线粒体与细胞核之间的代谢串扰对弓形虫感染至关重要-动脉网

Metabolic crosstalk between the mitochondrion and the nucleus is essential for Toxoplasma gondii infection

Nature 等信源发布 2025-03-07 11:21

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Abstract

摘要

Toxoplasma gondii

弓形虫

, an intracellular pathogenic protist with a remarkable ability to infect a wide range of host cells, displays an equally exceptional design of its carbon metabolism. There are, however, critical gaps in our understanding of the metabolic network in

，一种细胞内致病原生生物，具有感染多种宿主细胞的显著能力，其碳代谢设计同样异常独特。然而，我们对其代谢网络的理解仍存在关键的空白。

T. gondii

弓形虫

. We characterized the mito-nuclear metabolism and organelle coupling during its acute infection (lytic cycle). The major enzymes of the TCA cycle, i.e., citrate synthase (CS1), succinyl-CoA synthase alpha subunit (SCSα), succinate dehydrogenase (SDHA) and FAD malate dehydrogenase (MDH-FAD) located in the parasite mitochondrion support its asexual reproduction but are not needed for its survival.

我们对其急性感染（裂解周期）过程中的线粒体-核代谢和细胞器耦合进行了研究。三羧酸循环（TCA循环）的主要酶，如位于寄生虫线粒体中的柠檬酸合成酶（CS1）、琥珀酰辅酶A合成酶α亚基（SCSα）、琥珀酸脱氢酶（SDHA）和FAD苹果酸脱氢酶（MDH-FAD），支持其无性繁殖，但对其生存并非必需。

The SCSα and SDHA mutants are nearly avirulent in a mouse model, and they can protect the host against a lethal challenge infection. Genetic deletion of MDH-FAD dysregulated glucose-derived carbon flux, leading to a collapse of the mitochondrial membrane potential. The parasite also harbors a cytosolic isoform of MDH and a nuclear malic enzyme (ME) contributing to malate oxidation; however, only the latter is essential for the lytic cycle.

SCSα和SDHA突变体在小鼠模型中几乎无毒力，并且它们可以保护宿主免受致死性挑战感染。MDH-FAD的基因缺失会扰乱葡萄糖衍生的碳流，导致线粒体膜电位崩溃。该寄生虫还含有MDH的胞质同工型和一种核苹果酸酶（ME），二者共同参与苹果酸氧化；然而，只有后者对裂解周期至关重要。

Expression of ME in the nucleus is crucial for the parasite development. Besides, conditional knockdown of ME impairs the histone acetylation and disrupts the expression of several genes in tachyzoites. Our work discloses novel network design features of .

ME在细胞核中的表达对寄生虫的发育至关重要。此外，条件性敲低ME会损害组蛋白乙酰化并破坏速殖子中多个基因的表达。我们的工作揭示了新的网络设计特性。

T. gondii

弓形虫

and highlights the therapeutic and vaccination potential of the parasite metabolism.

并突出了寄生虫代谢的治疗和疫苗潜力。

Introduction

简介

Toxoplasma gondii

弓形虫

, a prevalent zoonotic parasite, infects many warm-blood organisms and can reproduce in several nucleated host cells

，一种普遍存在的**人畜共患寄生虫**，能够感染许多温血生物，并在多种有核宿主细胞中繁殖。

. It has a complex life cycle, with sexual reproduction occurring exclusively in felid hosts and asexual development in a wide range of intermediate hosts, such as pigs, cattle, sheep and humans

它有着复杂的生活周期，有性繁殖仅在猫科宿主中发生，而无性发育则在广泛的中间宿主中进行，例如猪、牛、羊和人类。

. The parasite exists in two infectious stages in its hosts: fast-replicating tachyzoites and slow-growing bradyzoites. Acute toxoplasmosis in pregnant individuals or animals can cause abortion, stillbirth or fetal deformities

寄生虫在其宿主体内存在两个感染阶段：快速复制的速殖子和缓慢生长的缓殖子。孕妇或动物的急性弓形虫病可导致流产、死产或胎儿畸形。

. In contrast, the chronic disease usually persists for the entire life of the infected host and can result in recrudescence upon impairment of the immunity. These outcomes seriously threaten public health and socioeconomic development. Currently, pyrimethamine and sulfadiazine are the only common drugs for treating acute toxoplasmosis.

相比之下，慢性病通常在受感染宿主的整个生命周期中持续存在，并且在免疫力受损时可能导致复发。这些结果严重威胁公共卫生和社会经济发展。目前，乙胺嘧啶和磺胺嘧啶是治疗急性弓形虫病仅有的常用药物。

，

and none are available for against chronic infection. Extended drug usage is also associated with adverse effects and drug resistance

并且没有可用于对抗慢性感染的药物。长期用药也与不良反应和耐药性有关。

，

. There is only one commercial vaccine (Toxovax), which is weakly virulent and used exclusively in sheep, with a potential risk of reverting to virulence. Therefore, there is an urgent need to explore additional drug and vaccine targets to control the disease

仅有一种商业疫苗（Toxovax），其毒性较弱，且仅用于绵羊，存在恢复毒力的潜在风险。因此，迫切需要探索更多的药物和疫苗靶点以控制该疾病。

，

. Although the parasite metabolism remains central to drug development, its limited knowledge has hindered the discovery of new therapeutic vulnerabilities in

尽管寄生虫代谢仍然是药物开发的核心，但对其有限的认识阻碍了新治疗弱点的发现。

T. gondii

弓形虫

。

Toxoplasma

弓形虫

’s acutely infectious tachyzoite stage utilizes glucose- and glutamine-derived carbon to fuel their carbon metabolism and, thereby, the lytic cycle

急性感染性的速殖子阶段利用葡萄糖和谷氨酰胺衍生的碳来推动其碳代谢，从而促进裂解周期。

，

. Glucose and glutamine are metabolized through glycolysis and the TCA cycle, respectively, and both nutrients cooperate to enable a rapid propagation of tachyzoites. Glucose enters the parasite through a high-affinity transporter located in the plasma membrane

葡萄糖和谷氨酰胺分别通过糖酵解和三羧酸循环代谢，这两种营养物质协同作用以促进速殖子的快速增殖。葡萄糖通过位于质膜上的高亲和力转运蛋白进入寄生虫体内。

and then converted to pyruvate

然后转化为丙酮酸

via

通过

glycolysis

糖酵解

，

. Pyruvate is imported into the mitochondrion

丙酮酸被导入线粒体

via

通过

the mitochondrial pyruvate carrier (MPC) complex, where it is converted to acetyl-coenzyme A (Ac-CoA) by a branched-chain α-ketoacid dehydrogenase E1 subunit (BCKDH-E1α), feeding into the TCA cycle

线粒体丙酮酸载体（MPC）复合物，其中它通过支链α-酮酸脱氢酶E1亚基（BCKDH-E1α）转化为乙酰辅酶A（Ac-CoA），进入三羧酸循环（TCA循环）。

，

. However, tachyzoite mutants lacking MPC or BCKDH-E1α are still viable and partially virulent

然而，缺乏MPC或BCKDH-E1α的速殖子突变体仍然具有活力且部分有毒力。

，

. Glutamine is first converted to glutamate, entering the TCA cycle as α-ketoglutarate or as succinate

谷氨酰胺首先转化为谷氨酸，以α-酮戊二酸或琥珀酸的形式进入TCA循环。

via

通过

a γ-aminobutyric acid (GABA) shunt

γ-氨基丁酸（GABA）分流途径

. Tachyzoites can also survive the deletion of glutamate decarboxylase (GAD), the first enzyme of the GABA shunt, although their growth and virulence are impaired

速殖子也可以在缺失谷氨酸脱羧酶（GAD）——GABA旁路的第一个酶——的情况下存活，尽管它们的生长和毒力会受到损害。

. These studies reveal significant vulnerabilities and plasticity in the parasite metabolism. The TCA cycle is a vital eukaryotic pathway, but its significance for the asexual growth of

这些研究揭示了寄生虫代谢中显著的脆弱性和可塑性。TCA循环是一个重要的真核生物途径，但它对无性生长的重要性在于

T. gondii

弓形虫

remained ambiguous. The tachyzoite stage harbors all enzymes expressed in the mitochondrion

仍然不明确。速殖子阶段包含线粒体中表达的所有酶。

, and stable isotopic labeling showed evidence of a canonical oxidative-type TCA cycle

，稳定的同位素标记显示了典型的氧化型TCA循环的证据

. The first step is catalyzed by citrate synthase 1 (CS1), forming citrate from acetyl-CoA and oxaloacetate. Subsequently, aconitase (ACO) isomerizes citrate to isocitrate, which is then oxidized to α-ketoglutarate by isocitrate dehydrogenase I (IDH1). α-ketoglutarate serves as a substrate for α-ketoglutarate dehydrogenase (KDH), producing succinyl-CoA, which is converted to succinate by succinyl-CoA-synthetase (SCS).

