Autophagy Protein Atg3 is Essential for Maintaining Mitochondrial Integrity and for Normal Intracellular Development of Toxoplasma gondii Tachyzoites
[['Proteolysis is very important to eukaryotic cells and occurs at a considerable constitutive rate .', 'Base line degradation regulates the levels of numerous proteins and removes misfolded proteins .', 'Mechanistically , this process can be separated into two main pathways: one pathway mediated by the proteasome and the other pathway by the lysosome [1] .', 'The proteasome relies on the ubiquitin system for selection of target proteins and plays a major role in the rapid degradation of short-lived proteins as well as abnormal proteins .', 'The lysosome , which represents the terminal compartment of the endosomal pathway , is a membrane-bound organelle that contains a diverse array of hydrolases for the degradation of plasma membrane proteins and endocytosed extracellular proteins .', 'Lysosomes are also involved in bulk degradation of cytoplasmic components , such as long-lived cytosolic proteins and organelles , and this is achieved through a process called autophagy .', 'Autophagy has been divided into several classes of pathways , such as macroautophagy , microautophagy , and chaperone-mediated autophagy , but macroautophagy has been studied most extensively and we will refer to this specific form of the process as “autophagy” in this manuscript for simplicity .', 'Autophagy is evolutionarily conserved in eukaryotes from yeast to mammals and has important roles in various cellular functions [2] .', 'The basal role of autophagy is in turnover and recycling of cellular constituents; these housekeeping functions include the elimination of defective proteins and organelles and the prevention of abnormal protein aggregates accumulation .', 'Autophagy also plays an important role in organelles and proteins ( but also lipids ) recycling under nutrient starvation conditions , as a nutrient source for the cell .', 'Finally , there are pleiotropic and more specialised roles for different eukaryotic cells ( in particular in mammals ) including cellular remodelling during differentiation and development , regulation of cell longevity and programmed cell death , elimination of invading pathogens and providing antigens to the immune system [2] , [3] .', 'In the autophagic process , cytosolic components are sequestered in a double-membrane vesicle known as the autophagosome .', 'The outer membrane of the autophagosome will then fuse with the lysosomal compartment to deliver the inner contents of the vesicle , which will be subsequently degraded .', 'Although autophagy has been initially identified in mammalian cells , the characterisation of the molecular machinery involved in this cellular process has been mainly done in yeast .', 'Due to the ease of genetic analyses in the yeast system , screening for mutants unable to survive nitrogen starvation , allowed the identification of more than thirty Atg genes involved in autophagy and the related cytoplasm to vacuole targeting pathway ( Cvt ) [4] .', 'Of these , some are only present in one organism and others represented by orthologues in different eukaryotic cell types .', 'Among all Atg proteins , Atg8 occupies a central position: it is essential to the process of autophagosome formation , especially for the membrane expansion step [5] , [6] and , possibly , the final membrane fusion steps [7] .', 'The protein is present as a soluble form in the cytosol of eukaryotic cells , and gets recruited to the autophagosomal membrane upon induction of autophagy .', 'Interestingly , the binding of Atg8 to the autophagosomal membrane involves two conjugation systems that resemble ubiquitin-conjugation systems [8] .', 'Toxoplasma gondii is an obligate intracellular protozoan parasite that is virtually able to infect all species of warm-blooded animals [9] .', 'T . gondii is a member of the phylum Apicomplexa , which also includes several other notable human pathogens such as Plasmodium and Cryptosporidium .', 'The secretory pathway of T . gondii is highly polarised and includes several unique organelles devoted to the invasion of the host cell ( the micronemes , rhoptries and dense granules ) [10] .', 'In addition the parasite harbours the apicoplast , a plastid-like organelle of endosymbiontic origin .', 'The micropore , a cytostome-like structure formed by the invagination of the plasma membrane , can be seen laterally [11] , which suggests