Identification of a novel interaction between Theileria Prohibitin (TaPHB-1) and bovine RUVBL-1

Bovine tropical theileriosis causes huge economic loss worldwide. It is a tick borne disease of bovine caused by the parasite Theileria annulata. T. annulata is an intracellular parasite that belongs to the phylum Apicomplexa. The sporozoites of T. annulata are released by the tick into the bloodstream of the host during the blood meal that invades bovine B cells, macrophages, or monocytes. This infection leads to the transformation of the host cells and brings cancer-like phenotype in the host cells. The parasite proteins play a vital role in the transformation of the host cell. However, the parasite factors involved in the host cell transformation are not well explored. Previously, TaPIN1, a peptidyl-prolyl isomerase of T. annulata, was shown to be secreted to the host cytosol and play a role in the host cell transformation. The present study was carried out to explore the parasite-host interactions that may play an important role in the host cell transformation. We identified the parasite proteins that are expressed in the schizont stage with a signal peptide. We narrow down our search to a parasite prohibitin. The in silico analysis of T. annulata prohibitin (TA04375, TaPHB-1) showed that TaPHB-1 shares homology with the mammalian prohibitin 1. With the localization experiments, we confirmed that TaPHB-1 is exported to the parasite surface and also to the host cell cytosol. Further, we observed that the localization of host prohibitin differs in the parasite-infected cells and could not be reverted back by the elimination of the parasite in the infected cells. We found through the yeast-two-hybrid studies that bovine RUVBL1 (BoRUVBL-1) interacts with TaPHB-1. The interaction between BoRUVBL1 and TaPHB-1 was predominantly observed on the parasite surface in the infected bovine cells. The interaction was further confirmed with immunoprecipitation and LC-MS/MS analysis. Further, the LC-MS/MS based TaPHB-1 interactome study reveals that it interacts with proteins that regulate actin cytoskeleton organization, protein folding, mRNA processing, and metabolic processes. Our finding suggests that the parasite releases prohibitin protein into the cytoplasm of the host cell where it interacts with the host RUVBL-1. This finding has implications not only in the understanding of Theileria parasite biology in greater depth but also in the cancer biology where previously differential localization of prohibitin proteins was observed but its interaction partner was not known. Author summary Theileria annulata, an apicomplexan, is a unique parasite which can transform host leucocytes. This parasite uses this strategy for its own multiplication. The cells infected with this parasite, when treated with buparvaquone, an anti-theilerial drug, cannot survive without the parasite. This observation suggests that the parasite derived factors are required to maintain the cancerous phenotype of the host cell. We mined the parasite proteome to find out the proteins with signal sequence that may be secreted to the host cell cytosol and being expressed in the schizont stage. The parasite prohibitin (TaPHB-1) chosen for this study was found to be secreted to the host cytoplasm and on the parasite surface. Interestingly, we observed a noticeable change in the localization of the host prohibitin in the parasite infected cells. The host prohibitin that is normally localized to the mitochondria in the uninfected cells was observed in the host cell nucleus similar to the cancerous cells. Since the parasite protein is exported to the host cytoplasm we looked for its interacting partner. We performed yeast-two-hybrid screening with TaPHB-1 with in-house prepared the cDNA library of the infected bovine leucocytes. We identified bovine RUVBL1 as the interacting partner of TaPHB-1. Interestingly, the interaction between parasite prohibitin and bovine RUVBL1 was observed on the parasite surface. Further, analysis of the parasite prohibitin interactome in the infected cells shows that it might be involved with those proteins which regulate actin cytoskeleton organization, protein folding, mRNA processing and metabolic process. Since parasite infected cells have cancer like phenotype, the identification of this novel interaction will open up new avenues not only in the arena of parasite biology but also in the domain of cancer biology.