第一步由柠檬酸合酶1（CS1）催化，将乙酰辅酶A和草酰乙酸转化为柠檬酸。随后，乌头酸酶（ACO）将柠檬酸异构化为异柠檬酸，然后异柠檬酸被异柠檬酸脱氢酶1（IDH1）氧化为α-酮戊二酸。α-酮戊二酸作为α-酮戊二酸脱氢酶（KDH）的底物，生成琥珀酰辅酶A，再由琥珀酰辅酶A合成酶（SCS）将其转化为琥珀酸。

Depletion of the SCSα subunit only partially (~30% reduction) impairs parasite growth, suggesting an alternative pathway for succinate production.

SCSα亚基的缺失仅部分（约30%的减少）损害了寄生虫的生长，表明琥珀酸生成存在替代途径。

. A succinic semialdehyde dehydrogenase (SSADH) is also present in the parasite genome, which can potentially produce succinate from succinic semialdehyde

寄生虫基因组中也存在琥珀酸半醛脱氢酶（SSADH），它可能从琥珀酸半醛生成琥珀酸。

. The latter may originate from γ-aminobutyric acid (GABA) shunt in tachyzoites

后者可能源自速殖子中的γ-氨基丁酸（GABA）旁路。

. However, the physiological role of SSADH in

然而，SSADH 的生理作用在于

T. gondii

弓形虫

remains unknown.

仍然未知。

Succinate is oxidized by succinate dehydrogenase (SDH) to form fumarate, which in turn is hydrated to produce malate by the catalytic action of fumarate hydratase (FH). Malate is oxidized to oxaloacetate by malate dehydrogenase (MDH). Oxaloacetate has multiple fates depending on the physiological status of the parasite.

琥珀酸被琥珀酸脱氢酶（SDH）氧化生成延胡索酸，延胡索酸在延胡索酸水合酶（FH）的催化作用下被水合成苹果酸。苹果酸被苹果酸脱氢酶（MDH）氧化为草酰乙酸。草酰乙酸的命运取决于寄生虫的生理状态。

It can fuse with acetyl-CoA, completing the TCA cycle.

它可以与乙酰辅酶A融合，完成TCA循环。

, and is used for aspartate synthesis

，用于合成天冬氨酸

via

通过

aspartate aminotransferase/glutamic oxaloacetic transaminase (AST/GOT)

天门冬氨酸氨基转移酶/谷草转氨酶 (AST/GOT)

. Additionally, during gluconeogenesis in glucose-starved conditions, oxaloacetate drives the formation of phosphoenolpyruvate by a mitochondrial isoform of phosphoenolpyruvate kinase (PEPCK)

此外，在葡萄糖饥饿条件下的糖异生过程中，草酰乙酸通过线粒体异构体磷酸烯醇式丙酮酸激酶（PEPCK）驱动磷酸烯醇式丙酮酸的形成。

. The latter two are cataplerotic reactions, which dispose of TCA cycle intermediates to other pathways. Glutamate dehydrogenase (GDH) catalyzing the formation of glutamate from α-ketoglutarate and the malic enzyme (ME) producing pyruvate from malate are other cataplerotic proteins encoded by the parasite, but their physiological relevance has not been studied.

后两种反应属于代谢物排出反应，这些反应将三羧酸循环中间体转移到其他途径。谷氨酸脱氢酶（GDH）催化α-酮戊二酸生成谷氨酸，苹果酸酶（ME）催化苹果酸生成丙酮酸，这是寄生虫编码的其他代谢物排出蛋白，但它们的生理相关性尚未得到研究。

The parasite also harbors two variants of malate dehydrogenases: MDH and MDH-FAD.

该寄生虫还携带两种苹果酸脱氢酶变体：MDH 和 MDH-FAD。

; however, neither has been examined in

；然而，两者都尚未被检查

T. gondii

弓形虫

。

The cataplerotic contribution of the TCA cycle to biosynthetic pathways necessitates anaplerotic replenishment of its intermediates to ensure continued operation. Pyruvate carboxylase (PyC), which converts pyruvate to oxaloacetate in the mitochondrial matrix, is the typical anaplerotic enzyme and is also present in .

TCA循环对生物合成途径的消耗作用需要通过回补反应来补充其中间体，以确保其持续运作。丙酮酸羧化酶（PyC）是一种典型的回补酶，可在线粒体基质中将丙酮酸转化为草酰乙酸，也存在于其中。

T. gondii

弓形虫

. However, it is dispensable in both glycolysis-competent and glycolysis-deficient tachyzoites

然而，在具备糖酵解能力和缺乏糖酵解能力的快速繁殖子中，它都是可有可无的。

. Hence, its metabolic role remains unknown. This study presents a systematic dissection of the central carbon metabolism in

。因此，它的代谢作用仍然未知。本研究系统地剖析了中心碳代谢在

T. gondii

弓形虫

by genetic mutagenesis of CS1, SSADH, SCSα, SDHA, ME, MDH-FAD and MDH. In-depth phenotyping of the mutants coupled with metabolomics discloses novel design features and therapeutic targets in the core metabolic network of

通过CS1、SSADH、SCSα、SDHA、ME、MDH-FAD和MDH的基因诱变，对突变体进行深入表型分析并结合代谢组学，揭示了核心代谢网络中的新设计特性和治疗靶点。

T. gondii

弓形虫

。

Results

结果

The CS1 is needed for the growth but not for the survival of tachyzoites

CS1 是速殖子生长所需要的，但不是其存活所必需的。

The genome of

基因组的

T. gondii

弓形虫

harbors three isoforms of citrate synthase (

包含三种柠檬酸合酶的同工型 (

www.ToxoDB.org

); however, only CS1 is located in the tachyzoite mitochondrion

); 然而，只有 CS1 位于速殖子线粒体中

. We started our work confirming the subcellular location of CS1 in the mitochondrion by 3’-genomic tagging (Fig.

我们通过3'-基因组标记确认了CS1在线粒体中的亚细胞定位（图。

). The

）。这个

CS1

计算机科学1

-specific CRISPR/Cas9 construct and the homology donor amplicon encoding YFP-

-特定的CRISPR/Cas9构建体和编码YFP的同源供体扩增子-

DHFR

二氢叶酸还原酶

* were transfected into tachyzoites to generate a

* 被转染到速殖子中以生成

cs1

计算机科学1

mutant (YFP

突变体 (YFP)

) (Supplementary Fig.

) （补充图。

). Surprisingly, we could isolate a

）。令人惊讶的是，我们可以分离出一个

CS1

计算机科学1

deletion strain despite its predicted essentiality in tachyzoites

尽管在速殖子中预测其具有必需性，但删除了该菌株

，

. Genomic PCR screening (Supplementary Fig.

. 基因组PCR筛选（补充图。

), genome sequencing (Supplementary Fig.

)，基因组测序（补充图。

), and indirect immunofluorescence assays (IFA) confirmed the deletion of the

），间接免疫荧光测定（IFA）证实了删除的

CS1

计算机科学1

locus in the mutant (Supplementary Fig.

突变体中的位点（补充图）。

). The

）。这个

cs1

计算机科学1

mutant grew slower in routine culture and produced smaller plaques due to poor replication than the parental strain (Fig.

突变体在常规培养中生长较慢，由于复制能力较差，产生的斑块比亲本菌株小（图。

1b–d

). The

）。这个

cs1

计算机科学1

mutant was severely attenuated in mice as judged by their higher survival rate at doses of 10

在小鼠中，通过其在10剂量下的较高存活率判断，突变体的毒性显著减弱。

parasites/mouse (84%, Fig.

寄生虫/小鼠（84%，图。

). Even at higher doses (up to 10

). 即使在较高剂量（高达10

parasites/mouse), 84% of animals survived the infection (Fig.

寄生虫/小鼠），84%的动物在感染中存活（图。

). The parasite load in mutant-infected animals was almost 10

）。感染变异型寄生虫的动物体内的寄生虫负荷几乎为10

times lower than the parental (

倍数低于亲本（

DiCre

迪克莱

) strain (Fig.

`) 应变 (图`

). These results reveal that CS1 is required for optimal growth and virulence but is not essential for tachyzoite survival.