the existence of an endocytic pathway within the parasite , although it has not been clearly defined at the molecular level .', 'Until very recently , the parasite was thought not to contain any morphological equivalent of “classic” lysosomes , as rhoptries appeared to be the only acidified organelles containing hydrolases in T . gondii [12] .', 'Two recent reports have nevertheless identified an acidified vacuole bearing a cathepsin-like peptidase in Toxoplasma tachyzoites that could fulfil that role [13] , [14] , but its precise physiological role remains to be characterised .', 'The autophagic pathway is even less well described in T . gondii , although it can reasonably be suspected to be involved in the survival of Apicomplexa , as well as for other parasites with complex life cycles .', 'For example , the importance of autophagy for the development and virulence of protozoan parasites such as the trypanosomatids has been shown previously [15] , [16] and recent data have shown an implication of autophagy in the cellular remodelling of Plasmodium liver stages [17] .', 'Here , we used the experimental opportunities afforded by Toxoplasma as a genetic model organism to investigate the autophagic machinery and assess its physiological role in the parasite .'], ['Many of the molecular components involved in autophagy have been identified in the budding yeast Saccharomyces cerevisiae .', 'We have thus used the known S . cerevisiae Atg protein sequences to identify T . gondii homologues through homology searches in the ToxoDB database ( [18] , Table S1 ) .', 'The core machinery for the assembly of the pre-autophagosomal structure appears to be present in T . gondii .', 'This includes the regulating kinase Atg1 , as well as proteins central to autophagosome formation itself , like Atg8 and its conjugating partners Atg7–Atg3 .', 'In several other eukaryotes , Atg8 requires the activity of two conjugation systems to bind the autophagosomal membrane: Atg7–Atg3 on the one hand and Atg5–Atg12 on the other ( see [8] , [19] for a review ) , the latter being dispensable in vitro [20] .', 'Interestingly , no clear orthologues of the members of the Atg5–Atg12 conjugation system were found in our homology search .', 'These are also absent from the genomes of several other parasitic protozoa [21]–[23] .', 'In Leishmania , distantly related proteins might have similar function [24] .', 'However , the orthologues of these Leishmania proteins cannot be found in the T . gondii genome .', 'Overall , this either suggests that the Atg5–Atg12 conjugation is not essential for autophagosome formation in T . gondii , or that Toxoplasma uses different ( possibly lineage-specific ) proteins to perform this task .', 'In fact , a significant part of the yeast Atg proteins seems to be missing from the T . gondii genome .', 'For instance , most of the Atg proteins involved in the Cvt pathway are not found in Toxoplasma .', 'This may not be surprising as this pathway is likely fungi-specific , and is also largely lacking in other eukaryotic systems that are clearly able to perform autophagy [19] .', 'The most reliable marker for autophagosomes is Atg8 ( or LC3 , its mammalian counterpart ) .', 'This protein is essential to autophagosome formation , and stays associated with the autophagosomal membrane from the early membrane recruitment step until the fusion with the lysosomal compartment [25] .', 'Hence , to further characterise the autophagic process , we raised a specific antibody against TgAtg8 ( TGME49_054120 , Table S1 ) .', 'Using this antibody on tachyzoite lysates , we detected by Western blot analysis a major protein band consistent with the predicted molecular weight of 14 kDa ( Figure 1 ) .', 'However , the rather weak signal that was observed suggested a low level of expression for endogenous TgAtg8 .', 'To observe the dynamics of autophagosomes formation we then cloned TgAtg8 in fusion with the green fluorescent protein ( GFP ) gene to generate a T . gondii cell line that stably expresses GFP-TgAtg8 in addition to its endogenous copy .', 'Stable clones of GFP-TgAtg8 transgenic parasites were obtained and showed no apparent difference in growth or morphology ( data not shown ) .', 'When GFP-TgAtg8 parasites were assessed by Western blot with anti-TgAtg8 and anti-GFP antibodies , both detected a fusion protein at the expected molecular weight of 42 kDa ( Figure 1 ) .', 'The strong tubulin promoter driving the expression of GFP-TgAtg8 