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Tropical theileriosis or Mediterranean fever is a tick-borne haemoprotozoan disease caused 73 by Theileria annulata, an intracellular parasite. Tropical theileriosis is geographically distributed 74 in various countries in Central Asia, Southern Europe, North Africa and Middle East. T. annulata 75 is transmitted by Hyalomma anatolicum. It causes disease in Bos taurus and Bubalus bubalis (1). 76 The symptoms of the disease include fever, enlarged peripheral lymph nodes, anemia, decline in which are involved in cellular processes like microtubule organization, DNA repair and cell 105 apoptosis (11). T. annulata cyclophilin1 interacts with host cell MED21 which is normally 106 involved in regulating the transcription of RNA polymerase II-dependent genes but knockdown of 107 MED21 in T. annulata-infected leucocytes had no effect on NF-κB signaling (12). T. annulata 108 surface protein (TaSP) interacts with host microtubulin (13). During host cell mitosis TaSP co-109 localizes and interacts with the spindle poles, which suggests that this interaction has a potential 110 role in parasite distribution into the host cells (13). Also, TaSP is phosphorylated by the host cell 111 kinase CDK1 which plays a crucial role in cell division (14). 112 Prohibitins are highly conserved proteins found in all eukaryotes. They belong to 113 stomatin/prohibitin/flotillin/HfIK/C (SPFH) family. They play a significant role in transcription, interacts with the host HSP70 present on macrophage surface which may help the parasite to 119 escape invasion by host macrophages (17). In the case of Plasmodium berghei, prohibitin regulates 120 mitochondrial membrane polarity and prohibitin deficient parasite causes mitochondrial 121 depolarization in the mosquito vector, which, in turn, leads to a block in transmission (18). The 122 role of prohibitin in the T. annulata parasite is unknown. However, previously it was speculated 123 that prohibitin may have a role in transformation of the host cell (19) . In this study, we 124 characterized the prohibitin of T. annulata and identified its interacting partner.

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Prohibitins are highly conserved proteins 127 The screening of T. annulata proteome for proteins possessing a signal peptide using SignalP3.0 128 led to the identification of 438 proteins (S1 sheet). Forty nine out of these 438 proteins were found 129 to be expressed in the schizont stage based on the available experimental T. annulata schizont 130 proteome data (20,21). Only 25 of these 49 proteins were non-hypothetical (S1 Fig and S1 sheet).

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Our analysis is consistent with the previously published data (19). We selected prohibitin(s) out of 132 these 25 proteins for further analysis. The phylogenetic analysis of prohibitin proteins suggests 133 that the prohibitins of phylum Apicomplexa branch away from the higher eukaryotes such as Bos 134 taurus and Homo sapiens (Fig 1A). Among apicomplexans, the T. annulata prohibitins are closely 135 related to Babesia bovis and distantly related to Cryptosporidium parvum. We also observed the 136 presence of an additional prohibitin (PHB-3) in apicomplexan parasites except in Cryptosporidium 137 parvum. Based on the phylogenetic tree analysis the three putative prohibitins of T. annulata were 138 named as; TA04375 (TaPHB-1), TA19320 (TaPHB-2) and TA08975 ((TaPHB-3) prohibitin like 139 protein). The multiple sequence alignment and the identity matrix of these three prohibitins of T. 140 annulata suggest that TaPHB-3 is the most divergent prohibitin (Fig S2A and S2B). The 141 subcellular localization prediction by the CELLO server of three putative prohibitin proteins of T. 142 annulata suggests that TaPHB-1 possesses a putative signal sequence that could localize this 143 protein to the cytoplasm of the host while other two prohibitins were predicted to be localized in 144 the mitochondria/chloroplast (Fig 1B). Thus, we hypothesized that if TaPHB-1 protein is 145 transported to the cytoplasm of the host then it could interact with the host protein(s) and may help 146 in the transformation of the host cell. Hence, we selected TaPHB-1 for further analysis.   All our efforts to express TaPHB-1 as a soluble protein remained unsuccessful. Thus, we 160 purified recombinant His-tagged TaPHB-1 under denatured condition (Fig 2A). The mice sera 161 raised against denatured TaPHB-1 detect recombinant His-tagged TaPHB-1 in western blot 162 analysis (Fig 2A). The mice sera against TaPHB-1 were affinity purified against recombinant His-163 tagged TaPHB-1. The expected size of TaPHB-1 is 30kDa. In the western blot analysis, the affinity 164 purified antibodies against TaPHB-1 could recognize TaPHB-1 protein at a higher molecular size 165 (~60kDa) in the lysates of Ana2014 cells (Fig 2B). The LC-MS/MS analysis of recombinant 166 TaPHB-1 confirmed that the protein used for raising antibodies was TaPHB-1 only (S4 Fig). 167 Further, the affinity purified antibodies against TaPHB-1 could not recognize any protein in the 168 BoMac cell lysate ( Fig 2B). As the signal was observed only from Ana2014 cell lysate in western 169 blotting, we concluded that the affinity purified antibodies were specific to TaPHB1 and did not 170 cross react with BoPHB-1.