这些结果表明，CS1 对于最佳生长和毒力是必需的，但对速殖子的存活并非必不可少。

Fig. 1: CS1 is required for parasite growth.

图1：CS1是寄生虫生长所必需的。

CRISPR/Cas9-assisted 3’-genomic tagging of the CS1 gene with a spaghetti monster HA (smHA) tag in the RH

在RH中使用CRISPR/Cas9辅助的3'-基因组标记，将CS1基因标记上意大利面怪兽HA（smHA）标签

Δku80

strain. HSP60 (magenta) was used as a mitochondrial marker. Scale bars, 5 μm.

菌株。HSP60（洋红色）用作线粒体标记。比例尺，5微米。

A 7-day plaque assay to evaluate the growth of

一个为期7天的斑块测定，用于评估

DiCre

迪克雷

(RH DiCre_T2A

Δku80Δhxgprt

) and

) 和

Δcs1

mutant (100 tachyzoites/well, 3 wells/strain).

突变体（100个速殖子/孔，3孔/株）。

The relative size of plaques from (

斑块的相对大小来自（

) (means ± SEM; ****

) （表示 ± SEM；****

≤ 0.0001, Student’s

≤ 0.0001，学生

test).

测试）。

Replication efficiency of

复制效率

DiCre

双Cre系统

and

和

Δcs1

strains. The number of vacuoles was counted 24 hours post-invasion (

菌株。在侵入后24小时计数液泡的数量（

= 4 independent experiments, means ± SEM; two-way ANOVA).

= 4次独立实验，平均值±标准误；双因素方差分析）。

Virulence test of ICR mice infected with

感染ICR小鼠的毒力测试

DiCre

双Cre系统

或

Δcs1

stains (10

污渍（10

or 10

或 10

parasites/mouse and 6–8 mice/group).

寄生虫/小鼠，6-8只小鼠/组。

Parasite burden in the peritoneal fluid of mice. ICR mice were infected with

小鼠腹腔液中的寄生虫负荷。ICR小鼠被感染了

DiCre

迪克雷

或

Δcs1

mutants (10

突变体 (10

tachyzoites/mouse and 5 mice/strain). Parasite load in peritoneal fluid was calculated 5 days post-infection by qPCR based on the non-coding fragment length of 529 bp.

速殖子/小鼠和5只小鼠/品系）。感染后5天，通过qPCR基于529 bp的非编码片段长度计算腹腔液中的寄生虫载量。

Full size image

全尺寸图像

Deletion of

删除

SCSα

compromises parasite growth, whereas SSADH is dispensable

抑制寄生虫生长，而SSADH是可有可无的

To gain additional insight into the importance of the TCA cycle, we investigated SCSα. The HA-tagged SCSα is also expressed in the mitochondrion (Fig.

为了更深入地了解TCA循环的重要性，我们研究了SCSα。带有HA标签的SCSα也在线粒体中表达（图。

). Earlier work has shown that ATc-mediated depletion of

). 早期的研究表明，ATc介导的消耗

SCSα

transcript inflicts only minor growth defects in tachyzoites

转录本在速殖子中仅造成轻微的生长缺陷

. Such an unexpected (poor) phenotype upon conditional knockdown can be attributed to a lack of stringent regulation by ATc or a nonessential role of SCSα. We, therefore, engineered a

这样的条件性敲减后出现意外（不良）表型，可以归因于ATc调控不严格或SCSα的角色非必需。因此，我们设计了

SCSα

deletion mutant by CRISPR/Cas9-assisted homologous gene replacement (Supplementary Fig.

通过CRISPR/Cas9辅助的同源基因替换获得的缺失突变体（补充图）。

). The

）。这个

Δscsα

(YFP

) mutant was isolated after pyrimethamine selection, diagnostic PCR screening and immunostaining (Supplementary Fig.

）突变体是在乙胺嘧啶筛选、诊断性PCR筛选和免疫染色后分离得到的（补充图）。

1g, h

1克，小时

). The

）。这个

Δscsα

strain was notably viable in prolonged culture despite severely impaired plaque formation and replication defect (Fig.

尽管斑块形成严重受损且复制存在缺陷，但在长时间培养中该菌株仍显著存活（图。

2b–d

). The data indicate a partial TCA cycle operation is sufficient for parasite survival.

). 数据表明，部分TCA循环操作足以维持寄生虫的生存。

Fig. 2: SCSα is vital for the lytic cycle of

图 2：SCSα 对裂解周期至关重要

T. gondii.

弓形虫。

Endogenous tagging of SCSα confirms its mitochondrial localization.

内源性标记SCSα证实了其线粒体定位。

A 7-day plaque assay to assess the growth of

一种为期7天的斑块测定法，用于评估

DiCre

双Cre系统

and

和

Δscsα

strains (100 tachyzoites/well and 3 wells for each strain).

菌株（每孔100个速殖子，每种菌株3孔）。

The relative size of plaques from (

斑块的相对大小来自 (

) (means ± SEM; ****

) (表示 ± SEM；****

≤ 0.0001, Student’s

≤ 0.0001，学生

test).

测试）。

Intracellular growth of

细胞内生长

Δscsα

in vitro. The strain was cultured for 24 h after infection with HFF cells, and the number of parasites in vacuoles was recorded by IFA (

体外。该菌株在感染HFF细胞后培养24小时，并通过IFA记录液泡内寄生虫的数量 (

= 3 independent experiments, means ± SEM; two-way ANOVA).

= 3次独立实验，平均值±标准误；双因素方差分析）。

Virulence tests of

毒力测试

DiCre

双Cre重组酶系统

and

和

Δscsα

strains in ICR mice (10

ICR小鼠品系（10

, 10

，10

, 10

，10

, 10

，10

, 10

，10

parasites/mouse and 5–8 mice/group).

寄生虫/小鼠，每组5-8只小鼠。

ICR mice (WT) or immunodeficient mice (BLAB/c nude) infected with

ICR小鼠（WT）或免疫缺陷小鼠（BLAB/c裸鼠）感染了

DiCre

双Cre系统

and

和

Δscsα

strains (10

菌株（10

tachyzoites/mouse, 5–6 mice/strain). 5 days after infection, the parasite load in the peritoneal fluid was analyzed by qPCR. (means ± SEM;

速殖子/小鼠，5-6只小鼠/品系）。感染后5天，通过qPCR分析腹腔液中的寄生虫载量。（均值±标准误；

-test).

-test)。

Naive or

天真或者

Δscsα

-immunized mice were infected with 10

- 免疫的小鼠被感染了10个单位

RH-

Luc

卢克

tachyzoites (4 mice in each group). The parasite load of mice was analyzed by the IVIS Spectrum imaging system 5 days after RH-

速殖子（每组4只小鼠）。小鼠的寄生虫载量在RH-处理后5天通过IVIS Spectrum成像系统进行分析。

Luc

吕克

infection.

感染。

The bioluminescence signal intensity of mice from (

小鼠的生物发光信号强度来自 (

) was calculated and plotted.

) 已计算并绘制。

我

The survival of naive or

天真或

Δscsα

immunized mice infected with RH-

感染RH-的免疫小鼠

Luc

吕克

(**

= 0.0084, simple survival analysis).

= 0.0084，简单生存分析）。

The survival of naive or

天真或

Δscsα

-immunized mice infected with 10

-接种疫苗的小鼠感染了10

ME49 tachyzoites. Statistical significance was examined by log-rank Mantel–Cox test (*

ME49速殖子。统计学意义通过log-rank Mantel-Cox检验进行分析（*

= 0.0144).

= 0.0144)。

Full size image

全尺寸图像

We also investigated SSADH, which can produce succinate from succinic semialdehyde

我们还研究了SSADH，它能够从琥珀酸半醛生成琥珀酸。

. The 3’HA-tagged SSADH was expressed in the mitochondrion, co-localizing with HSP60 – a known organelle marker (Fig.

带有3’HA标签的SSADH在线粒体中表达，并与已知的细胞器标记物HSP60共定位（图。

). We next engineered a

）。我们接下来设计了一个

Δssadh

strain using CRISPR/Cas9-mediated homologous recombination (Supplementary Fig.

使用CRISPR/Cas9介导的同源重组进行菌株筛选（补充图）。

1天

), pyrimethamine selection and PCR screening (Supplementary Fig.

)，乙胺嘧啶筛选和PCR筛选（补充图。

). The gene deletion had only a minor impact on plaque formation (Fig.

）。基因缺失仅对斑块形成有轻微影响（图。

3b, c

3b，c

), replication (Fig.

