretion, TgROM5 was often colocalized with MIC2 at the extreme posterior end (arrow). (Scale bar: 1.0 ␮m.) (D) MICROBIOLOGY CryoimmunoEM labeling by using anti-HA9 antibodies revealed that TgROM5 was localized in patches at the surface of the parasite. (Scale bar: 0.25 ␮m.)

Brossier et al. PNAS ͉ March 15, 2005 ͉ vol. 102 ͉ no. 11 ͉ 4149 Downloaded by guest on September 25, 2021 Fig. 6. Cleavage of MIC adhesins by TgROMs. (A) Schematic comparison of full-length MIC2 with the MIC6 and MIC12 synthetic (SYN) substrates used for activity analysis. (B) Full-length TgMIC2 (containing its integrin-like and six thrombospondin domains) was GFP tagged at its N terminus and cotransfected into COS cells with TgROMs and Drosophila Rho-1. Although Drosophila Rho-1 could cleave the MIC2 adhesin weakly, cleavage by TgROM5 was very efficient (arrowhead). No cleavage was detected with the other TgROMs or when the active site serine of TgROM5 was mutated to alanine (SA). 3,4-Dichloroisocoumarin (DCI) at 50 ␮M blocked MIC2 cleavage by TgROM5 (assay time was limited to2htoavoid DCI indirect effects, as in ref. 11). The weak upper band in media lanes is due to some shedding of MIC2 by cellular proteases (although these analyses were performed in the presence of a metalloprotease inhibitor). (C and D) The TMDs of MIC6 and MIC12 were encoded in a synthetic substrate comprised of GFP as its ectodomain and the TGF␣ tail as its cytoplasmic domain (12). TgROM5 efficiently cleaved the TMDs corresponding to MIC6 (C) and MIC12 (D) Note that TgROM3 could also weakly cleave the TMD from MIC12 (arrowhead) and MIC6 (data not shown).

data not shown) and localized by immunofluoresence in transiently yet, no cleavage occurs there. The activity of TgROM5 was transfected parasites. completely abolished by mutating the catalytic serine to alanine Expression from their endogenous promoters revealed that (GSSG3GSAG) (Fig. 6B), establishing that the release depends TgROM1 colocalized with MIC2 in micronemes in intracellular on its proteolytic activity. The presence of the MIC2-associated and extracellular tachyzoites (Fig. 5A). TgROM4 was uniformly protein, which forms a complex with the MIC2 ectodomain, had distributed at the surface of intracellular and extracellular parasites no effect on the cleavage of MIC2 by TgROM5 (data not shown). (Fig. 5B). TgROM5 was primarily on the surface at the posterior Isocoumarins are the only inhibitors that are end of intracellular parasites, in an opposite pattern from MIC2, known to inhibit all rhomboids tested (24), and MIC2 cleavage which is confined to apical secretory organelles (Fig. 5C). Similarly, by TgROM5 was inhibited by 3,4-dichloroisocomarin (Fig. 6B). in extracellular parasites, TgROM5 was distributed along the length Previous studies have indicated that treatment of intact parasites of the cell but was primarily at the back of the parasite and only with 3,4-dichloroisocomarin reduces the release of MIC2 into rarely located at the front. We also examined the distribution of the supernatant, although this effect may also be due in part to TgROM5 in parasites after induction of microneme secretion by decreased secretion of MIC2 from the micronemes (7). treatment with ethanol as described in refs. 7 and 8 (Fig. 5C Middle TgROM5 was also efficiently able to cleave the TMDs from and Bottom). Under these conditions, TgROM5 was often concen- MIC6 and MIC12 when they were contained within a chimeric trated at the extreme back end in a prominent dot that colocalized substrate (Fig. 6 C and D). These observations suggest that with MIC2 (arrow in Fig. 5C). TgROM5 is the protease primarily responsible for cleaving MIC2, Whereas TgROM4 was evenly distributed along the parasite and also indicate that TgROM5 processes multiple microneme surface, the distribution of TgROM5 was more heterogeneous. To adhesins. further evaluate the distribution of TgROM5, we examined its localization by cryoimmunoEM. Immunogold labeling revealed Discussion that TgROM5 was distributed in clusters and that it was exposed at Our biochemical and cell biological analyses of T. gondii rhom- the surface of the parasite (Fig. 5D). boid proteases suggest that rhomboids fulfill several distinct developmental functions during the parasite lifecycle. Although Activity of TgROMs on Microneme Adhesins. TgROM1 colocalized with MIC2 in micronemes while TgROMs 4 and 5 were at the cell surface; however, cleavage of MIC protein TMDs does not occur in micronemes, but only once they reach the cell surface. Because the ectodomains of MIC adhesins are very large (Fig. 6A) and could prevent interaction with rhomboid proteases, as is common with other intramembrane proteases, we examined the capacity of TgROMs to process MIC TMDs directly within a native adhesin. Full-length MIC2 was tagged at its N terminus with GFP, and its ability to be cleaved by TgROMs was investigated in the heterol- ogous assay. TgROM5 proved to be the only parasite protease capable of Fig. 7. Model of MIC adhesin cleavage during parasite invasion. TgROM5 (green) is on the parasite posterior surface and in patches more laterally, processing this substrate efficiently, as detected by the release whereas MIC adhesins (red) are in micronemes. The sequence of events from of GFP-MIC2 into the medium (Fig. 6B). Note that TgROM1 left to right is as follows: unattached parasite, microneme secretion and apical did not have the capacity to cleave MIC2, providing a possible attachment, translocation of adhesins, colocalization of TgROM5 and MICs at explanation of how they are able to colocalize in micronemes, the posterior and MIC cleavage, adhesin release and parasite internalization.

