A VPS15-like kinase regulates apicoplast biogenesis and autophagy by promoting PI3P generation in Toxoplasma gondii

Phosphoinositides are important second messengers that regulate key cellular processes in eukaryotes. While it is known that a single phosphoinositol-3 kinase (PI3K) catalyses the formation of 3’-phosphorylated phosphoinositides (PIPs) in apicomplexan parasites like Plasmodium and Toxoplasma, how its activity and PI3P formation is regulated has remained unknown. Present studies involving a unique Vps15 like protein (TgVPS15) in Toxoplasma gondii provides insight into the regulation of phosphatidyl-3-phosphate (PI3P) generation and unravels a novel pathway that regulates parasite development. Detailed investigations suggested that TgVPS15 regulates PI3P formation in Toxoplasma gondii, which is important for the inheritance of the apicoplast-a plastid like organelle present in most apicomplexans and parasite replication. Interestingly, TgVPS15 also regulates autophagy in T. gondii under nutrient-limiting conditions as it promotes autophagosome formation. For both these processes, TgVPS15 uses PI3P-binding protein TgATG18 and regulates trafficking and conjugation of TgATG8 to the apicoplast and autophagosomes, which is important for biogenesis of these organelles. TgVPS15 has a protein kinase domain but lacks several key residues conserved in conventional protein kinases. Interestingly, two critical residues in its active site are important for PI3P formation and parasitic functions of this kinase. Collectively, these studies unravel a signalling cascade involving TgVPS15, a novel effector of PI3-kinase in T. gondii and possibly other Apicomplexa, that regulate critical processes like apicoplast biogenesis and autophagy.

. PI3P typically executes its cellular functions via specific PI3P-68 binding domain containing proteins like FYVE or PX domains (Lemmon, 2003). Several PIP 69 binding proteins have been identified in apicomplexa but function of most of these has 70 remained unknown (Wengelnik et al, 2018). Recently, we demonstrated that autophagy 71 related protein ATG18, which interacts with PI3P, is critical for apicoplast biogensis (Bansal 72 et al, 2017). Autophagy related protein 8 (ATG8), which is localized to the apicoplast, in 73 steady state conditions is indispensable for apicoplast biogenesis (Leveque et al, 2015). 74 These proteins are typically associated with autophagy in yeast and mammals. Autophagy is a  In our efforts to understand the mechanisms involved in the generation of PIPs, which are not 88 understood in apicomplexan parasites, we investigated the role of Vps15 like protein from 89 Toxoplasma. Vps15 has been reported to increase the activity of Vps34 like class III PI3-90 kinases in yeast and other organisms (Stack & Emr, 1994). While it is classified as a 91 "pseudokinase", the yeast homologue has been reported to exhibit autophosphorylation 92 (Stack & Emr, 1994) but no substrates of this kinase have been reported. However, the 93 human homologue p150 does not exhibit autophosphorylation (Panaretou et al, 1997). While 94 it is unclear if it regulates PI3Ks by phosphorylation, Vps15 is part of two major multi-95 protein complexes comprising of Vps34 (PI3K) and several other proteins like ATG14L, 96 Beclin1 in Complex I and VPS38/UVRAG replaces ATG14 in Complex II. Complex I is 97 important for autophagosome formation as it promotes PI3P generation at the phagophore. 98 Complex II regulates various other cellular processes like endosome sorting, lysosome   111 We were interested in understanding the regulation of PI3P in apicomplexan parasites 112 Toxoplasma and Plasmodium. In our quest to identify putative Vps15 homologues, in silico 113 sequence based search revealed VPS15 orthologues in Toxoplasma gondii (ToxoDB ID: 114 TGGT1_310190) and a P. falciparum (PlasmoDB ID: PF3D7_0823000). These observations 115 were consistent with previous studies, which also indicated the presence of these genes as 116 putative VPS15 orthologues in these parasites (Navale et al, 2014) (Smith et al, 2021). 117 However, VPS15 has not been analyzed and characterized and its function in parasite biology 118 has remained unknown. The yeast ScVps15 has a kinase domain at the N-terminus followed 119 by Armadillo like helical repeats (HEAT) in the middle that are connected to a six WD40 120 repeats at the C-terminus, which form a -propeller structure (Backer, 2016;Rostislavleva et 121 al, 2015). The domain analysis of TgVPS15 suggested that it possesses the kinase and the 122 Helical or HEAT domain (Fig. 1A). However, various algorithms could at best predict two 123 highly degenerate WD40 repeats with very low scores in the case of TgVPS15. Therefore, it 124 is possible that TgVPS15 does not contain WD40 repeats. Alternatively, it is possible that the 125 corresponding C-terminal region may lack sequence homology but may fold into -propeller 126 structure observed in the yeast VPS15. Detailed structural analysis will only be able to test 127 these possibilities. In the case of PfVPS15, the WD40 repeats could not be predicted with 128 certainty. Moreover, PfVPS15 is a much shorter protein and ends with the Helical or HEAT 129 domain (Fig. 1A). These observations suggested that VPS15 in these parasites has a highly 130 divergent domain arrangement when compared to the yeast and mammalian counterparts.