showed an overexpression of ∼20 fold of the GFP-fused TgAtg8 compared with the native protein , which greatly facilitated its detection .', 'We studied the localisation of the GFP-TgAtg8 fusion protein by fluorescence microscopy on living tachyzoites .', 'In extracellular tachyzoites freshly released from host cells , the GFP-TgAtg8 signal was generally distributed throughout the cytoplasm and also occasionally found in one or more punctate vesicles of various sizes that could correspond to autophagic vesicles ( Figure 2A ) .', 'Using the anti-TgAtg8 antibody , both on the parental cell line and on the GFP-TgAtg8-expressing parasites , we verified by immunofluorescence assay ( IFA ) that GFP-TgAtg8 and anti-TgAtg8 labelled identical cellular compartments ( Figure S1 ) .', 'The GFP-TgAtg8 signal was convenient to follow; we thus conducted our subsequent microscopic observations on the GFP-TgAtg8 cell line .', 'To establish whether the observed structures are autophagic vesicles , we performed starvation experiment on GFP-TgAtg8-expressing extracellular parasites .', 'Free tachyzoites allow a more direct control of environmental conditions and nutrient access , compared with intracellular parasites sheltered within their host cells .', 'Strong induction through starvation is a hallmark of autophagosomes in many eukaryotic systems .', "To induce starvation , we incubated GFP-TgAtg8-expressing extracellular tachyzoites for increasing intervals of time in an isotonic solution devoid of amino acids ( Hank's balanced salt solution , HBSS ) .", 'We imaged the parasites and quantified our findings .', 'We noticed that the number of parasites bearing GFP-labelled puncta increased over the incubation time ( increasing from 15±3% to 79±8% after 8 hours , Figure 2B ) as did the number of puncta per parasite ( Figure S2A ) .', "As a control , incubation of the tachyzoites in complete Dulbecco's modified Eagle medium ( DMEM , supplemented with 10% fetal bovine serum ) for similar time periods induced no appearance of these puncta ( 17±2% of parasites bearing GFP-labelled puncta versus 21±2% after 8 hours ) .", 'These changes in the GFP-TgAtg8 signal were induced quickly and reached a plateau after ∼8 hours of starvation .', 'The re-localisation of GFP-TgAtg8 from the cytosol to vesicular structures during starvation suggested that these puncta were indeed autophagosomal structures .', 'The presence of punctate GFP-TgAtg8 signal in ∼20% of the extracellular tachyzoites at the start of the experiment might reflect basal autophagy , or a proportion of the population that have egressed early and are already in a relative nutrient stress condition .', 'The majority of these extracellular tachyzoites showed a single relatively large vesicle , which appeared prominently localised in the anterior part of the parasite , in the Golgi apparatus/apicoplast region ( Figure S2B ) .', 'Similarly , we performed microscopic observations on intracellular tachyzoites and found that they also occasionally displayed vesicular GFP-TgAtg8 in normal growth conditions ( Figure S2B ) .', 'Additional vesicles were found to be localised in a non-polarised manner throughout the cytoplasm of the tachyzoites .', 'Although mostly present as a punctate signal , the GFP-positive structures were found to display some heterogeneity as previously described for GFP-TgAtg8 in other eukaryotes [26] .', 'We sought to morphologically characterise these autophagic vesicles .', 'Tachyzoites , either freshly released or starved for 8 hours , were fixed and processed for electron microscopy analysis .', 'Compared to the control parasites , the starved tachyzoites displayed a higher number of large cytoplasmic vacuoles ( Figure 3A , V ) .', 'Additionally , in the starved parasites we identified several membrane-bound vesicles of about 300–900 nm in diameter containing cytoplasmic material or organelles .', 'These structures were usually bounded by two membranes ( Figure 3 , Ap ) .', 'These are commonly observed structural features of autophagosomes [27] .', 'As an example of organelles that could be found in these vesicles , part of the unique mitochondrion of a tachyzoite was seen segregated inside a membranous compartment ( Figure 3A ) and co-labelling of GFP-TgAtg8-decorated autophagosomes and mitochondrion showed partial co-localisation ( Figure 3B ) , which is suggestive of mitophagy occurring in Toxoplasma .', 'When the autophagosome has fused with the lysosomal compartment