4150 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407918102 Brossier et al. Downloaded by guest on September 25, 2021 further investigation is required to decipher these roles, the PbTRAP, the MIC2 homolog in P. berghei, was correctly processed analyses presented here allow us to reach several conclusions when expressed in T. gondii (9). The genome of P. falciparum about their function during invasion. contains eight rhomboid-like genes, three of which are clear ho- TgROM5 was the only parasite rhomboid protease to be highly mologs of the TgROMs (see Fig. 1), whereas several others are active against the full-length MIC2 adhesin and the TMDs of MIC6 predicted to be mitochondrial. Intriguingly, parasite rhomboids and MIC12. TgROM5 could thus be involved in processing most form distinct clades that are not closely similar to any of the MIC TMDs during invasion, especially because other MICs, in- previously characterized groupings (27). This divergence may re- cluding MIC7, 8, and 9, also share a similar TMD and are likely to flect the ancient origin of the phylum Apicomplexa (28, 29) along serve as good substrates for TgROM5. Consistent with this role, with the relative absence of molecular sequence data from closely TgROM5 was strongly expressed in tachyzoites, but was also related taxa (i.e., dinoflagellates and ciliates). expressed in sporozoites and at low levels in bradyzoites, and was There is a serious need for identifying new targets for combating clustered in patches at the surface of tachyzoites. A similar patchy apicomplexan infections, and proteases have proven to be very distribution has previously been reported for cell surface-exposed attractive targets for therapeutic intervention in many other dis- MIC2 (25), and these two proteins were observed to colocalize at eases. Based on our findings here, we predict that the Plasmodium the posterior end of the parasite after secretion. Although rhomboid homologous to TgROM5 (i.e., PfROM4, NP࿝703414.1) TgROM5 may also exist as an isoform with a longer N terminus is the primary rhomboid protease involved in P. falciparum throm- (AY634626), this form is also active (S.U., unpublished observa- bospondin-related anonymous protein (PfTRAP) processing dur- tions), and the construct we used to localize TgROM5 is sufficiently ing invasion. PfTRAP is essential for sporozoite entry into the long to encode either variant, although it remains unclear which salivary glands and invasion of hepatocytes during infection in the form predominates in the parasite. vertebrate host (30–32). Thus, our work provides a role for rhom- Somewhat surprisingly, although TgROM4 is most similar to boid proteases in processing adhesins important for host-pathogen TgROM5, it was not active against substrates tested here. TgROM4 recognition and suggests a therapeutic target for interrupting was uniformly distributed on the cell surface, consistent with a role infection by sporozoites. Additionally, it is possible that rhomboids in processing a cell surface substrate. However, based on this play key developmental roles at other stages in the life cycle, and uniform distribution, it would be expected that if TgROM4 were interruption of their functions might offer additional means of active on MICs, that this result would be a premature release of the interrupting infection by this important group of parasites. adhesins. In contrast, TgROM5 provides an attractive candidate for T. gondii rhomboid proteases differed in their expression pat- microneme protein protease 1 because its activity would be regu- lated by spatial distribution at the back of the organism. Whether terns, subcellular localization, and activities and are thus likely to be TgROM4 and TgROM5 fulfill unique or overlapping functions will involved in multiple functions during the complex life cycle of the require further analysis, including removing their respective activ- parasite. TgROM1 was the most widely expressed rhomboid and, ities in vivo by targeted gene disruption. yet, its location within micronemes would appear to preclude its role Taken together, these data suggest a model for MIC adhesin in processing of MIC protein TMDs. TgROM2 and TgROM3 were cleavage during invasion (Fig. 7). Adhesins are secreted from largely or exclusively confined to the sporozoite stage. Although the micronemes to the apical surface of the parasite at the start of function of these TgROMs remains uncertain, sporozoites undergo invasion. MIC adhesins bind host-cell receptors, and these com- a complex program of cell development after meiosis in the oocyst plexes are translocated from the anterior to the posterior end of the (33–35), thus these proteases may play roles in cell signaling or parasite. TgROM5 is concentrated at the posterior surface, where development. it cleaves the MIC adhesins upon their translocation, thus releasing the adhesin complexes and allowing parasite entry. Remarkably, We thank Wandy Beatty for performing the cryoimmunoEM, this process is strikingly reminiscent of EGF ligand cleavage in Michael White and Jay Radke (Montana State University, Bozeman) for Drosophila; EGF ligands are sequestered in the ER, whereas the generous gift of total RNA from sporozoites, Kelinag Tang for construction of full-length cDNA, Julie Suetterlin for expert technical rhomboid proteases are localized in the Golgi apparatus (26). The assistance, Roy Black (Immunex) for providing BB1101, and Mike Wolfe transport factor Star is required to translocate EGF ligands to the for generously providing space and resources to S.U. S.U. was supported Golgi apparatus, allowing them to be cleaved efficiently by rhom- by the International Human Frontier Science Program. F.B. was sup- boids and released as active ligands for signaling. ported in part by National Institutes of Health Grant AI 34036. L.D.S. Cleavage of the transmembrane microneme proteins occurs is a recipient of a Scholar Award in Molecular Parasitology from the through a mechanism that is conserved within apicomplexans. Burroughs Wellcome Fund.

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