bioRxiv preprint doi: https://doi.org/10.1101/2021.02.02.429490; this version posted February 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Full Title: Proximity-labeling reveals novel host and parasite proteins at the 2 Toxoplasma parasitophorous vacuole membrane 3 4 Running Title: Proximity-labeling identifies Toxoplasma PVM proteins 5 6 Alicja M. Cygana, Pierre M. Jean Beltranb, Tess C. Branonc,d, Alice Y. Tingc, Steven A. 7 Carrb, John C. Boothroyda 8 9 Author Affiliations: 10 aDepartment of Microbiology and Immunology, Stanford School of Medicine, Stanford 11 CA, USA 12 bBroad Institute of MIT and Harvard, Cambridge MA, USA 13 cDepartment of Chemistry, Stanford University, Stanford CA, USA 14 dCurrent address: Department of Molecular and Cell Biology, University of California, 15 Berkeley CA, USA 16 17 18 19 #Address correspondence to John C. Boothroyd, [email protected] 20 21 Word count, abstract: 178 22 Word count, text: 3440 23 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.02.429490; this version posted February 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 24 Abstract 25 Toxoplasma gondii is a ubiquitous, intracellular parasite that envelopes its 26 parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for 27 interactions with the infected host cell such as nutrient transport and immune defense. 28 Only a few parasite and host proteins have so far been identified on the host-cytosolic 29 side of the PVM. We report here the use of human foreskin fibroblasts expressing the 30 proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the 31 PVM, in combination with quantitative proteomics to specifically identify proteins present 32 at this crucial interface. Out of numerous human and parasite proteins with candidate 33 PVM localization, we validate three novel parasite proteins (TGGT1_269950, 34 TGGT1_215360, and TGGT1_217530) and four new host proteins (PDCD6IP/ALIX, 35 PDCD6, CC2D1A, and MOSPD2) as localized to the PVM in infected human cells 36 through immunofluorescence microscopy. These results significantly expand our 37 knowledge of proteins present at the PVM and, given that three of the validated host 38 proteins are components of the ESCRT machinery, they further suggest that novel 39 biology is operating at this crucial host-pathogen interface. 40 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.02.429490; this version posted February 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 41 Importance 42 Toxoplasma is an intracellular pathogen which resides and replicates inside a 43 membrane-bound vacuole in infected cells. This vacuole is modified by both parasite 44 and host proteins which participate in a variety of host-parasite interactions at this 45 interface, including nutrient exchange, effector transport, and immune modulation. Only 46 a small number of parasite and host proteins present at the vacuolar membrane and 47 exposed to the host cytosol have thus far been identified. Here we report the 48 identification of several novel parasite and host proteins present at the vacuolar 49 membrane using enzyme-catalyzed proximity-labeling, significantly increasing our 50 knowledge of the molecular players present and novel biology occurring at this crucial 51 interface. 52 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.02.429490; this version posted February 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 53 Introduction 54 Toxoplasma gondii, the prototypical model organism for the phylum 55 Apicomplexa, is uniquely capable of infecting virtually any nucleated cell in almost any 56 warm-blooded animal. This ubiquitous intracellular parasite can be especially 57 dangerous for immunocompromised individuals, the developing fetus, and is also a 58 significant veterinary pathogen (1). In addition to its medical importance, Toxoplasma is 59 a powerful model organism for studying the biology of intracellular parasitism, due to its 60 genetic tractability and ease of culture. 