Mitochondrial ADP/ATP Carrier 1 Is Important for the Growth of Toxoplasma Tachyzoites

ABSTRACT Metabolism associated with energy production is highly compartmentalized in eukaryotic cells. During this process, transporters that move metabolites across organelle membranes play pivotal roles. The highly conserved ADP/ATP carrier (AAC) involved in ATP and ADP exchange between the mitochondria and cytoplasm is key to linking the metabolic activities in these 2 compartments. The ATP produced in mitochondria can be exchanged with cytoplasmic ADP by AAC, thus satisfying the energy needs in the cytoplasm. Toxoplasma gondii is an obligate intracellular parasite with a wide range of hosts. Previous studies have shown that mitochondrial metabolism helps Toxoplasma to parasitize diverse host cells. Here, we identified 2 putative mitochondria ADP/ATP carriers in Toxoplasma with significant sequence similarity to known AACs from other eukaryotes. We examined the ATP transport function of TgAACs by expressing them in Escherichia coli cells and found that only TgAAC1 had ATP transport activity. Moreover, knockdown of TgAAC1 caused severe growth defects of parasites and heterologous expression of mouse ANT2 in the TgAAC1 depletion mutant restored its growth, revealing its importance for parasite growth. These results verified that TgAAC1 functions as the mitochondrial ADP/ATP carrier in T. gondii and the functional studies demonstrated the importance of TgAAC1 for tachyzoites growth. IMPORTANCE T. gondii has an efficient and flexible energy metabolism system to meet different growth needs. ATP is an energy-carrying molecule and needs to be exchanged between organelles with the assistance of transporters. However, the function of TgAACs has yet to be characterized. Here, we identified 2 putative AACs of T. gondii and verified that only TgAAC1 had ATP transport activity with expression in the intact E. coli cells. Detailed analyses found that TgAAC1 is critical for the growth of tachyzoites and TgAAC2 is dispensable. Moreover, complementation with mouse ANT2 restored the growth speed of iTgAAC1, further suggesting TgAAC1 functions as a mitochondrial ADP/ATP carrier. Our research demonstrated the importance of TgAAC1 for tachyzoites growth.