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VPS15 from yeast and mammals has been classified as a pseudokinase as it lacks key 132 catalytic residues present in protein kinases (Fig. 1B) (Herman et al, 1991;Stack et al, 1993). 133 We analyzed the sequence of the kinase domain of Tg/PfVPS15 by comparing it with that of  (Johnson et al, 1996;Nolen et al, 2004) is also missing from both 139 ScVPS15 and TgVPS15. However, the catalytic aspartate D216 in TgVPS15 is conserved in 140 these kinases; c. The aspartate in the DFG motif of the catalytic loop of protein kinases is 141 critical for chelating divalent cations like Mn 2+ or Mg 2+ and is conserved in TgVPS15 (D234) 142 and ScVPS15, whereas the following phenylalanine is absent in TgVPS15 (Fig. 1C); c. APE 143 motif, which is part of P+1 loop (Nolen et al, 2004), is well conserved in these kinases. The  In order to investigate the function of TgVPS15, an inducible knockdown was generated in T. 154 gondii, which was achieved by using a tetracycline regulated transactivator system (Salamun     203 Our results suggested that defects in parasite replication were observed only in second and 204 subsequent cycle after TgVPS15 depletion (Fig. 2 E). Such delayed death phenotype in 205 apicomplexans like Toxoplasma and Plasmodium has been associated with defective parasite 206 division after the first lytic cycle and is attributed to the loss of apicoplast (Fichera & Roos,207 1997). Therefore, we analyzed the status of apicoplast formation, which is inherited from the 208 mother to budding daughter cells during endodyogeny, by performing IFA for apicoplast 209 resident protein Cpn60. Cpn60 is a nuclear encoded protein and contains a N-terminal 210 apicoplast targeting signal, which is cleaved after its trafficking to the apicoplast. A 211 significant loss of apicoplast was progressively observed after ATc treatment, which was 212 indicated by either diffused or missing Cpn60 (Fig. 3A, B). Furthermore, the mature form of 213 Cpn60-obtained after processing of its apicoplast targeting signal at the apicoplast was 214 reduced and predominantly the unprocessed Cpn60 was observed in TgVPS15 depleted 215 parasites (Fig. 3C). Consistent with these observations, apicoplast genome replication was 216 also significantly impaired upon TgVPS15 depletion (Fig. 3D). Collectively, these data 217 established a role of TgVPS15 in apicoplast biogenesis or inheritance.  TgATG8 localization in remaining parasites-that possessed the apicoplast-was determined.

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More than half of these parasites did not exhibit TgATG8 at the apicoplast (Fig. 4A). In order 234 to be functionally active, TgATG8 needs to be conjugated to phosphatidylethanolamine (PE), 235 which is critical for its localization at the apicoplast membrane and subsequent segregation .  286 As mentioned above, the interaction of TgATG18 with PI3P and PI(3,5)P2 has been 287 demonstrated (Bansal et al, 2017). However, its role in autophagy has remained unknown.