to deliver its content for degradation , it becomes an autolysosome , or autophagic vacuole .', 'These degradative vacuoles containing partially degraded cytoplasmic material could also be identified in starved parasites ( Figure 3 , Av ) .', 'Importantly , similar structures could be identified in a starved cell line expressing GFP-TgAtg8 , by immuno-electron microscopy with an anti-GFP antibody ( Figure 3C ) , providing a link to the observations we made by fluorescence microscopy .', 'Our results thus show that nutrient starvation triggers the appearance of autophagosomes in T . gondii tachyzoites through the recruitment of autophagosomal marker TgAtg8 , indicating that autophagy is operating and functional in this parasite .', 'The conjugation process of Atg8 to the autophagosomal membrane is fairly well understood at the molecular level in yeast and mammalian cells and resembles ubiquitination [8] .', 'In order to bind the autophagosomal membrane , Atg8 must first undergo proteolytic maturation by the cysteine peptidase Atg4 [28] , [29] .', 'This exposes a C-terminal glycine that is then conjugated to a phosphatidylethanolamine ( PE ) lipid moiety through the action of the Atg7–Atg3 system .', 'Accordingly , most eukaryotic Atg8 orthologues described so far bear one or several amino acids after the C-terminal glycine .', 'However , to our surprise , translation of the putative TgAtg8 gene as annotated by the ToxoDB genome database results in a protein that ends in a glycine in the absence of post-translational maturation .', 'This feature was conserved in other predicted apicomplexan Atg8 proteins , with the exception of several Cryptosporidium species ( Figure S3 ) .', 'To confirm this , we performed a 3′-RACE experiment using T . gondii cDNA as template and primers specific for Atg8 to map the stop codon .', 'In all the clones that we obtained and sequenced , the penultimate codon was confirmed to be coding for a glycine ( data not shown ) .', 'It is conceivable that this peculiar feature may result in constitutive lipidation of Atg8 in T . gondii and that maturation by the Atg4 peptidase may not be required .', 'However , analysis of the T . gondii genome reveals the presence of a protein with some similarity to Atg4 from yeast ( Table S1 ) , and a conserved active site catalytic triad appears to be present .', 'This peptidase , which appears to be expressed in T . gondii tachyzoites according to the proteomic data available in the ToxoDB database , could be involved in TgAtg8 recycling from the autophagosomal membrane , but this remains to be investigated .', 'Atg8 and its lipidated form can be separated by SDS-PAGE in the presence of urea [25] .', 'We thus used this technique to separate proteins from GFP-TgAtg8 lysates , followed by Western blot analysis with an anti-GFP antibody .', 'In these conditions , the anti-GFP antibody revealed a major protein band with a molecular size consistent with the prediction for a GFP-TgAtg8 fusion protein ( 42 kDa ) and an additional faster migrating protein ( Figure 4A ) .', 'We used the anti-Atg8 antibody on the parental cell line: we were also able to separate two isoforms of the native protein by urea SDS-PAGE at around 14 kDa ( Figure 4A ) .', 'Lipidated Atg8 typically migrates more rapidly than the non-lipidated form [29] , thus the faster migrating protein possibly corresponds to the GFP-TgAtg8-PE form .', 'To confirm that the faster migrating protein was indeed a membrane-associated form of GFP-TgAtg8 , we performed cell fractionation experiments that showed the faster migrating form was exclusively present in the 100 000 g insoluble pellet ( in contrast , the slower migrating protein was present in both fractions , but appeared to be enriched in the soluble fraction ) ( Figure 4B ) .', 'Similar results were obtained on parental cells with the anti-TgAtg8 antibody ( Figure S4 ) .', 'Moreover , only a treatment by a detergent ( DOC ) , but not by chemicals disrupting low energy bonds ( NaCl and urea ) , could lead to the complete solubilisation of TgAtg8 proteins present in the membrane fraction ( Figure 4C ) .', 'This has been described for other Atg8s [30] and suggests a tight association of TgAtg8 to membranes .', 'Metabolic labelling using 3H-ethanolamine ( as a precursor for PE ) and subsequent immunoprecipitation of TgAtg8 , revealed that TgAtg8 indeed incorporated the label , thus confirming that the membrane association was likely to be mediated by PE ( Figure 4D ) .', 'Also , the abundance of this membrane-associated form increased with the duration of the starvation period ( Figure 4A ) .', 'This confirmed that in T . gondii tachyzoites TgAtg8 becomes increasingly lipidated , and hence , potentially , more is recruited to the autophagosomal membranes during starvation ( as more autophagosomes are formed ) .', 'It is to note that proportions of soluble versus membrane-associated forms of TgAtg8 are higher in the GFP-TgAtg8-expressing cell line , compared to what is generally observed for TgAtg8 in the parental cell line .', 'One explanation is that while the global pool of GFP-TgAtg8 is higher in the GFP-TgAtg8 parasites , the number of autophagosomal vesicles harbouring the membrane-bound form is limited , leaving a significant part of GFP-TgAtg8 in the cytosol .', 'We then constructed , by site-directed mutagenesis , a variant of GFP-TgAtg8 were we replaced the C-terminal glycine by an alanine .', 'We expected that this should abolish the lipidation of the protein [31] .', 'Tachyzoites were transfected with the GFP-TgAtg8-G/A construct and GFP-positive clones were selected .', 'Autophagy was induced by starvation in amino acids-depleted medium as described above and the appearance of autophagosomes was monitored by fluorescence microscopy ( Figure 5A ) .', 'While the proportion of autophagosome-bearing cells increased along time in the GFP-TgAtg8 control cell line , we did not observe the formation of GFP-labelled autophagosomes in the GFP-TgAtg8-G/A mutant .', 'This finding suggests that the mutated protein is not recruited to autophagosomes ( Figure 5A and B ) , confirming the essential role of the glycine for the recruitment .', 'This finding further validates the interpretation of the GFP-TgAtg8 positive vesicular compartment as autophagosomal .', 'We also followed by Western blot the lipidation of GFP-TgAtg8-G/A following starvation .', 'Consistent with the microscopy results , no lipidated form could be detected for GFP-TgAtg8-G/A by Western blot analysis following urea SDS-PAGE ( Figure 5C ) .', 'Overall , in spite of a constitutively exposed C-terminal glycine , our results demonstrate that TgAtg8 exists both in soluble and membrane-associated forms in the tachyzoites .', 'Using GFP-TgAtg8 as a marker we were able to demonstrate that autophagy occurs in extracellular parasites under starvation conditions .', 'However , these are not necessarily reflecting physiological conditions and we wondered whether the parasite might encounter the need for this process during its normal intracellular development .', 'After invasion of the host cell , T . gondii tachyzoites replicate inside a parasitophorous vacuole by a process called endodyogeny [32] .', 'This process is composed of single gap phase ( G1 ) preceding a synthesis ( S ) phase , which is then followed by mitosis and cytokinesis through budding [33] .', 'Daughter cells are assembled within the mother and , as they form , encapsulate most of the maternal cell contents , leaving only a small residual body behind .', 'We hypothesized that some of the maternal material could be digested by autophagy to the benefit of the daughter cells .', 'We thus sought to follow the presence of autophagy in intracellular parasites developing within their host cells in tissue culture .', 'GFP-TgAtg8-expressing tachyzoites were used to synchronously infect host cells [34] and the proportion of intracellular tachyzoites displaying autophagic vesicles was measured as previously at different time points following infection .', 'It is to note that intracellular parasites were found to display a less intense and a more diffuse GFP-TgAtg8 labelling than the autophagic vesicles induced by starvation , yet slightly more intense than the cytosolic background ( Figure 6B , compare 4h and 48 h timepoints ) .', 'Similar structures were also seen in intracellular parasites expressing non functional GFP-TgAtg8-G/A and were partly co-localising with Golgi apparatus marker GRASP visualised with a red fluorescent protein ( RFP ) fusion ( Figure S5 ) , allowing us to rule out that they were autophagic vesicles .', 'Taking this into account for our quantification experiments , it appeared that there was an initial slight increase in the proportion of cells displaying intensely labelled GFP-TgAtg8 autophagic vesicles , peaking at 2 hours post-invasion .', 'However , such labelling generally decreased in following hours of intracellular development ( Figure 6A ) , where parasites could still be found to display one or several GFP-TgAtg8 autophagic vesicles , but the majority showed a rather homogenous cytosolic signal ( Figure 6B ) .', 'Also , the GFP-TgAtg8 vesicular signal was usually not found at the residual body occasionally formed within the vacuole after parasite division ( only in ∼2 . 