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TaPHB-1 is exported out to the host cytoplasm by the parasite 172 As mentioned earlier, the signal sequence analysis of the prohibitins of T. annulata suggests that 173 TaPHB-1 has a signal sequence that may export this protein to the host cytoplasm. We performed 174 confocal microscopy with affinity purified antibodies against TaPHB-1 to localize this protein in 175 the infected cells. The parasite surface was labelled with TaSP antibodies. The TaPHB-1 protein 176 was found to be localized in the host cell cytosol (Fig 2C). We also observed TaPHB-1 protein on 177 the parasite surface ( Fig 2C). We could not observe any signal from the BoMac cells when probed 178 with affinity purified TaPHB-1 antibodies. This suggests that the TaPHB-1 protein can cross the 179 parasite cell membrane to reach to the host cytoplasm.  182 We used commercial antibodies against PHB-1 protein (anti-PHB-1) for localization of host PHB-183 1. First, we analyzed the ability of the anti-PHB-1 to distinguish host PHB-1 and TaPHB-1. We 184 observed that the anti-PHB-1 could recognize host PHB-1 as a band at 30kDa was observed in 185 western blot analysis with both BoMac cells and Ana 2014 cells ( Fig 3A). Further, to confirm that 186 anti-PHB-1 could recognize TaPHB-1, we probed recombinant TaPHB-1 with anti-PHB-1 187 antibodies. Anti-PHB-1 was able to recognize the parasite PHB-1 also ( Fig 3B). Thus, we 188 concluded that anti-PHB-1 recognizes both parasite and host PHB-1. We labelled host PHB-1 with 189 PHB-1 antibodies for confocal microscopy while we used TOMM antibodies, a mitochondrial 190 marker, to label the mitochondria. We observed that bovine prohibitin-1 was majorly localized 191 with in the host cell nucleus in Ana2014 cells (Fig 3C). We could not observe any co-localization 192 of PHB-1 with TOMM suggesting that bovine PHB-1 was not present in the mitochondria (Fig   193   3C) of Ana2014 cells. However, in the uninfected cells, i.e., BoMac cells, the bovine PHB-1 was 194 majorly observed in the cytosol which co-localizes with TOMM ( Fig 3C) suggesting that in the 195 uninfected cells bovine PHB-1 is present in the mitochondria. Thus, we conclude that the T. 196 annulata infection leads to the export of host PHB-1 to the host cell nucleus from the mitochondria.

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Further, we observed that the exported TaPHB-1 was present in the host cytosol but did not localize

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Gal/AbA agar plate after 3 days of incubation confirmed that there was no auto-activation of 232 reporter genes by TaPHB-1 in Y2HGold yeast cells ( Fig 5B). Also, the size of the colonies on 233 SD/-Trp agar plate were observed to be normal which suggests that TaPHB-1 has no toxic effect 234 on Y2HGold yeast cells.

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Yeast two-hybrid library screening led to the identification of bovine 236 RUVBL1 as an interacting partner of TaPHB-1 237 The yeast two-hybrid library screening was performed as mentioned in the methods section. Sixty   251 The interaction of TaPHB-1 and bovine RUVBL1 (Bo-RUVBL1) was reconfirmed by the analysis 252 of interaction of these two proteins by co-expression in vitro. We used pETDuet-1 vector to co- TaPHB-1 led to the identification of S-tagged Bo-RUVBL1 as a co-eluate which confirmed the 258 interaction between TaPHB-1 and bovine RUVBL1 (Fig 6A).