2. Line 75. "while TgAAC2 neither transports ATP" -can the authors rule this out entirely? Their data do not provide evidence for transport activity, but this could be due to the conditions of the experiment. Perhaps be clearer: "while TgAAC2 neither transports ATP under the conditions tested ..." 3. Line 86. "TgAACs had high homology compared with ..." What is meant by "high homology" here? Either specify the degree of similarity or just state that they had homology. 4. Figure 1. How were the alpha-helices predicted? Please clarify. 5. Line 161. "It can be assumed that the parasite will almost completely lose its growth capacity under attack from the host immune system." Maybe, but this is not something the authors test. Perhaps stick to a conclusion that is supported by the data -TgAAC1 is important for parasite growth in vitro. 6. Figure 3 legend. "Means {plus minus} SD from two replicates ... three independent experiments repeated, a representative one is shown here." I don't understand what the replicates are in this experiment. Are the "two replicates" mentioned two out of the three independent experiments (and if so, why wasn't the third replicate included in the analysis?). Or did the authors undertake two technical replicates for each independent experiment? Which data were used in the statistical test (the independent experiments or the two replicates)? This needs clarification. 7. Figure 4 legend. Along the same lines as the previous comment, how can the graph depict both the "means {plus minus} SD from three independent experiments" and show only a "representative one"? This needs clarification. (same comment for Figure  6E). 8. Line 171. "the replication rate of Com-TgAAC1 was slower than iTgAAC1 without ATc treatment". The statistical test for this is not shown in Figure 4E -i.e. comparing the -ATc conditions in Figure 4E. (same comment for Line 186 and Figure 5D) 9. Line 190. "We also studied the physiological roles of TgAAC2" -the authors investigate whether TgAAC2 is important for parasite growth, which is not the same as addressing its physiological role. Consider re-wording. 10. Line 202. "The blockage of mitochondrial ATP transport to the cytoplasm by inhibiting AAC1 expression led to severe growth defects of T. gondii ... This further suggests a more prominent contribution of ATP production by T. gondii mitochondrion to parasite growth". The authors assume that AAC1 acts by transporting ATP from the mitochondrion into the cytosol. While this is certainly possible (and perhaps likely), can the authors rule out that this transporter can (instead, or in some physiological contexts) import ATP into the mitochondrion in exchange for ADP? For example, to drive reverse activity of ATP synthase in establishing a proton gradient across the inner mitochondrial membrane, as occurs in bloodstage Trypanosoma brucei parasites (PMID 16270030)? Given that the authors don't measure mitochondrial ATP synthesis or the membrane potential of the inner mitochondrial membrane in the absence of TgAAC1, the authors should interpret their data more critically, and consider alternative possibilities. 11. Line 217. "the knockdown of AAC1 caused severe phenotype defects because of the inhibition of ATP export" -see previous comment. The authors don't measure ATP export from the mitochondrion, so need to be more cautious in their conclusions here.
12. Line 232. "is consistent with previous research that overexpression of ANT1 inactivated NF-κB activity and increased Bax expression accompanied by the disruption of mitochondrial membrane potential, which induced cardiomyocyte death, cancer cells apoptosis, and suppressed nude mouse tumor growth". I don't understand the logic here. NF-κB and Bax are not found in T. gondii. Perhaps the point to make here is that ANT1 overexpression in mice leads to changes in mitochondrial physiology (such as changes to mito membrane potential), which leads to outcomes such as inactivated NF-κB activity and increased Bax expression. Overexpression of AAC in T. gondii may result in similar physiological effects in the parasite mitochondrion that result in impaired parasite proliferation, although this is not something the authors test.
13. Line 251. "indicating that TgAAC1 is equally significant in bradyzoites." Without investigating bradyzoites directly, the authors can't conclude this. Perhaps "suggests that TgAAC1 may also function in bradyzoites"? 14. Line 274. "which linearized by upstream and downstream primers into TATi strain". It isn't clear to me what this means. Have the authors amplified the ATc-regulatable upstream region and DHFR selectable marker by PCR, using primers containing flanks for integration into the AAC1 locus? This needs clarification.
15. Line 290. The authors should specify the concentration of ATP and the amount of radiolabel used in the uptake assays.
16. Line 309. The authors should list the publications where the primary antibodies are first described, and the catalogue numbers of the commercial secondary antibodies.
Grammatical suggestions -overall, the paper is well written and the results clearly described. I have numerous comments and suggestions for improving clarity of some aspects of the manuscript that the authors may consider taking on board before publication.
Line 119. "which means TgAAC2 was unable to transport ATP(S-1B)" -under the conditions of the experiment.
Line 127. "We doubted these differences might be the reason made TgAAC2 disable the transportation of ATP" -convoluted sentence, meaning unclear. I think: "We hypothesised that these differences might be the reason why TgAAC2 was unable to transport ATP" Line 132. "the results showed none differed from uninduced groups" -none differed in what sense? Specify that "none of the mutant TgAAC2s were able to transport ATP" Line 135. "investigated by conducting out competition experiments in the presence or absence of 100-fold nucleotide (ADP, ATP, AMP, CTP, GTP, UTP)." This needs a clearer description. "... by conducting 32P-ATP transport assays in the presence or absence of 100-fold molar excess of a range of unlabelled nucleotides (ADP ...)" Line 138. What is meant by "input" here? "Transport" or "import" perhaps? Line 148. "regulate" instead of "repression of" Line 152. "disappeared" instead of "was disappeared" Line 155. "culture" rather than "cultural" Line 159. "growth defects were still stacked" -meaning unclear. "growth defects were still apparent"? Line 230. "replication" rather than "replicate" Line 242. "mutated" instead of "mutant". Later in this sentence "non-functional" Reviewer #2 (Comments for the Author): In this manuscript, the authors characterized two ATP/ADP carriers, TgAAC1 and TgAAC2. By epitope tagging, they localized both AACs to the parasite's mitochondria. A functional assay expressing AAC individually in E. coli determined that TgAAC1 can primarily transport ATP and ADP. Then they knock down the TgAAC1 and found that it is important for parasite growth, whereas TgAAC2 is dispensable in the parasites. In addition, they functionally complemented mouse ANT2 protein, an ortholog of AAC, into TgAAC1 knockdown strain. Overall, this manuscript revealed the roles and functions of two putative Toxoplasma ATP/ADP carriers in parasite growth. Several comments are listed below.
1. The author may consider changing the "essential" to "important" in the title. The knockdown parasites can still form very tiny plaques. Therefore, the straight knockout can be generated and show a viable phenotype.
2. Line 55-56, based on the data in this study, TgAAC1 still can transport AMP but not as efficiently as ATP and ADP.
2. In the knockdown parasites, the morphology of their mitochondria is normal, although their mitochondrial function is impaired. This phenomenon is similar to the TgCPOX knockout published in the previous work listed below. I suggest the authors talk about this in the discussion. Due to heme deprivation, the mutant does not have functional mitochondria. Still, it forms tiny plaques, suggesting that the parasites use other pathways to acquire heme to keep their mitochondria functional, albeit not to a full extent.