5% of the residual bodies ) ( Figure 6B , arrowed ) .', 'To better follow the timing and localisation of the GFP-TgAtg8 signal , we used the inner membrane complex protein IMC1 [35] as a marker to track the progress of cytokinesis .', 'This protein is a component of the subpellicular network that defines the periphery of both the mature tachyzoite and of the daughter cells developing with the mother cell .', 'We imaged GFP-TgAtg8/IMC1-RFP co-expressing tachyzoites , and observed that the vesicular GFP-TgAtg8 signal was usually present in dividing cells ( Figure 6C ) .', 'More precisely , live imaging observation of these parasites showed that autophagosomes were usually present until the cytokinesis process , but disappeared shortly after ( data not shown ) .', 'Altogether , our data argue against a continuous autophagic activity in intracellular parasites , yet vesicle formations could occasionally be observed and transient autophagy seemed to occur .', 'Autophagy is a multistep process that can be modulated by upstream kinase effectors and so can be interfered with using specific pharmacological agents .', 'The “target of rapamycin” ( TOR ) kinase is a serine/threonine kinase which is central in regulating cellular growth by promoting anabolic processes and antagonising autophagy .', 'Consequently , treatment with TOR inhibitor rapamycin mimics nutrient starvation and is known to increase the level of autophagy in several eukaryotes [36] .', 'On the other hand , the class III phosphoinositide 3-kinase ( PI3K ) signalling cascade has been shown to promote autophagy , thus specifically inhibiting this kinase is thought to reduce autophagy [37] .', 'Both a TOR kinase and a class III PI3K are predicted to be present in the T . gondii genome ( Table S1 ) ; we have thus sought to use specific kinase inhibitors to evaluate the biological significance of autophagy in Toxoplasma .', 'We drug-treated GFP-TgAtg8-expressing extracellular parasites and evaluated the effect of treatment on autophagy .', 'Increasing concentrations of rapamycin in the starvation medium resulted in higher proportions of GFP-TgAtg8 vesicles in extracellular parasites , suggesting an increased level of autophagic activity ( Figure S6A ) .', 'However , the effective concentration was significantly higher than routinely observed in yeast or mammalian cells ( 5 µg/ml instead of 0 . 5 µg/ml ) .', 'This suggests a modest effect of rapamycin on the modulation of T . gondii autophagy , like previously observed with the TOR kinase of plants such as rice , tobacco or Arabidopsis [38] and the parasitic protist Trypanosoma brucei [23] .', 'We also evaluated the effects of class III PI3K on autophagy by incubating extracellular tachyzoites in the starvation solution in the presence of 3-methyladenine ( 3-MA ) or wortmannin , two known PI3K inhibitors .', 'In these conditions , there were lesser proportions of GFP-TgAtg8 vesicles-positive cells amongst the tachyzoites treated with the two inhibitors ( particularly wortmannin ) , which suggested a role for the PI3K in promoting autophagy in Toxoplasma ( Figure S6B ) .', 'However , again the concentrations of wortmannin and 3-MA we had to use were higher than those used for mammalian and yeast cells , similarly to plants were these PI3K inhibitors have to be used at greater concentrations to inhibit autophagy [39] , [40] .', 'In conclusion , the use of inhibitors specific for kinases known to regulate autophagy in other eukaryotic systems allowed us to show that they generally follow the same trends in T . gondii ( inhibition of autophagy by the TOR kinase and activation by the class III PI3K ) , although the use of these drugs at relatively high concentrations can alter their specificity and would preclude a use on intracellular parasites to investigate the autophagic function .', 'As we could not use kinase inhibitors to satisfyingly investigate the function of autophagy in Toxoplasma tachyzoites , we sought to genetically produce an autophagy-deficient cell line .', 'To this end , we chose Atg3 as a target , a gene coding for a protein involved in the