Article Cite This: J. Proteome Res. XXXX, XXX, XXX−XXX
pubs.acs.org/jpr
Identification of Novel Dense-Granule Proteins in Toxoplasma gondii by Two Proximity-Based Biotinylation Approaches Ming Pan,† Mingjun Li,† Longjiao Li,† Yongle Song,† Lun Hou,† Junlong Zhao,†,‡,§ and Bang Shen*,†,‡ †
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State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, PR China, 430070 ‡ Key Laboratory of Preventive Medicine, Wuhan, Hubei, PR China § Hubei Cooperative Innovation Center for Sustainable Pig Production, Wuhan, Hubei, PR China, 430070 S Supporting Information *
ABSTRACT: Toxoplasma gondii is an opportunistic pathogen infecting humans and a variety of vertebrate animals. Secretory dense-granule proteins (GRAs) play diverse roles in the mediation of host−parasite interactions and facilitate parasitism, but many of them still remain to be identified. Here, we used two proximity-based protein labeling techniques to identify novel GRA proteins. Taking GRA1 as bait, transgenic strains expressing GRA1-BirA* or GRA1-APEX were constructed to biotinylate GRAs. Using these methods, a total of 46 proteins were identified, 20 of which were known GRA proteins. Among these 46, 17 were identified by both strategies, and 14 out of the 17 were known GRAs. The other three were all confirmed to localize to dense granules. Nonetheless a significant portion of the proteins were only identified by either APEX or BirA*, indicating that there are differences between these methods. Of the 26 novel GRAs, 5 were validated as bona f ide GRAs by localization studies. The majority of these novel GRAs are only present in coccidian parasites and are likely dispensable for parasite growth in vitro; they may play roles during animal infections. The identification of novel GRAs laid the foundation for further studies investigating the mechanisms underlying parasite−host interactions. KEYWORDS: Toxoplasma gondii, dense-granule proteins, GRA1, APEX, BirA*
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cope with diverse hosts.10,13 Once released from corresponding organelles, these secretory proteins are localized to host− parasite interfaces or injected into host cells to counteract the host’s immune clearance or alter the host’s gene-expression activities.1,14 The best-studied proteins of this kind include ROP16 and ROP18, which are polymorphic kinases that affect the outcomes of T. gondii infections.15−17 In the mice infection model, ROP5, ROP18, and ROP17 are well-studied rhoptry proteins that form complexes on the parasitophorous vacuole membrane (PVM) to defeat the accumulation of immunerelated GTPase (IRGs) and guanylate-binding proteins (GBPs), which are key IFN-γ stimulated factors in mice to clear acute infection of T. gondii.18−21 GRA proteins also participate in immune evasion, such as GRA7, which stimulates the rapid turnover of IRGs and prevents their accumulation on PVM.22 Other GRA proteins penetrate further to enter host cytosol or even the nucleus to manipulate host cell signaling and gene expression.10 For example, TgIST is a recently discovered GRA protein that is secreted into host nucleus to silent the expression of STAT1-regulated genes by recruiting the repressive Mi-2/NuRD complex to STAT1 on the
INTRODUCTION Toxoplasma gondii is an obligate intracellular protozoan belonging to the phylum Apicomplexa, the members of which are important pathogens (such as Plasmodium and Eimeria species) for humans and animals.1 T. gondii is an extremely successful parasite, infecting one-third of the world’s population and numerous animals.2,3 As an opportunistic pathogen, its infections in the vast majority of healthy animals and humans are asymptomatic or subclinical.4,5 However, in immunocompromised individuals such as AIDS patients, T. gondii infection can lead to severe complications even death.5,6 Infection during pregnancy may lead to abortion and stillbirth, causing socioeconomic problems.7 Current medications to treat toxoplasmosis are limited and are not ideal, with strong side effects and effectiveness only against acute infections.8,9 Therefore, new intervention strategies are urgently needed to control this widely spread parasite. To survive and establish infections in diverse hosts, T. gondii needs complex strategies to manipulate each host for successful parasitism.1 Secretory proteins released from specialized secretory organelles such as dense granules (GRA) and rhoptries (ROP) are actively involved in mediating host− parasite interactions.10−12 T. gondii expresses a wide variety and large amounts of such secretory proteins and is likely to © XXXX American Chemical Society
Received: August 15, 2018 Published: October 17, 2018 A
DOI: 10.1021/acs.jproteome.8b00626 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research
Figure 1. Schematic illustration of proximity-dependent biotinylation of dense-granule proteins using GRA1-APEX and -BirA* fusions. Expression of GRA1-APEX and -BirA* fusions in parasites allows the selective biotinylation of proteins proximate to GRA1. Biotinylated proteins are then affinity-purified by streptavidin-coated beads and identified by mass spectrometry. Biotinylation in the GRA1-APEX parasites was triggered by biotin−phenol and H2O2 treatment, whereas biotin supplementation allows biotinylation in GRA1-BirA* expressing parasites. The omission of biotin−phenol or biotin served as controls for the corresponding labeling experiments.