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List of Responses
Dear Editor: Thank you for your letter and for the reviewers' comments concerning our manuscript entitled "Mitochondrial ADP/ATP carrier 1 is essential for the growth of Toxoplasma tachyzoites" (Spectrum00040-23). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied the comments carefully and have made corrections which we hope meet with approval. Revised portions are marked in yellow in the manuscript. The main corrections in the paper and the responses to the reviewer's comments are as follows: 1. Figure 1B. The authors need to verify that integration of the HA tag into the 3' region of the TgAAC1 and TgAAC2 loci were successful (e.g. via PCR screening approaches, as they have done for their other genetically modified parasite strains).
They should also undertake western blotting of proteins extracted from the TgAAC1-HA and TgAAC2-HA strains to determine whether proteins of the expected mass were correctly tagged. Western blotting may also provide an indication of the relative abundances of these proteins.
Response: Many thanks for your constructive comments and suggestions, we feel sorry that we did not provide enough information to identify the correct integration of HA tag in 3' region of TgAAC1 and TgAAC2. We have added PCRs to verify the true integration of HA in the two strains (Fig. 1C, line 96) and tested the correct protein band sizes by western blot (see below). However, as we did not select single clones when constructing the locus strains, the library alone would not allow a comparison of the relative abundance of the two proteins. Therefore, we detected the relative abundance of TgAACs by RT-qPCR in the RHΔku80 with the expression of the β-actin used as an internal reference. The results showed that both TgAACs were expressed but TgAAC1 was more highly expressed than TgAAC2.
These results are shown below. 3. Line 86. "TgAACs had high homology compared with ..." What is meant by "high homology" here? Either specify the degree of similarity or just state that they had homology.

Response:
We apologize for the unclear statement here. Thanks again for pointing this out. We have modified that "TgAACs had homology compared with….".
Response: Thanks very much for pointing it out, and we are sorry for our omission.
We predicted the structure and distinguished the alpha-helices of TgAACs in ToxoDB (https://toxodb. org/toxo/app/), and we have added these results to the manuscript. (Fig. 1B) 5. Line 161. "It can be assumed that the parasite will almost completely lose its growth capacity under attack from the host immune system." Maybe, but this is not something the authors test. Perhaps stick to a conclusion that is supported by the data -TgAAC1 is important for parasite growth in vitro.