conjugation of Atg8 and essential for autophagy [41] .', 'In other eukaryotes , Atg8 is activated by Atg7 to form an Atg8/Atg7 thioester intermediate and is then transferred to Atg3 to form an Atg8/Atg3 thioester intermediate , before being finally conjugated to the amino group of PE for binding to the autophagosome membrane .', 'TgAtg3 ( TGME49_036110 , Table S1 ) was identified in the T . gondii genomic sequence based on protein sequence homology searches with eukaryotic orthologues and was found to have a conserved predicted active site region with , in particular , a conserved active site cysteine ( Figure S7 ) .', 'T . gondii genomic database predicts a 397 amino acids-long TgAtg3 protein .', 'Yet , when aligned to yeast or human orthologues , predicted TgAtg3 shows a N-terminal extension bearing a poly-serine motif ( Figure S7 ) .', 'The corresponding putative mRNA region has a polypyrimidine tract ( promotes the assembly of the spliceosome ) , with a putative downstream AG splice acceptor site and a potential downstream start codon that would produce a shorter version of TgAtg3 , closer in size to other eukaryotic orthologues .', 'The regions corresponding to the 3′ and 5′ untranslated regions ( UTRs ) of T . gondii TgAtg3 gene were cloned in a plasmid on either side of a selection marker gene to use for a knock-out strategy by gene replacement through a double recombination event .', 'We tried to transfect the RHΔHX strain [42] and the RH-derived ΔKu80 strain , which is more amenable to targeted gene deletion [43] .', 'Despite numerous independent attempts , we were unable to obtain clones in which TgAtg3 had been deleted ( data not shown ) .', 'The inability to delete the TgAtg3 gene with these different strategies suggested an essential role for the corresponding protein in tachyzoites .', 'We thus sought to produce a conditional null mutant cell line for TgAtg3 .', 'We introduced an ectopic copy of TgAtg3 into the TAti tet-transactivator line by stable transformation [44] .', 'The ectopic copy was placed under the control of the tetracycline-regulatable promoter 7tetOSag1 .', 'To aid detection of the corresponding protein , the transgene was tagged with a sequence encoding an N-terminal c-myc epitope .', 'We cloned both a long and short versions of putative TgAtg3 into the expression vector ( named imyc-lTgAtg3 and imyc-sTgAtg3 , respectively ) .', 'After transfection of tachyzoites of the TAti cell line , clonal parasites were obtained .', 'Both cell lines showed an anhydrotetracycline ( ATc ) -regulatable TgAtg3 expression by Western blot and by IFA with anti-myc antibody ( data not shown ) .', 'IFA also demonstrated a cytosolic localisation for myc-tagged TgAtg3 ( long and short versions alike ) , which is compatible with a functional role in autophagy , where it should be conjugating cytosolic Atg8 to the autophagosomal membrane ( data not shown ) .', 'To replace TgAtg3 , we used a cosmid-based gene disruption strategy , allowing the use of large flanking regions to increase the chance to recombine at the appropriate locus [45] .', 'A single cosmid clone was available for the TgAtg3 locus ( TOXOU62 ) .', 'In this cosmid the TgAtg3 open reading frame was replaced with a chloramphenicol acetyl transferase marker by recombineering ( Figure 7A ) .', 'Parasites expressing a regulatable copy of TgAtg3 were transfected with the resulting cosmid and stable clones were isolated by chloramphenicol selection .', 'Clones were tested by PCR for 5′ and 3′ disruption of the TgAtg3 locus , and presence of the resistance cassette ( Figure 7B ) .', 'We note that we successfully obtained a null mutant clone in the imyc-sTgAtg3 background , but not in the cell line expressing the longer version of TgAtg3 .', 'This suggests that this short transcript likely encodes the functional protein .', 'Further analysis by Southern blot confirmed the correct integration of the cosmid-derived cassette and the disruption of native TgAtg3 locus ( Figure 7C ) .', 'Conditional mutant clone displayed a tight regulation of TgAtg3 expression by ATc , as shown by Western blot and IFA with anti-myc antibody ( Figure 8A and B ) : Western blot analysis showed that a 2 days treatment with ATc was reducing almost completely the expression of the protein and that a 4 days treatment led to undetectable levels of the extra copy .', 'We next assessed whether the Atg8-conjugation function of Atg3 was conserved in T . gondii .', 'To do so , we used the