)，复制（图。

三维

) and virulence (Fig.

）和毒力（图。

), ruling out SSADH as a primary source for succinate entry into the TCA cycle. Functional redundancy of SSADH and SCSα was examined by deleting

），排除了SSADH作为琥珀酸进入TCA循环的主要来源。通过删除SSADH和SCSα的功能冗余性进行了研究。

SSADH

in the

在

Δscsα

strain, resulting in a double mutant (

菌株，由此产生双突变体 (

ΔscsαΔssadh

) (Supplementary Fig.

) （补充图。

2a–c

). Phenotyping revealed a further reduction in the replication of

). 表型分析显示复制进一步减少，

ΔscsαΔssadh

(Fig.

（图。

); however, the mutant could be maintained in cultures (Fig.

）；然而，该突变体可以在培养物中维持（图。

3克

). This finding consolidates our other data on the metabolic flexibility in the TCA cycle.

）。这一发现巩固了我们关于TCA循环中代谢灵活性的其他数据。

Fig. 3:

图 3：

SSADH

deletion only mildly affects parasite development.

删除仅轻微影响寄生虫的发育。

SSADH was localized in the mitochondria of

SSADH 定位于线粒体中

T. gondii

弓形虫

, co-localized with the mitochondrial marker protein HSP60.

，与线粒体标志蛋白HSP60共定位。

A 7-day plaque assay using the

使用7天的斑块测定

DiCre

迪克莱

and

和

Δssadh

strain (100 tachyzoites/well, 3 wells/strain).

菌株（100个速殖子/孔，3孔/菌株）。

The relative size of plaques from panel (

斑块的相对大小来自面板（

）。

The replication of

复制

Δssadh

. The number of parasites in the vacuole was recorded after 24 h of culture in HFF cells (mean ± SEM; two-way ANOVA).

在HFF细胞中培养24小时后，记录了液泡内寄生虫的数量（平均值±标准误；双因素方差分析）。

Virulence test of ICR mice infected with

感染ICR小鼠的毒力测试

DiCre

迪克雷

and

和

Δssadh

parasites (100 tachyzoites/mouse and 8 mice/strain).

寄生虫（每只小鼠100个速殖子，每种品系8只小鼠）。

Replication efficiency of the

复制效率

Δscsα

and

和

ΔscsαΔssadh

strains. The number of vacuoles was counted 24 h post-infection.

菌株。感染后24小时计数液泡数量。

A 10-day plaque assay of the

10天的斑块测定

Δscsα

and

和

ΔscsαΔssadh

strains (100 tachyzoites/well, 3 wells/strain).

菌株（每孔100个速殖子，每株3孔）。

Full size image

全尺寸图像

The

Δscsα

mutant is avirulent, and it can protect mice from challenge infection

突变体无毒力，且能保护小鼠免受挑战性感染。

Our further work evaluated the in vivo relevance of the

我们的进一步工作评估了体内的相关性

Δscsα

strain by intraperitoneal injection into the ICR mice. As expected, the parental (

通过腹腔注射到ICR小鼠体内的菌株。正如预期的那样，亲本（

DiCre

迪克雷

) strain killed almost all animals within 2 weeks (Fig.

）菌株在两周内几乎杀死了所有动物（图。

). In contrast, mice infected with

). 相比之下，感染了

Δscsα

survived and exhibited no clinical signs. Even at much higher doses of the mutant (~10

存活下来且没有表现出临床症状。即使在更高剂量的突变体（~10）下也是这样。

tachyzoites/mouse), no apparent virulence was observed (Fig.

速殖子/小鼠），未观察到明显毒性（图。

). The

）。这个

Δscsα

and

和

DiCre

迪克里

strains (10

菌株（10

tachyzoites/animal) were also quantified in the peritoneal fluid by qPCR five days post-infection. The parasite burden in mice parasitized by the mutant was at or below the detection threshold of qPCR (Fig.

五天后，通过qPCR还对腹腔液中的速殖子/动物进行了定量。被突变体寄生的小鼠的寄生虫负荷处于或低于qPCR的检测阈值（图。

). We tested whether the host immune response underpins the attenuated growth of the

）。我们测试了宿主免疫反应是否是减弱生长的基础。

Δscsα

strain in mice. Immunodeficient Balb/c-nu mice were infected (10

小鼠体内的菌株。免疫缺陷的Balb/c-nu小鼠被感染（10

parasites/animal), and the parasite load was quantified. The outcome resonated with the immune-competent ICR mice, as the

寄生虫/动物），并量化了寄生虫负荷。结果与免疫活性ICR小鼠一致，因为

Δscsα

tachyzoites were barely detectable in the Balb/c-nu mice (Fig.

在Balb/c-nu小鼠中，几乎检测不到速殖子（图。

）。

In the next step, we explored the potential of genetically attenuated

在下一步中，我们探索了基因减毒的潜力

Δscsα

strain as a whole-cell vaccine because it could be cultured in vitro but failed to proliferate in vivo. Indeed, the

作为一个全细胞疫苗的菌株，因为它可以在体外培养，但无法在体内增殖。确实，

Δscsα

-immunized mice rapidly cleared the challenge infection by the RH-

-免疫的小鼠迅速清除了RH-的挑战感染

Luc

吕克

tachyzoites, as evident by bioimaging of luminescence (Fig.

速殖子，通过发光生物成像明显可见（图。

2g, h

2克，h

). Furthermore, all

). 此外，所有

Δscsα

-vaccinated animals survived infection of RH-

-接种疫苗的动物在RH-感染中存活下来-

Luc

卢克

and ME49 tachyzoites (Fig.

和ME49速殖子（图。

2i, j

). In conclusion, our data highlight the therapeutic and vaccination potential of SCSα protein and

）。总之，我们的数据突显了SCSα蛋白的治疗和疫苗潜力。

Δscsα

mutant, respectively. The inability of the

突变体，分别。无法

Δscsα

strain to propagate itself in mice also suggests a critical in vivo role of SCSα in the mitochondrial metabolism.

在小鼠中努力繁殖自己的菌株也表明了SCSα在线粒体代谢中的关键体内作用。

SDHA and FH are vital, but MDH and OMT are dispensable in tachyzoites

SDHA 和 FH 在速殖子中至关重要，但 MDH 和 OMT 可有可无。

In the next step, succinate is oxidized to fumarate by SDH, also known as complex II, because it delivers TCA cycle-derived electrons to the mitochondrial electron transport chain (mETC)

接下来，琥珀酸被SDH（也称为复合物II）氧化为延胡索酸，因为它将TCA循环衍生的电子传递给线粒体电子传递链（mETC）。

。

Toxoplasma

弓形虫

SDH is a large complex of over 500 kDa, broadly divided into a membrane-anchoring domain and an enzymatic core. The latter, localizing in the matrix, comprises SDHB and SDHA. The SDHA contains the succinate-binding site and the FAD cofactor

SDH 是一个超过 500 kDa 的大复合体，大致分为膜锚定域和酶核心。后者位于基质中，包含 SDHB 和 SDHA。SDHA 包含琥珀酸结合位点和 FAD 辅因子。

. We generated an

。我们生成了一个

SDHA

琥珀酸脱氢酶复合体黄素蛋白亚基A

mutant confirmed by PCR screening and immunostaining (Supplementary Fig.

通过PCR筛选和免疫染色确认的突变体（补充图）。

1i–k

). The

）。这个

Δsdha

was severely compromised compared to the parental strain (Fig.

与亲本菌株相比，严重受损（图。

4a–c

). Accordingly, the mutant could not propagate in vivo (Fig.

). 因此，该突变体无法在体内传播（图。

4天

), resulting in the survival of all infected mice (Fig.

），结果所有受感染的小鼠都存活了下来（图。

). Even at much higher doses of the

). 即使在更高剂量下

Δsdha

mutant (~10

突变体 (~10

tachyzoites/mouse), no significant virulence was observed (Fig.

速殖子/小鼠），未观察到显著的毒力（图。

). Similar to the

）。类似于

Δscsα

mutant, immunization of mice with

突变体，用小鼠免疫接种

Δsdha

induced effective host immune protection against challenge with wild-type RH-

诱导有效的宿主免疫保护以对抗野生型RH的挑战

Luc

吕克

and ME49 (Fig.

和 ME49（图。

4g–j

4克–丁

). Next, we tried to ablate the fumarate hydratase (FH) protein, converting SDHA-derived fumarate to malate. However, our efforts to delete the

)。接下来，我们尝试消融延胡索酸水合酶 (FH) 蛋白，将SDHA衍生的延胡索酸转化为苹果酸。然而，我们试图删除

gene were unsuccessful, suggesting its necessity for parasite survival.