Response:
We apologize for the unclear descriptions here. What we meant to say was "A total of three independent experiments were conducted, with two technical replications for each independent experiment. The representative one from three independent experiments is shown here." Thank you for pointing it out, and we have revised it in the manuscript. (Fig. 3 legend) 7. Figure 4 legend. Along the same lines as the previous comment, how can the graph depict both the "means {plus minus} SD from three independent experiments" and show only a "representative one"? This needs clarification. (same comment for Figure 6E).
Response: Again, we apologize for the unclear descriptions. Many thanks for pointing it out. We have revised it to "The same method with Fig. 3G." in the manuscript. (Fig. 4, 5, 6 legends) 8. Line 171. "the replication rate of Com-TgAAC1 was slower than iTgAAC1 without ATc treatment". The statistical test for this is not shown in Figure 4E  suggestions, and we are sorry for our omission. We have done the statistical comparison of Com-TgAAC1(and Com-MusANT2) vs iTgAAC1 without ATc treatment according to the Reviewer's comments, which indeed showed significant differences. (Fig. 4E, 5D) 9. Line 190. "We also studied the physiological roles of TgAAC2" -the authors investigate whether TgAAC2 is important for parasite growth, which is not the same as addressing its physiological role. Consider re-wording.

Response:
We are sorry that this part is incorrectly phrased in the original manuscript. We have revised the contents from "physiological roles" to "physiological importance" of this part. Thank you for your advice. (Line 191) 10. Line 202. "The blockage of mitochondrial ATP transport to the cytoplasm by inhibiting AAC1 expression led to severe growth defects of T. gondii ... This further suggests a more prominent contribution of ATP production by T. gondii mitochondrion to parasite growth". The authors assume that AAC1 acts by transporting ATP from the mitochondrion into the cytosol. While this is certainly possible (and perhaps likely), can the authors rule out that this transporter can (instead, or in some physiological contexts) import ATP into the mitochondrion in exchange for ADP? For example, to drive reverse activity of ATP synthase in establishing a proton gradient across the inner mitochondrial membrane, as occurs in bloodstage Trypanosoma brucei parasites (PMID 16270030)? Given that the authors don't measure mitochondrial ATP synthesis or the membrane potential of the inner mitochondrial membrane in the absence of TgAAC1, the authors should interpret their data more critically, and consider alternative possibilities.

Response:
We sincerely appreciate the valuable comments. Sorry about the inaccurate conclusion and we are unable to exclude other possibilities for damage to the parasite. We have revised this section to be more accurate which is "Moreover, inhibiting AAC1 expression led to severe growth defects of T. gondii, which indicated AAC1 is critical for tachyzoites growth", and deleted "The blockage of mitochondrial ATP transport to the cytoplasm by inhibiting AAC1 expression led to severe growth defects of T. gondii ... This further suggests a more prominent contribution of ATP production by T. gondii mitochondrion to parasite growth". (Line 204) The inactivity of AAC causes an adenine nucleotide imbalance in mitochondria, resulting in the uncoupling of the inner membrane, which affects the mitochondrial gene expression, electron transport chain assembly, mtDNA integrity, and cell viability. And whether the growth inhibition in the iTgAAC1 strain is also a result of these problems needs further study. This part has been discussed in line 222.
11. Line 217. "the knockdown of AAC1 caused severe phenotype defects because of the inhibition of ATP export" -see previous comment. The authors don't measure ATP export from the mitochondrion, so need to be more cautious in their conclusions here.
Response: It is a giant mistake to the whole quality of our article. We feel sorry for our carelessness. We have corrected it into "In T. gondii, the knockdown of AAC1 also caused severe phenotype defects", and we also feel great thanks for your pointer. (Line 217) 12. Line 232. "is consistent with previous research that overexpression of ANT1 inactivated NF-κB activity and increased Bax expression accompanied by the disruption of mitochondrial membrane potential, which induced cardiomyocyte death, cancer cells apoptosis, and suppressed nude mouse tumor growth". I don't understand the logic here. NF-κB and Bax are not found in T. gondii. Perhaps the point to make here is that ANT1 overexpression in mice leads to changes in mitochondrial physiology (such as changes to mito membrane potential), which leads to outcomes such as inactivated NF-κB activity and increased Bax expression.
Overexpression of AAC in T. gondii may result in similar physiological effects in the parasite mitochondrion that result in impaired parasite proliferation, although this is not something the authors test.