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Since TgVPS15 regulates PI3P formation, we tested the involvement of TgATG18 in 289 autophagy. For this purpose, TgATG18 was HA-tagged at the C-terminus in TgVPS15-iKD TgATG18 were in distinct locations in complete medium, nutrient deprivation resulted in 302 formation of bigger puncta with TgVPS15 staining, which was also the case with TgATG18. 303 Interestingly, in almost 40% of parasites TgVPS15 and TgATG18 co-localized in these 304 puncta, which as described above may be a pre-autophagosome compartment (Fig. 5E,F).  324 As mentioned above (Fig. 1), several key elements present in proteins kinases are either 325 absent from TgVPS15 or are divergent. Therefore, it may be a pseudokinase like ScVPS15. 326 However, it is known that ScVPS15 autophosphorylates and some of the key residues may be 327 important for its function (Herman et al, 1991). We investigated if TgVPS15 activity is 328 important for its parasitic functions. Our attempts to perform kinase assays with the kinase 329 domain of TgVPS15 (TgVPS15-KD) in E.Coli were inconsistent as expression of the 330 recombinant was poor. Therefore, we used an independent approach to address this issue by 331 ectopically expressing either WT TgVPS15 or two of its mutants. These mutants were 332 generated by replacing D216 with A, which is part of the divergent HGD motif and may act 333 as a catalytic base (Nolen et al, 2004). A E268A mutant of the conserved APE motif , which 334 is important for key interactions necessary for stabilization of the activation loop (Nolen et al,335 2004), was also made. D216A and E268A mutants were ectopically expressed in TgVPS15-336 iKD with a Ty-tag as described above for the WT TgVPS15 complementation line 337 (TgVPS15-iC WT ) (Supp. Fig. S5).

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First, the role of these residues in the ability of TgVPS15 to promote PI3P was assessed. For 339 this purpose, the 2xFYVE reporter construct described above (Fig. 2G) was expressed in 340 these parasite lines. The loss of PI3P at the apicoplast in TgVPS15-iKD parasites upon ATc 341 addition (~15% parasites with PI3P at apicoplast) was efficiently reverted in TgVPS15-iC 342 parasites (~44%) in which WT TgVPS15 were complemented. In contrast, both D216A and 343 E264A mutants (~15%) were unable to restore PI3P formation efficiently. Based on these 344 data, it is reasonable to conclude that TgVPS15 catalytic activity may be critical for the 345 regulation of TgPI3K and promote the formation of PI3P in the parasite. 346 Next, the role of TgVPS15 activity on its parasitic functions was investigated. As described 347 above, the complementation with WT TgVPS15 prevented any defects in parasite replication 348 that were observed in TgVPS15-iKD parasites upon ATc addition. However, the expression 349 of D216A as well as E268A mutants of TgVPS15 did not prevent the arrest in parasite 350 replication (Fig. 7B). Next, we assessed apicoplast biogenesis in these parasite lines. As 351 reported above (Fig. 3A-B), only ~20% of ATc treated TgVPS15-iKD parasites exhibited the 352 presence of apicoplast, which significantly increased (~40%) when WT TgVPS15 was 353 ectopically expressed (TgVPS15-iC WT ). In contrast, parasites expressing the D216A and 354 E268A in TgVPS15-iKD parasites also exhibited only ~20% parasites with an apicoplast 355 (Fig. 7C, Supp. Fig. S6). These data suggested that D216 and E268 were critical for 356 TgVPS15 function in parasite replication, which is dependent on its ability to regulate 357 apicoplast biogenesis. 358 We also investigated the involvement of these TgVPS15 active site residues in autophagy by 359 using the above mentioned parasite lines. A significant increase in autophagosome formation 360 was observed in TgVPS15-iC parasites was observed when compared to ATc-treated 361 TgVPS15-iKD parasites. In contrast, the expression of both D216A and E268A mutants did 362 not cause a similar increase. These data suggested that catalytic site residues play a critical 363 role in autophagy as well (Fig. 7D, Supp. Fig. S7) and hint at the possible involvement of 364 TgVPS15 activity even though it is not a conventional kinase. unknown. In addition, a putative PIKfyve kinase homologue, which is likely to be involved 379 in PI(3,5)P2 formation is coded only in Toxoplasma and is also involved in apicoplast 380 homoeostasis (Daher et al, 2015). In the present study, we demonstrate that TgVPS15 is a 381 regulator of TgPI3K activity as its abrogation results in depletion of PI3P from the parasite 382 (Fig. 2G). Although biochemical characterization of TgPIKfyve has not been done, it is likely 383 that it may use PI3P as a substrate to convert it to PI(3,5)P2 like the yeast enzyme. Therefore, 384 it is very likely that TgVPS15 may also regulate PI(3,5)P2 generation as PI3P serves as a 385 precursor for PI(3,5)P2 formation, which we could not test in the present study. Typically,