基因未能成功，表明其对寄生虫生存的必要性。

Fig. 4: Knockout of

图4：敲除

SDHA

琥珀酸脱氢酶复合体黄素蛋白亚基A

is detrimental for the parasite.

对寄生虫有害。

A 6-day plaque assay to assess the growth of

6天斑块测定以评估生长情况

DiCre

迪克里

and

和

Δsdha

strains (100 tachyzoites/well and 3 wells for each strain).

菌株（每孔100个速殖子，每种菌株3孔）。

The relative size of the plaques from (

斑块的相对大小来自 (

）。

The replication efficiency of

复制效率

DiCre

迪克莱

and

和

Δsdha

strains in HFF cells were compared. The parasized HFF cells were cultured for 24 h, and the number of parasites in the vacuoles was determined by IFA. (

在HFF细胞中比较了菌株。将寄生的HFF细胞培养24小时，并通过IFA测定液泡内寄生虫的数量。（

= 3 independent experiments, means ± SEM; ****

= 3 次独立实验，均值 ± 标准误；****

≤ 0.0001, two-way ANOVA).

≤ 0.0001，双因素方差分析）。

WT mice (ICR) or immune-deficient mice (BLAB/c nude) were infected with

野生型小鼠（ICR）或免疫缺陷小鼠（BLAB/c裸鼠）被感染了

DiCre

迪克莱

and

和

Δsdha

mutants (10

突变体 (10

tachyzoites/mouse and 5 mice/strain). Parasite loads in peritoneal fluid were calculated by qPCR after 5 days of infection. The results are mean ± SEM. ****

速殖子/小鼠和5只小鼠/菌株）。感染5天后通过qPCR计算腹腔液中的寄生虫载量。结果为平均值±标准误。****

≤ 0.0001, Student’s

≤ 0.0001，学生

test.

测试。

Virulence test of ICR mice infected with

感染ICR小鼠的毒力测试

DiCre

双Cre系统

或

Δsdha

parasites (100 tachyzoites/mouse and 10 mice/strain).

寄生虫（每只小鼠100个速殖子，每种品系10只小鼠）。

Virulence test of ICR mice infected with a dose of 10

感染剂量为10的ICR小鼠毒力测试

parasites of the specified strains (8 mice). The

指定菌株的寄生虫（8只小鼠）。

Δsdha

mutant was also inoculated at higher doses (10

突变体还以更高剂量（10

, 10

，10

, 10

，10

parasites/mouse; 5 mice/group).

寄生虫/小鼠；每组5只小鼠）。

Naive or

天真或

Δsdha

-immunized mice were infected with 10

- 免疫的小鼠被感染了10个单位

RH-

Luc

吕克

tachyzoites (3 mice in each group). The parasite load of mice was analyzed by the IVIS Spectrum imaging system 5 days after RH-

速殖子（每组3只小鼠）。RH-感染后5天，使用IVIS Spectrum成像系统分析小鼠的寄生虫负荷。

Luc

卢克

infection.

感染。

The bioluminescence signal intensity of mice from (

小鼠的生物发光信号强度来自 (

) was calculated and plotted (means ± SEM; ****

）被计算并绘制（平均值±标准误；****

≤ 0.0001, Student’s

≤ 0.0001，学生

test).

测试）。

我

The survival of naive or

天真或

Δsdha

immunized mice infected with RH-

感染RH-的免疫小鼠

Luc

吕克

。

The survival of naive or

天真或

Δsdha

-immunized mice infected with ME49 tachyzoites (10

-接种疫苗的小鼠感染了ME49速殖子（10

）。

Full size image

全尺寸图像

Our following work focused on MDH, oxidizing malate into oxaloacetate. It was earlier shown to localize on the parasite mitochondrion

我们接下来的工作集中在MDH上，它将苹果酸氧化为草酰乙酸。早前的研究表明它定位于寄生虫的线粒体中。

. However, 3’-genomic tagging of MDH with an HA tag revealed its expression in the cytoplasm, localizing with ALD (Fig.

然而，用HA标签对MDH进行3'-基因组标记揭示了其在细胞质中的表达，并与ALD共定位（图。

). To assess its physiological importance, we engineered a knockout strain using the CRISPR/Cas9 system (Supplementary Fig.

为了评估其生理重要性，我们使用CRISPR/Cas9系统构建了一个敲除菌株（补充图）。

). PCR screening (Supplementary Fig.

). PCR筛选（补充图。

) and genome sequencing (Supplementary Fig.

) 和基因组测序（补充图。

) verified the deletion of MDH. The plaque (Fig.

）验证了MDH的删除。斑块（图。

5b, c

5b，c

), replication (Fig.

)，复制（图。

5天

) and virulence (Fig.

）和毒力（图。

) assays displayed that loss of MDH had no discernible effect on the parasite. We were, therefore, promoted to consider alternative pathways that could supply malate to the cytoplasm. Tachyzoites encode a 2-oxoglutarate/malate translocase (OMT, TGGT1_274060), which may transport malate from the mitochondrion to the cytoplasm.

）测定显示，MDH 的缺失对寄生虫没有明显影响。因此，我们进一步考虑了其他可能为细胞质提供苹果酸的替代途径。速殖子编码一种 2-氧戊二酸/苹果酸转运酶（OMT，TGGT1_274060），该酶可能将苹果酸从线粒体转运到细胞质中。

Indeed, the HA-tagged OMT was expressed in the mitochondrion. Subsequently, the .

确实，带有HA标签的OMT在线粒体中表达。随后，。

Δomt

mutant was isolated by CRISPR/Cas9, pyrimethamine selection, screening PCR and immunostaining (Supplementary Fig.

通过CRISPR/Cas9、乙胺嘧啶筛选、PCR筛选和免疫染色分离出突变体（补充图）。

5a, b

). Deleting the

). 删除

OMT

gene only slightly affected the tachyzoite growth, as evaluated by plaque, replication, virulence and parasite load assays (Supplementary Fig.

基因仅轻微影响了速殖子的生长，通过斑块、复制、毒力和寄生虫负荷试验评估（补充图）。

5c–f

). The data imply a functional redundancy between OMT and MDH for supplying malate to the parasite cytosol.

）。数据表明，OMT和MDH在为寄生虫细胞质提供苹果酸方面存在功能冗余。

Fig. 5: MDH is not required for the lytic cycle.

图5：MDH对于溶解周期不是必需的。

Construction of the MDH-HA localization strain from RH

从RH构建MDH-HA定位菌株

Δku80

. MDH was found in the cytoplasm of

MDH存在于细胞质中

T. gondii

弓形虫

, co-localized with the characteristic cytoplasmic protein ALD.

，与特征性的细胞质蛋白ALD共定位。

The plaque size between parental and

亲本与子代之间的斑块大小

Δmdh

was compared (7 d, 100 tachyzoites were used in each well, and 3 wells for each strain).

进行了比较（7天，每孔使用100个速殖子，每株3孔）。

The relative size of the plaques from (

斑块的相对大小来自（

) (means ± SEM; Student’s

) (均值±标准误；学生氏

test).

测试）。

The replication assays of parental and

亲本和子代的复制测定

Δmdh

strains in HFF cells were compared. The strains infected with HFF cells were cultured for 24 h, and the number of parasites in the vacuoles was determined by IFA.

在HFF细胞中比较了不同菌株。将感染HFF细胞的菌株培养24小时，并通过IFA测定液泡内寄生虫的数量。

ICR mice were injected intraperitoneally with wild-type and

ICR小鼠被腹腔注射野生型和

Δmdh

tachyzoites (100 parasites/mouse and 10 mice/strain).

速殖子（每只小鼠100个寄生虫，每种品系10只小鼠）。

Full size image

全尺寸图像

Toxoplasma

弓形虫

encodes a functional MDH-FAD located in mitochondria

编码位于线粒体中的功能性 MDH-FAD

Our data above describing cytoplasmic expression and the nonessential nature of MDH indicated the presence of an isoform for malate oxidation in the parasite mitochondrion. The database search identified a FAD-dependent malate-dehydrogenase (MDH-FAD). 10×HA fusion of the native MDH-FAD by 3′-genomic tagging disclosed a mitochondrial expression, co-localizing with HSP60 (Fig. .

