A spatially localized rhomboid protease cleaves cell 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 microneme protein 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 serine proteases that are unique in being able to identified, cloned, and characterized the five nonmitochondrial cleave within the first few residues of their substrate 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 Toxoplasma gondii. 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 catalytic triad 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 Drosophila 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 ligand 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 malaria; 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. rhomboid protease 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 Apicomplexa, 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- micronemes (2). Microneme proteins 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