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As there are no reports of interaction between TaPHB-1 and bovine RUVBL1 we 275 speculated that the interaction between these two proteins might involve other interactions also. In 276 order to identify the interactome, we performed co-immunoprecipitation using affinity purified 277 TaPHB-1 antibodies against the Ana2014 cell lysate. The bovine RUVBL1 was observed in the 278 eluate through western blotting which confirms the interaction between TaPHB-1 and bovine 279 RUVBL1 ( Fig 8A). Pre-immune sera was used as negative control in co-immunoprecipitation. 280 Further, the eluate of co-immunoprecipitation was analyzed by mass spectrometry. The mass 281 spectrometry led to the identification of 45 bovine proteins and 2 parasite proteins with more than 282 ten unique peptides with less than 0.05 false discovery rate (Fig 8B, S2 sheet). We confirmed the 283 result of LC-MS/MS by western blot analysis of one of the proteins, RPL7A, identified in the mass 284 spectrometry analysis (Fig 8C). The interactome analysis of TaPHB-1 by STRING suggests that 285 the proteins involved in protein folding, cellular metabolic process, mRNA processing, catalytic 286 activity, transporter activity and actin cytoskeleton organization are involved in the TaPHB-1 287 interactome ( Fig 8B).   The observation that the TaPHB-1 is secreted to the host cytoplasm led to speculate that it 326 should be involved in protein-protein interaction with host protein(s). The yeast two-hybrid system 327 is a well-established method for screening of interacting partners of interested protein (bait protein) 328 against a library of proteins (prey library) (31). Thus we prepared a prey library with the cDNA of has shown that RUVBL-1 can interact with PHB-1. The present study is the first ever report to 348 identify the interactions between bovine RUVBL1 and T. annulata prohibitin (TaPHB-1). Further, 349 LC-MS/MS analysis deciphered the interactome of these two proteins. We speculate that 350 TaHSP70 is the link between bovine RUVBL1 and T. annulata prohibitin (TaPHB-1).

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In conclusion, we showed in the present study that the localization of bovine PHB-1 in 352 Theileria infected bovine leucocytes changes from the host mitochondria to the host nucleus, while 353 parasite prohibitin (TaPHB-1) is exported out to the host cytoplasm and is also present on parasite 354 surface. Further, we demonstrated that TaPHB-1 interacts with bovine RUVBL1. The TaPHB-1 355 interactome analysis suggests that TaPHB-1 involves with those proteins which regulate actin 356 cytoskeleton organization, protein folding, mRNA processing and metabolic process. We 357 speculate that the interaction between PHB-1 and RUVBL1 will have implications not only in the 358 arena of parasite biology but also in the field of cancer biology.  In-silico analysis of TA04375 (TaPHB-1) 394 The SignalP 3.0 server was used for searching proteins with signal sequence in the proteome of T.  The total RNA was extracted from Ana2014 cells using TRIzol® reagent (Invitrogen) following 439 the manufacturer's protocol. One microgram of total RNA after DNase I (Invitrogen) treatment 440 was used for cDNA library preparation using Make Your Own "Mate & Plate™" Library System 441 (TaKaRa). All the procedures were followed according to the manual provided. Briefly, the first-442 strand cDNA was synthesized using SMART MMLV reverse transcriptase using CDS III primer 443 also SMART oligo (S1 table), which finally results in known sequences at both ends of cDNA.

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Next, the cDNA was amplified by long distance PCR amplification using Advantage polymerase 445 mix (TaKaRa). The amplified ds cDNA was purified using CHROMA SPIN TE-400 column 446 which retains and traps the DNA molecules of less than 200 bp size. The ds cDNA after CHROMA 447 SPIN TE-400 column purification was used for library construction by in vivo homologous 448 recombination in yeast. The purified ds cDNA and SmaI linearized pGADT7-Rec were co-449 transformed into Y187 yeast strain using Yeastmaker Yeast Transformation System 2 (TaKaRa).

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The transformation mix was spread on to the SD/-Leu agar plates and incubated at 30ºC for 3-4 451 days. All the transformants were harvested and pooled in freezing medium (YPDA/25% glycerol), 452 aliquoted and stored at -80ºC. Also, the number of independent clones was determined.