Response:
We apologize for the unclear descriptions here. From the results, we found that the growth speeds of Com-TgAAC1 and Com-MusANT2 were slower than iTgAAC1 which both were without ATc treatment, and this is very confusing.
We considered that this is because the overexpressed AAC is harmful to the parasite and therefore cite the reference of "mice ANT1 overexpression part" to support this idea. The point we were trying to make is indeed that ANT1 overexpression in mice leads to changes in mitochondrial physiology. Thanks very much for pointing it out. We have revised this part's contents. (Line 229) 13. Line 251. "indicating that TgAAC1 is equally significant in bradyzoites." Without investigating bradyzoites directly, the authors can't conclude this. Perhaps "suggests that TgAAC1 may also function in bradyzoites"? Line 36. "ATP as energy molecular needs to be exchanged ..." -meaning unclear. "ATP is an energy carrying molecule and needs to be exchanged ..." Response: Thanks very much for pointing it out, and we are sorry for the unclear representation. We have remanded this part according to the Reviewer's comments.

(Line 36)
Line 41. "Moreover, complement[ion] with mouse ANT2 restored growth of iTgAAC1, 1. The author may consider changing the "essential" to "important" in the title. The knockdown parasites can still form very tiny plaques. Therefore, the straight knockout can be generated and show a viable phenotype.
Response: Thank you very much for your pertinent comments. We have carefully considered and discussed this issue and have unanimously agreed to replace the "essential" with "important". (Line 1) 2. Line 55-56, based on the data in this study, TgAAC1 still can transport AMP but not as efficiently as ATP and ADP.
Response: Thank you for raising this critical issue. Indeed, we have noted the issue.
We discussed and speculated that "the decline of uptake" may be due to AMP occupying the binding site, resulting in a decrease in ATP uptake by AAC. After reviewing the literature, we found that human ANT1 also has a similar substrate preference [1] and this part has also been added to the manuscript (Line 144, 211).
Considering that possible, all we can conclude is that ADP and ATP are substrates of TgAAC1. For clarity of presentation, we have changed "These results proved that TgAAC1 has substrate specificity with ADP and ATP" to "These results proved that ADP and ATP are substrates of TgAAC1" in the manuscript. (Line 145) 3. In the knockdown parasites, the morphology of their mitochondria is normal, Response: Special thanks to you for your good comments. We think this is an excellent suggestion. But we politely clarify that we haven't test whether the morphology of the mitochondrion is normal in the absence of iTgAAC1. The morphology of the mitochondrion requires further analysis by transmission electron microscopy and the IFA results presented in original manuscript cannot allow for this analysis. Besides, we also noticed that iTgAAC1formed tiny plaques with Atc treatment and in our unpublished study, iTgAAC1+ATc strain has higher ATP level than iTgAAC1-ATc strain. We speculated that there is another AAC or other pathways in parasites to supplement ATP. Considering we only blasted two AACs in T. gondii, we ruled out the first option. Then we assumed the compensatory activity of glycolysis is the reason. In line with this possibility, we tried to culture the parasite with glucose-free medium. The replication assay in glucose-free conditions showed that replication speed of iTgAAC1 with ATc treatment was significantly impaired, with only 1, 2 parasites in each parasitophorous vacuole, while the replication rate of strain without ATc treatment was unaffected (see Figure below). However, this study is unpublished because we haven't finished. So sincerely sorry that we couldn't add this part to the discussion part. Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
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