chromatin.23,24 Similarly, GRA16 and GRA24 also traffic to the host-cell nucleus and alter the expression of host genes involved in metabolism, cell cycle progression, and immune responses.25,26 In addition, GRA proteins may also facilitate parasitism by importing nutrients from host cells, such as GRA17.27 T. gondii genome encodes a variety of GRA proteins. Although many GRA proteins are secreted into the parasitophorous vacuole (PV) and contribute to the maturation and structure integrity of the PV, the majority of them are not essential for parasite growth in vitro.28,29 Except for GRA1 and GRA10, which are refractory to gene deletion, many other GRAs can be knocked out without significant impact on parasite growth in vitro.28 Similarly, only a small subset of GRA proteins, including IST, GRA15, GRA16, GRA24, and GRA25, were shown to affect host gene expression, metabolism, or immune responses during mice infection.1,14,23,29 The dispensability of the majority of GRA proteins under standard conditions or during mice infection does not necessary mean a lack of function, and they may mediate parasite−host interactions during the infection of animals other than mice. Genomic analysis revealed the presence of hundreds of potential secretory pathogenesis determinants (SPDs) in T. gondii.13 Such SPDs include GRA proteins, but the majority of them have not been characterized so far. Some of the hypothetical SPDs may also be GRA proteins because currently known GRAs consist only a small subset of the total proteins in these organelles. Indeed, the promiscuous biotin ligase BirA* (a mutant form of E. coli BirA that loses substrate specificity) based proximity-dependent protein labeling was recently used to identify more GRA proteins.30,31 A number of novel GRAs were identified by this approach, and the dense-granule localization of 13 of these GRAs was confirmed by endogenous gene tagging.31 Despite this effort, the discovery of GRA proteins has not been exhausted, and more GRAs are to be identified. GRA1 is the first identified dense-granule protein in T. gondii,32 yet its function is still completely unknown. The disruption of the GRA1 gene has been tried but was never successful, suggesting that it might be essential.28 In fact, GRA1 is one of the few GRAs that are refractory to gene deletion, indicating a distinct functional mode. Early studies have reported that GRA1 was a soluble protein secreted into the lumen of PV, and some GRA1 molecules were also peripherally associated with the tubulo-vesicular network
inside PV.33 Because of these unique features of GRA1, we used it as a bait to screen for novel GRA proteins using two proximity-dependent protein labeling techniques. In addition to the BirA*-based approach described above,30 we also used the ascorbate peroxidase (APEX) fusion for proximitydependent protein labeling.34 APEX is able to oxidize phenol derivatives (such as phenol−biotin) to phenoxyl radicals that covalently react with electron-rich amino acids such as Tyr and Trp in proteins.34 Because phenoxyl radicals are short-lived (