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The total RNA was extracted from Ana2014 cells as mentioned above. The cDNA was synthesized 455 using SuperScript™ First-Strand Synthesis System (Invitrogen) following the manufacturer's 456 protocol. Theileria annulata putative prohibitin (TA04375, GenBank accession no.

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XM_949999.1, TaPHB-1) was amplified by PCR using its specific primers: TA04375-F and 458 TA04375-R (S1 table). The PCR product was purified using PCR purification kit (Macherey- interactions were sequenced. The prey plasmid was sequenced using the respective primers which 511 were used for PCR. The sequence obtained was analyzed using BLAST tool of NCBI.

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Both the clones were confirmed by double digestion and sequencing as well. The recombinant 537 constructs were transfected into HEK293 cells either individually or together using 538 Lipofectamine® 3000 reagent (Invitrogen) following the instructions provided. After 24 h of 539 transfection, the fluorescence images of the HEK293 cells were captured using Axio Observer 7 540 microscope Apotome 2 (Carl Zeiss). The coding sequence of TaPHB-1 was PCR amplified from Ana2014 cells cDNA using specific 544 primers (S1 table). The full length TA04375 was digested with NdeI and NotI, and cloned into the The antibodies specific to TaPHB-1 were purified from mice sera by immunoblotting as described 561 previously (51). Briefly, 500 μg of the purified TaPHB-1 protein was separated on SDS-PAGE gel 562 and, transferred onto PVDF membrane. The membrane was blocked with blocking buffer (5% 563 BSA in TBST) for 2 h at room temperature. The protein band was stained by Ponceau S staining, 564 the protein band was cut and washed with TBST until the membrane was destained. The membrane 565 strip was incubated with 500 µl of mice sera for overnight at 4ºC. The strip was washed thrice with 566 TBST. Finally, the bound antibodies were eluted in 2 M glycine (pH-2.5) and the eluate was 567 immediately neutralized with 1M Tris-HCl buffer (pH-9.0). The Co-IP eluate was processed for LC-MS/MS analysis by in-solution trypsin digestion method.

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The eluate was subjected to reduction with 20 mM DTT at 56ºC for 1 h. Next, alkylation was 580 carried out with 20 mM iodoacetamide (IAA) for 1 h at the room temperature in dark. The proteins 581 were digested with trypsin/LysC (1:30 w/w) overnight at 37ºC. The digestion reaction was stopped 582 by adding 0.1% trifluoroacetic acid. The digested peptides were purified with C-18 spin columns.

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The purified peptides were concentrated by a vacuum evaporator and finally re-suspended in 0.1% 584 formic acid. The peptides were analyzed by Q Exactive HF-Orbitrap mass spectrometer 585 (Thermofisher Scientific) coupled with Ultimate 3000 RSLCnano LC system (Thermofisher 586 Scientific). The peptides were injected into a reverse phase C-18 column (PepMap RSLC C18, 2 587 µm, 100 Å, 75 µm X 50 cm, Thermofisher Scientific) and separated by a gradient flow of solvent 588 B (0.1% formic acid in 80/20 acetonitrile/water) from 5% to 90% in 180 mins. A parent ion scan 589 was performed with a scan range of 375 to 1600 m/z with a resolution of 60,000. The top 25 intense 590 peaks were fragmented by higher energy collision induced dissociation (HCD) fragmentation in 591 MS/MS with the resolution of 15,000. The obtained spectra was analyzed using Thermo Proteome 592 Discoverer software version 2.5. The database search was carried out using SEQUEST HT with 593 the following parameters: maximum missed cleavage sites of 2, fragment mass tolerance of 0.02 594 kDa and precursor mass tolerance of 10 ppm. Fixed modification, carbamidomethylation of Cys 595 (Cysteine) and dynamic modification, oxidation of Met (Methionine) were added. The resulting 596 peptides were validated using percolator at a 5% False Discovery Rate (FDR), which validates 597 using PEP (Psterior Error Probability) and q value. The interactome analysis was performed using 598 STRING tool and Cytoscape ver 3.8.2 software.