我们的上述数据描述了胞质表达和 MDH 的非必需性质，表明寄生虫线粒体中存在一种负责苹果酸氧化的同工酶。数据库搜索鉴定出一种 FAD 依赖性的苹果酸脱氢酶 (MDH-FAD)。通过 3′ 基因组标记将天然 MDH-FAD 与 10×HA 融合，揭示了其线粒体表达，并与 HSP60 共定位（图。

）。

电报

MDH-FAD was further examined by functional complementation of a

MDH-FAD通过功能互补进一步检验了

Saccharomyces cerevisiae

酿酒酵母

mutant (

突变体 (

ScΔmdh1

), which cannot grow on a non-fermentable carbon source

），它不能在非发酵碳源上生长

. As shown (Fig.

。如图所示（图。

), the parental (BY4741) and

），亲本（BY4741）和

ScΔmdh1

strains transformed with the empty plasmid (

转化了空质粒的菌株（

pRS416

) or construct (

）或构造（

pRS416

-MDH-FAD) were all able to grow on the fermentable carbon source. As expected, the mutant growth was restored by expressing

-MDH-FAD）都能够利用可发酵的碳源生长。正如预期的那样，通过表达恢复了突变体的生长。

电报

MDH-FAD, and

MDH-FAD，以及

pRS416

failed to rescue its growth on glycerol (YPG, non-fermentable carbon source) (Fig.

未能在甘油（YPG，非发酵碳源）上挽救其生长（图。

). We constructed an

）。我们构建了一个

ScΔmdh

电报

MDH-FAD strain expressing the parasite enzyme from chromosome XI to validate our results. Consistently,

表达来自第十一号染色体的寄生虫酶的MDH-FAD菌株，以验证我们的结果。一致地，

电报

MDH-FAD rescued the

MDH-FAD 恢复了

ScΔmdh1

mutant on YPG plates (Fig.

YPG平板上的突变体（图。

), showing its functionality in yeast.

），展示了其在酵母中的功能。

Fig. 6:

图6：

Toxoplasma

弓形虫

expresses MDH-FAD in the mitochondrion.

在线粒体中表达MDH-FAD。

Immunofluorescence assays were performed on the 10×HA-tagged strain to determine the localization of MDH-FAD. HSP60 was used as a marker specific to the mitochondrion for colocalization.

对10×HA标签的菌株进行了免疫荧光测定，以确定MDH-FAD的定位。HSP60被用作线粒体特异性标记，用于共定位分析。

Toxoplasma

弓形虫

MDH-FAD cDNA was amplified and cloned into the

MDH-FAD cDNA 被扩增并克隆到

pRS416

. The parental (BY4741) and

. 亲本（BY4741）和

ScΔmdh1

strains transformed with the empty plasmid (

转化了空质粒的菌株（

pRS416

) or construct (

）或构造（

pRS416

-MDH-FAD) were characterized on medium with glucose or glycerin as a sole carbon source by serial dilution assay. The plates were grown at 30 °C for 3 days.

在以葡萄糖或甘油为唯一碳源的培养基上，通过系列稀释法对-MDH-FAD）进行了表征。平板在30°C下培养3天。

The

电报群

MDH-FAD expressing mini gene was reintroduced into the chromosome of the

MDH-FAD 表达的迷你基因被重新引入到染色体中

ScΔmdh1

. Growth of parental (BY4741),

. 亲本（BY4741）的生长，

ScΔmdh1

and

和

ScΔmdh1-Tg

MDH-FAD strains was tested either on glucose (YPD, fermentable carbon source) or on glycerol (YPG, non-fermentable carbon source) for 3 days at 30 °C.

MDH-FAD菌株在30°C下，分别在葡萄糖（YPD，可发酵碳源）或甘油（YPG，不可发酵碳源）上培养3天。

Full size image

全尺寸图像

The MDH-FAD is required for the asexual reproduction of tachyzoites

MDH-FAD 是速殖子无性繁殖所必需的。

In the next step, we investigated the metabolic relevance of MDH-FAD in tachyzoites. A pool of transgenic parasites expressing rapamycin-inducible dimerizable Cre (

在下一步中，我们研究了速殖子中MDH-FAD的代谢相关性。我们使用了一个表达雷帕霉素诱导的可二聚化Cre重组酶的转基因寄生虫池 (

DiCre

双Cre系统

) and harboring loxP-flanked MDH-FAD (

) 并且携带 loxP 侧翼的 MDH-FAD (

电报

MDH-FAD-cKD) was generated. Subsequently, a

MDH-FAD-cKD）被生成。随后，一个

Δmdh-fad

strain was produced by treating the

菌株是通过处理产生的

电报

MDH-FAD-cKD strain with rapamycin (Supplementary Fig.

使用雷帕霉素的MDH-FAD-cKD菌株（补充图）。

). The mutant was validated by PCR (Supplementary Fig.

）。通过PCR验证了该突变体（补充图。

3天

), genome sequencing (Supplementary Fig.

), 基因组测序（补充图。

) and immunofluorescence (Supplementary Fig.

`) 和免疫荧光（补充图 `

) assay. Although the

）测定。尽管

Δmdh-fad

strain formed notably smaller plaques (Fig.

菌株形成的斑块明显较小（图。

7a, b

) and exhibited much smaller parasitophorous vacuoles (Fig.

）并显示出小得多的寄生泡（图。

), the

)，这个

Δmdh-fad

mutant could be maintained in routine culture for a long time. In extended work, we infected mice with the

突变体可以在常规培养中维持很长时间。在后续工作中，我们用

Δmdh-fad

strain and quantified the parasite load in the peritoneal fluid (Fig.

菌株并量化了腹腔液中的寄生虫负荷（图。

7天

). Indeed, the deletion of MDH-FAD significantly reduced the parasite proliferation. Besides, mice parasitized by the

). 实际上，MDH-FAD的删除显著减少了寄生虫的增殖。此外，被寄生的小鼠

Δmdh-fad

mutant survived longer than those infected with the parental strain (Fig.

突变体的存活时间比感染亲本菌株的个体更长（图。

), indicating a reduced virulence upon deletion of

），表明在删除后毒力降低

MDH-FAD

. These results emphasize the crucial function of MDH-FAD in promoting tachyzoite development in vitro and in vivo.

这些结果强调了MDH-FAD在促进速殖子体外和体内发育中的关键功能。

Fig. 7: MDH-FAD is required for

图7：MDH-FAD是必需的

T. gondii

弓形虫

growth.

增长。

The growth of

增长

Δmdh-fad

and

和

DiCre

迪克雷

tachyzoites in vitro was compared by plaque assay (7 d, 100 tachyzoites/well and 3 wells for each strain).

通过斑块测定法比较了速殖子在体外的情况（7天，每孔100个速殖子，每种菌株3个孔）。

The relative size of plaques from (

斑块的相对大小来自 (

) (means ± SEM; ****

) （表示平均值±标准误；****

≤ 0.0001, Student’s

≤ 0.0001，学生

test).

测试）。

The replication assays of

复制实验

DiCre

迪克雷

and

和

Δmdh-fad

in vitro. After 24 h of infection with HFF cells, the number of parasites in vacuoles was determined by IFA.

体外。感染HFF细胞24小时后，通过IFA测定空泡内寄生虫的数量。

Measurement of parasite burden in ICR mice. ICR mice were infected by intraperitoneal injection of

测量ICR小鼠的寄生虫负荷。ICR小鼠通过腹腔注射感染。

DiCre

迪克雷

and

和

Δmdh-fad

tachyzoites (10

速殖子 (10

tachyzoites/mouse and 5 mice/strain). Parasite loads in peritoneal fluid were calculated by qPCR after 5 days of infection.

速殖子/小鼠和5只小鼠/品系）。感染5天后，通过qPCR计算腹腔液中的寄生虫载量。

ICR mice were injected intraperitoneally with wild-type and

ICR小鼠被腹腔注射了野生型和

Δmdh-fad

tachyzoites (100 parasites/mouse and 10 mice/strain).

速殖子（每只小鼠100个寄生虫，每种品系10只小鼠）。

DiCre

迪克莱

and

和

Δmdh-fad

tachyzoites (5 × 10

速殖子 (5 × 10

) were harvested and cultured for 4 h in a glucose-free medium supplemented with 8 mM

）被收获并在补充了8 mM的无葡萄糖培养基中培养4小时

-glutamine. The levels of

-谷氨酰胺。水平的

C incorporation in glycolysis and TCA cycle metabolites were determined by the LC-MS. M0-M5 represents the number of carbons in the

通过LC-MS测定糖酵解和TCA循环代谢物中的C掺入情况。M0-M5代表碳的数量。

C-labeled metabolites. Values are mean ± SEM of five independent experiments (

C标记的代谢物。数值为五次独立实验的平均值±标准误 (

= 5, two-way ANOVA).