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VPS15 from most organisms is classified as a pseudokinase due to the following reasons: a. 387 several key motifs that are absent from TgVPS15 kinase domain when compared to typical 388 protein kinases (Fig. 1B). For example, the glycine rich loop, HRD and DFG motifs are either   (Herman et al, 1991). It is possible that TgVPS15 406 may be autophosphorylated, which may promote its association with TgVPS34 and other 407 regulatory partners, which has been reported for ScVPS15 (Stack et al, 1995).

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TgVPS15 is present in vesicular structures in the parasite which may be suggestive of its role 409 in membrane/protein trafficking (Fig. 2B, 4C). Its depletion resulted in a loss of the 410 apicoplast (Fig. 3) and explained the defects in parasite replication observed after the first 411 replication cycle (Fig. 2E), which is typical of "delayed death" observed due to the loss of the formation and autophagy (Fig. 8).

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These studies unravel a dual role of TgVPS15 in Toxoplasma gondii and both are dependent 455 on its ability to facilitate PI3P formation; it regulates apicoplast inheritance during parasite 456 lytic cycle and autophagy under nutrient-limiting conditions (Fig. 8).    Table S1.   vector. Subsequently, an amplicon (I) from the kinase domain portion was amplified using 508 primers 33/30-with an NcoI site in the reverse primer and using the pET construct described 509 above containing mutant TgVPS15. Another amplicon (II) was generated using primers 29/34 510 from amplified from pCT-TyTgVPS15-iC WT in which NcoI site was introduced at the 5' end.

511
The two fragments (I and II) were assembled using Gibson assembly kit after NcoI digestion 512 of the pCT-TgVPS15-iC WT described above. The replacement of the WT TgVPS15 with the 513 desired mutant fragment from this construct was confirmed by DNA sequencing. 50μg of the 514 circular plasmid was digested using PmeI (linearized) and transfected in TgVPS15-iKD and 515 selection was done using 20μM chloramphenicol.

545
Transfected parasites were treated with pyrimethamine and phleomycin as described and 546 followed by limiting dilution cloning.  Freshly egressed T. gondii tachyzoites were used for the preparation of lysate, which was 566 done in a buffer containing 10 mM Tris (pH 7.5), 100 mM NaCl, 5 mM EDTA, 1% Triton X-567 100, and Complete protease inhibitor mixture (Roche Applied Science) or in 2% SDS. SDS-568 PAGE was performed, followed by transfer of proteins to nitrocellulose membranes.

569
Immunoblotting was performed using various primary antibodies and antisera, and HRP          E. Myc-TgVPS15-iKD/TgATG18-HA extracellular tachyzoites were incubated in complete 713 medium (0h) or HBSS (8h). Subsequently, parasites were fixed to perform IFA using anti-714 HA and anti-myc antibody, which revealed that TgATG18-HA as well as TgVPS15 715 localization changed to a larger puncta in HBSS. In some cases, they co-localized to these 716 larger puncta (arrows).  A. DD-GFP-2xFYVE was expressed in TgVPS15-iKD parasites or these parasites 735 complemented with a copy of Ty-tagged WT TgVPS15, or its D216A or E268A mutants.