conditional mutant to deplete intracellular tachyzoites of TgAtg3 by a 2 days treatment with ATc , we then isolated extracellular parasites and triggered autophagy by exposing them to starvation medium for increasing intervals of time as described before .', 'Parasites extracts were prepared and analysed by urea SDS-PAGE to visualise the lipid-conjugated form of TgAtg8 .', 'As described above , in the presence of TgAtg3 we observed an increase in the TgAtg8 lipidated form , as autophagy was induced ( Figure 8C ) .', 'In contrast , when TgAtg3 was repressed by ATc treatment:', 'i ) a significant proportion of the slower migrating form was present even before autophagy was induced and', 'ii ) the lipidated form was not upregulated by starvation ( Figure 8C ) .', 'This shows that reducing the levels of TgAtg3 leads to a reduced capacity in TgAtg8-conjugation to the autophagosomes and thus likely impairs the putative autophagic function in the parasite .', 'We examined the effect of TgAtg3 depletion on parasite growth using plaque assays .', 'The conditional TgAtg3 null mutant formed markedly smaller plaques when repression of the imyc-sTgAtg3 copy was induced by ATc ( Figure 9A and B ) .', 'Longer repression periods increased the effect on growth , as no plaque was visible with mutant parasites pre-incubated with ATc for 4 days prior to the start of the plaque assay ( Figure 9A ) .', 'No viable parasite coul
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Autophagy is a cellular process that is highly conserved among eukaryotes and permits the degradation of cellular material .', 'Autophagy is involved in multiple survival-promoting processes .', 'It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles , but it also plays a role in cell differentiation and cell development .', 'Equally important is its function for survival in stress-related conditions such as recycling of proteins and organelles during nutrient starvation .', 'Protozoan parasites have complex life cycles and face dramatically changing environmental conditions; whether autophagy represents a critical coping mechanism throughout these changes remains poorly documented .', 'To investigate this in Toxoplasma gondii , we have used TgAtg8 as an autophagosome marker and showed that autophagy and the associated cellular machinery are present and functional in the parasite .', 'In extracellular T . gondii tachyzoites , autophagosomes were induced in response to amino acid starvation , but they could also be observed in culture during the normal intracellular development of the parasites .', 'Moreover , we generated a conditional T . gondii mutant lacking the orthologue of Atg3 , a key autophagy protein .', 'TgAtg3-depleted parasites were unable to regulate the conjugation of TgAtg8 to the autophagosomal membrane .', 'The mutant parasites also exhibited a pronounced fragmentation of their mitochondrion and a drastic growth phenotype .', 'Overall , our results show that TgAtg3-dependent autophagy might be regulating mitochondrial homeostasis during cell division and is essential for the normal development of T . gondii tachyzoites .']
['Autophagy is a catabolic process involved in maintaining cellular homeostasis in eukaryotic cells , while coping with their changing environmental conditions .', 'Mechanistically , it is also a process of considerable complexity involving multiple protein factors and implying numerous protein-protein and protein-membrane interactions .', 'The cellular material to be degraded by autophagy is contained in a membrane-bound compartment called the autophagosome .', 'We have characterised the formation of autophagosomes in the protozoan parasite Toxoplasma gondii by following the relocalisation of autophagosome-bound TgAtg8 .', 'Thus , exploiting GFP-TgAtg8 as a marker , we showed that it is a process that is regulated and can be induced artificially by amino acid starvation .', 'Autophagic vesicles were also observed in normally dividing intracellular parasites .', 'Depleting Toxoplasma of the TgAtg3 autophagy protein led to an impairment of TgAtg8 conjugation to the autophagosomal membrane and , at the cellular level , to a fragmentation of the single mitochondrion of the parasite and to a severe growth arrest .', 'We have thus found that TgAtg3-dependent autophagy is essential for normal intracellular development of T . gondii tachyzoites .']
['parastic protozoans', 'toxoplasma gondii', 'protozoology', 'biology', 'microbiology']