61 As an obligate intracellular organism, Toxoplasma relies on host cell resources in 62 order to replicate. Like other major pathogenic species of the phylum Apicomplexa (e.g., 63 Cryptosporidium, Plasmodium), Toxoplasma has evolved to extensively modify its 64 intracellular environment in order to establish a replicative niche, the parasitophorous 65 vacuole (PV), in its host. The PV is necessary to facilitate Toxoplasma’s intracellular 66 development and for the avoidance of host defenses. Among other pathogenic 67 processes, Toxoplasma tachyzoites, the rapidly dividing forms of the parasite 68 responsible for acute infection, remodel the host cytoskeleton (e.g., vimentin and 69 microtubule organizing center (2–4)), recruit host organelles (e.g., mitochondria and 70 endoplasmic reticulum (5, 6)), scavenge essential nutrients and host resources (e.g., 71 tryptophan, cholesterol, and iron (7–9)), actively manipulate the host transcriptome to 72 support increased metabolism and inhibit the innate immune response (e.g., 73 accumulation of the host “master regulator” c-Myc (10, 11)), and even physically 74 inactivate host protein defenses mounted at the PV (e.g., inactivation of IFNγ -induced 75 immunity-related GTPases in murine cells (12, 13)). 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.02.429490; this version posted February 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 76 Much of this host cell manipulation relies on parasite effector proteins (e.g., 77 ROP18, GRA15, GRA17/23, MAF1) introduced into the host during infection (recently 78 reviewed in (14)), and located at the parasitophorous vacuole membrane (PVM), the 79 interface between the developing parasites and host. Exposed to the host cytosol, these 80 parasite effectors can interact with various host processes (recently reviewed in (15)). 81 Despite the critical importance of the parasite-host interactions occurring at the PVM, 82 only a small number of proteins at this boundary have thus far been identified. This lack 83 of knowledge has hampered our ability to mechanistically study interesting and 84 potentially disease-relevant aspects of biology, including processes that might be 85 amenable to parasite- and host-targeted drugs. 86 To address this lack, we have utilized advances in spatial proteomics to identify 87 host and parasite proteins specifically present at the host cytosolic side of the PVM. 88 Proximity labeling-based methods have successfully been used to identify proteins 89 within various Toxoplasma cellular compartments, including the PV lumen (16, 17), 90 relying on expression of the crucial biotin-ligase enzymes fused to parasite-expressed 91 proteins. To date, however, no study has used this approach to identify novel proteins 92 specifically present on the host-cytosolic face of the PVM. By fusing the promiscuous 93 biotin-labeling protein, miniTurbo (18), to a domain that targets it to the PVM and 94 engineering host cells to express this fusion, we have been able to generate high 95 confidence lists of candidate host and parasite proteins present at the host-cytosolic 96 face of the PVM. From a subset of this candidate pool, we have validated three parasite 97 and four host proteins as so localized, including several that indicate previously 98 undescribed processes occurring at this all-important interface. 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.02.429490; this version posted February 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 99 Results 100 Validation of the miniTurbo system in infected HFFs 101 To identify host and parasite proteins exposed to the host cytosol at the PVM, we 102 utilized enzyme-catalyzed proximity-labeling via the promiscuous biotin ligase, 103 miniTurbo, a recently developed variant of BioID (18, 19). To target miniTurbo to the 104 PVM, we fused V5-tagged miniTurbo-NES (NES = nuclear export signal) to the 105 arginine-rich-amphipathic helix (RAH) domain (amino acids 104-223) of the Toxoplasma 106 ROP17 protein (Fig. 1A), which had previously been shown to be capable of localizing 107 mCherry to the PVM when heterologously expressed in human cells (20). We stably 108 introduced this construct, as well as V5-tagged miniTurbo-NES without the RAH domain 109 as a control, into human foreskin fibroblasts (HFFs) using lentiviral transduction to 110 generate cell lines stably expressing one or other of the miniTurbo proteins (hereafter 111 referred to as “HFF miniTurbo-RAH” and “HFF miniTurbo”, respectively). 112 We assessed the localization of miniTurbo in these cells infected with 113 Toxoplasma tachyzoites (RH strain) using immunofluorescence assay (IFA). The results 114 (Fig. 1B) show a diffuse, cytosolic staining pattern in HFF miniTurbo cells, consistent 115 with the localization of this construct in other human cell lines (18), and with no apparent 116 association with parasite vacuoles.