= 5，双因素方差分析）。

Incorporation of

包含

C into glycolysis and TCA cycle metabolites, as determined by UHPLC-HRMS. The extracellular tachyzoites of the

C进入糖酵解和TCA循环代谢物，由UHPLC-HRMS测定。

DiCre

迪克莱

and

和

Δmdh-fad

strains were incubated with 8 mM

菌株与8 mM一起孵育

-glucose for 4 h (

-葡萄糖持续4小时（

= 5 biologically independent samples, means ± SEM; Student’s

= 5个生物学独立样本，平均值±标准误；学生氏

test).

测试）。

Effect of

效果

MDH-FAD

deletion on malate formation (data from panel (

删除对苹果酸形成的影响（数据来自面板（

)). The peak area represents the sum of M0-M4 peak areas (Student’s

)). 峰面积代表 M0-M4 峰面积的总和（学生的

test).

测试）。

我

Effect of

效应

MDH-FAD

deletion on malate formation (data from panel (

删除对苹果酸形成的影响（数据来自面板（

)). The peak area represents the sum of M0-M4 peak areas (Student’s

)). 峰面积代表 M0-M4 峰面积的总和（学生的

test).

测试）。

The total of NAD

NAD的总数

and NADH in

和NADH在

DiCre

双Cre系统

and

和

Δmdh-fad

parasites was measured using a NAD

使用NAD测量寄生虫

/NADH assay kit. Means ± SEM from two independent experiments (

/NADH检测试剂盒。两次独立实验的平均值±标准误（

= 2), each with two replicates (****

= 2)，每个有两个重复（****

≤ 0.0001, Student’s

≤ 0.0001，学生

test).

测试）。

Mitotracker staining was used to examine the effect of

使用Mitotracker染色来检查效果

MDH-FAD

depletion on the membrane potential (Δ

膜电位的消耗（Δ

ψm

）。

The number of Δ

Δ 的数量

ψm

positive tachyzoites in

速殖子阳性在

DiCre

迪克莱

and

和

Δmdh-fad

strains was counted (

菌株被计数 (

****

≤ 0.0001, Student’s

≤ 0.0001，学生

test). The parental tachyzoites showed the typical intense staining of the mitochondrion and were classified as Δ

测试）。亲本速殖子显示出线粒体典型的强烈染色，并被归类为 Δ

ψm

-positive.

-积极的。

Full size image

全尺寸图像

The

Δmdh-fad

strain displays impaired metabolism and membrane potential

菌株表现出代谢和膜电位受损

To investigate whether deletion of

为了调查是否删除

MDH-FAD

impacted the intermediates of the central carbon metabolism, we labeled extracellular tachyzoites of the

影响了中心碳代谢的中间体，我们标记了细胞外的速殖子

Δmdh-fad

and parental strains with [

和具有 [ 的亲本菌株

C]-glutamine for 4 h. We determined the inclusion of glutamine-derived carbon (

C]-谷氨酰胺持续4小时。我们确定了谷氨酰胺衍生碳的包含情况 (

C) into glycolytic and TCA metabolites using LC-MS. A change in

C) 使用LC-MS转化为糖酵解和TCA代谢物。一个变化

C labeling of α-ketoglutarate and succinate was not apparent (Fig.

C标记的α-酮戊二酸和琥珀酸不明显（图。

). However, the tracer incorporation into malate was significantly increased (Fig.

）。然而，示踪剂掺入苹果酸的量显著增加（图。

). We also noted that the inclusion of glucose-derived

）。我们还注意到，包含葡萄糖衍生的

C into fumarate and malate was increased upon deleting

删除后，C 转化为延胡索酸和苹果酸增加。

MDH-FAD

(Fig.

（图。

7克

). As expected, the abundance of malate was considerably increased in the mutant (Fig.

). 如预期的那样，突变体中苹果酸的丰度显著增加（图。

7小时

). We observed a difference in malate labeling of the

）。我们观察到苹果酸标记存在差异。

Δmdh-fad

strain fed with [

应变喂养 [

C]-glutamine and [

C]-谷氨酰胺和[

C]-glucose (Fig.

C]-葡萄糖（图。

7h, i

7小时，我

). The glucose-derived

）。葡萄糖衍生的

C into the TCA cycle requires oxaloacetate, whereas the influx of [

C进入TCA循环需要草酰乙酸，而[

C]-glutamine-derived carbon into malate is not dependent on oxaloacetate. A deletion of

C]-谷氨酰胺衍生的碳进入苹果酸并不依赖于草酰乙酸。删除

MDH-FAD

may reduce the availability of oxaloacetate in the mitochondrion, resulting in a lower glucose-derived

可能会降低线粒体中草酰乙酸的可用性，从而导致葡萄糖衍生的

C inclusion into malate. Our attempts to detect oxaloacetate in the parasites were unsuccessful, likely due to its rapid conversion to aspartate by aspartate aminotransferase (AST). We identified a mitochondrion-localized AST in

C并入苹果酸。我们尝试在寄生虫中检测草酰乙酸，但未成功，可能是因为它被天冬氨酸氨基转移酶（AST）迅速转化为天冬氨酸。我们鉴定了一种线粒体定位的AST。

T. gondii

弓形虫

, as shown by the 10×HA gene fusion of the native AST (Supplementary Fig.

，如原生AST的10×HA基因融合所示（补充图）。

). The deletion of

）。删除

AST

抽象语法树

in the

在

DiCre

迪克莱

strain resulted in a notable reduction in plaque formation, replication, virulence, and parasite load (Supplementary Fig.

菌株导致斑块形成、复制、毒力和寄生虫载量显著减少（补充图）。

6b–g

）。

Interconversion of malate and oxaloacetate by MDH-FAD depends on NAD

MDH-FAD依赖NAD进行苹果酸和草酰乙酸之间的相互转化。

加号

/NADH. We, therefore, measured the total NAD

/NADH。因此，我们测量了总的NAD

and NADH levels in the mutant (Fig.

以及突变体中的NADH水平（图。

), which declined by about 80% upon loss of MDH-FAD expression. Because NADH is an electron donor in the inner mitochondrial membrane, we examined the membrane potential (Δ

），在MDH-FAD表达丧失时下降了约80%。由于NADH是线粒体内膜中的电子供体，我们检测了膜电位（Δ

ψm

) using mitotracker staining (Fig.

) 使用 mitotracker 染色 (Fig.

). Intracellular tachyzoites of the parental strain had well-stained mitochondrion, classified as Δ

）。亲本菌株的细胞内速殖子具有染色良好的线粒体，分类为 Δ

ψm

(Fig.

（图。

). In contrast, mitochondrial staining was not detectable in over 80% of the

）。相反，在超过80%的

Δmdh-fad

parasites (Fig.

寄生虫（图。

), signifying a collapse of Δ

），标志着 Δ 的崩溃

ψm

in the organelle upon deletion of

在细胞器中，删除后

MDH-FAD

。

The ME, localized in the nucleus, is essential for parasite development

位于细胞核中的ME对寄生虫的发育至关重要。

Our follow-up work focused on ME, which catalyzes malate to pyruvate while producing NADPH. We first determined its subcellular localization using antisera and by ectopic expression of epitope-tagged ME (Fig.

我们的后续工作集中在ME上，它催化苹果酸转化为丙酮酸，同时产生NADPH。我们首先使用抗血清并通过对表位标记的ME进行异位表达来确定其亚细胞定位（图。

8a, b

). Immunofluorescent staining showed that ME co-localized with Hoechst (DNA dye, Fig.

免疫荧光染色显示，ME与Hoechst（DNA染料，图）共定位。

8a, b

). In the next step, we made a conditional mutant of ME using the auxin-inducible degron (AID) system (Supplementary Fig.

）。在下一步中，我们使用了生长素诱导的降解子（AID）系统制作了ME的条件突变体（补充图。

) because its knockout

`) 因为它令人震撼`

via

通过

CRISPR/Cas9-mediated homologous replacement was not feasible. PCR screening of the drug-resistant parasite clones identified a miniAID-ME mutant (Supplementary Fig.

CRISPR/Cas9介导的同源替换不可行。对药物抗性寄生虫克隆的PCR筛选鉴定出一个miniAID-ME突变体（补充图）。

3克

), in which the protein could be rapidly degraded by indole-3-acetic acid (IAA, Fig.

)，蛋白质可以被吲哚-3-乙酸 (IAA，图) 迅速降解。

). A knockdown of ME resulted in reduced plaque size (Fig.

）。ME的敲除导致斑块尺寸减小（图。

8天

) and smaller vacuole size distribution (Fig.

）和较小的液泡大小分布（图。

). Besides, the ICR mice infected by the miniAID-ME tachyzoites (10

). 此外，感染了miniAID-ME速殖子的ICR小鼠（10

, i.p.), followed by treatment with IAA in drinking water, demonstrated a highly-reduced parasite load compared to the untreated control (-IAA) and the parental strain (Fig.

，腹腔注射），随后在饮水中使用IAA进行治疗，结果显示与未处理的对照组（-IAA）和亲本菌株相比，寄生虫载量显著减少（图。

). These results underline the critical role of ME in tachyzoite propagation.

这些结果强调了ME在速殖子传播中的关键作用。

Fig. 8: ME is critical for the lytic cycle of tachyzoites.

图8：ME 对速殖子的裂解周期至关重要。

Immunofluorescent microscopic analysis of ME. Hoechst is a cell nucleus marker.

免疫荧光显微分析ME。Hoechst是细胞核标记物。

Immunofluorescence detection of co-localization of native ME with the nucleus marker, Hoechst.

免疫荧光检测天然ME与细胞核标记Hoechst的共定位。

Immunofluorescent localization and regulation of ME by indole-3-acetic acid (IAA). Intracellular miniAID-ME tachyzoites cultured in the absence or presence of 500 μM IAA were stained by α-HA and α-ALD antibodies.

通过吲哚-3-乙酸（IAA）对ME进行免疫荧光定位和调控。在不存在或存在500 μM IAA的情况下，细胞内miniAID-ME速殖子用α-HA和α-ALD抗体染色。

The growth of TiR1 (RH

TiR1（RH）的增长

Δku80

-TiR1) and miniAID-ME tachyzoites was compared by plaque assay (−/+500 μM IAA, 7d, 100 tachyzoites/well and 3 wells for each strain).

通过斑块测定法比较了TiR1和miniAID-ME速殖子（−/+500 μM IAA，7天，每孔100个速殖子，每种菌株3个孔）。

Intracellular growth of TiR1 and miniAID-ME strains (−/+500 μM IAA, 24 h, means ± SEM,

TiR1和miniAID-ME菌株的细胞内生长（−/+500 μM IAA，24 h，平均值±SEM，

= 3 independent experiments.

= 3 次独立实验。

****

≤ 0.0001, two-way ANOVA).

≤ 0.0001，双因素方差分析）。

ICR mice were infected with TiR1 and miniAID-ME tachyzoites by intraperitoneal injection (10

ICR小鼠通过腹腔注射感染了TiR1和miniAID-ME速殖子（10

tachyzoites/mouse and 5 mice/strain). IAA was added to the drinking water or not. After 5 days of infection, parasite loads in peritoneal fluids were calculated by qPCR based on the 529 gene,

速殖子/小鼠和5只小鼠/品系）。IAA被添加到饮用水中或不添加。感染5天后，通过基于529基因的qPCR计算腹腔液中的寄生虫载量。

****

≤ 0.0001, Student’s

≤ 0.0001，学生

test.

测试。

，

Immunostaining and quantification of histone acetylation in the parasite nucleus. Analyses were performed using rabbit anti-H3K9ac antibody, and the fluorescence intensity was quantified. Each symbol in panel (

免疫染色和寄生虫细胞核内组蛋白乙酰化的定量分析。分析使用了兔抗H3K9ac抗体，并对荧光强度进行了量化。面板中的每个符号 (

) marks the pixel density of a parasite nucleus (means ± SEM,

）标记了寄生虫细胞核的像素密度（平均值±标准误，

= 3 assays.

= 3 次测定。

****

≤ 0.0001, Student’s

小于等于0.0001，学生的

test).

测试）。

Full size image

全尺寸图像

The ME-depleted strain exhibits a selective perturbation of transcriptome

ME耗尽菌株表现出选择性的转录组扰动。

ME produces pyruvate from malate, a crucial precursor for several metabolic pathways in the cytoplasm, apicoplast, and mitochondrion

ME利用苹果酸生成丙酮酸，这是细胞质、顶复体和线粒体中多种代谢途径的关键前体。

. While not known in

. 虽然不为人知

T. gondii

弓形虫

, pyruvate metabolism in the nucleus is critical for histone acetylation and chromatin remodeling in mammalian cells

，细胞核中的丙酮酸代谢对哺乳动物细胞的组蛋白乙酰化和染色质重塑至关重要

，

. To determine whether the ME depletion impacts histone acetylation in tachyzoites, we conducted an immunofluorescence analysis using the specificity of the H3K9ac-directed antibodies (Fig.

为了确定ME耗竭是否影响速殖子的组蛋白乙酰化，我们使用H3K9ac导向抗体的特异性进行了免疫荧光分析（图。

8克

). Our findings revealed that ME depletion markedly decreased the H3K9ac signal intensity in the parasite nucleus (Fig.

）。我们的研究结果表明，ME的缺失显著降低了寄生虫细胞核中的H3K9ac信号强度（图。

8小时

), indicating that ME-derived pyruvate plays a pivotal role in acetyl-CoA synthesis for epigenetic regulation in tachyzoites.

），表明ME衍生的丙酮酸在速殖子的乙酰辅酶A合成中对表观遗传调控起着关键作用。

The miniAID-ME strain was grown with or without IAA for 12 h, and then samples were collected for RNA sequencing to assess the global consequences of ME knockdown. The ME-depleted strain exhibited a lower expression of 2342 transcripts, while 1731 were more abundant (Supplementary Fig.

miniAID-ME 菌株在有或没有 IAA 的情况下生长 12 小时，然后收集样品进行 RNA 测序，以评估 ME 敲低的全局影响。ME 缺失菌株表现出 2342 个转录本的表达降低，而 1731 个转录本的表达更高（补充图）。

). Several histone deacetylases and histone acetyltransferases, including HDAC1, HDAC2, HDAC5, SiR2, MYST, GCN5, and GNAT family protein, displayed dysregulated expression (Supplementary Fig.

）。包括HDAC1、HDAC2、HDAC5、SiR2、MYST、GCN5和GNAT家族蛋白在内的几种组蛋白去乙酰化酶和组蛋白乙酰转移酶显示出表达失调（补充图。

). The qPCR verified the lower expression of SIR2 and more abundance of GCN5-B, HDAC5 and MYST-B transcripts in the ME-depleted mutants (Supplementary Fig.

）。qPCR验证了SIR2的较低表达以及GCN5-B、HDAC5和MYST-B转录本在ME缺失突变体中更为丰富的表达（补充图。

). Furthermore, KEGG enrichment analysis revealed that DNA replication, mismatch repair and amino sugar and nucleotide sugar metabolism were the most significantly affected in instances where ME expression was lacking (Supplementary Fig.

此外，KEGG富集分析显示，在ME表达缺失的情况下，DNA复制、错配修复以及氨基糖和核苷酸糖代谢受到的影响最为显著（补充图）。

7天

）。

Nuclear localization of ME is imperative for tachyzoite growth

ME的核定位对速殖子生长至关重要

ME comprises the N-terminal, Tudor, and C-terminal domains (Fig.

ME 包含 N 端、Tudor 和 C 端结构域（图。

). Notably, the C-terminal contains two nuclear localization signals (Fig.

). 值得注意的是，C端包含两个核定位信号（图。

). To determine the functional relevance of these domains, we complemented the miniAID-ME strain by the N-terminal domain, both the N- and C-terminal domains, or the complete open reading frame of ME at the

为了确定这些结构域的功能相关性，我们通过ME的N端结构域、N和C端结构域、或完整的开放阅读框在miniAID-ME菌株中进行了互补。

UPRT

locus (Fig.

位点（图。

九号电池

). A diagnostic PCR of each strain confirmed the integration of the respective insert (Supplementary Fig.

）。每个菌株的诊断性PCR证实了相应插入片段的整合（补充图）。

8a–c

, comp-ME, comp-ME-N/C, comp-ME-N). Immunostaining revealed a nuclear localization in the comp-ME and comp-ME-N/C strains (Fig.

，comp-ME，comp-ME-N/C，comp-ME-N）。免疫染色显示在comp-ME和comp-ME-N/C菌株中呈现核定位（图。

). In contrast, the comp-ME-N strain showed a cytosolic expression (Fig.

）。相反，comp-ME-N 菌株显示出胞质表达（图。

九乘

), indicating a role of the C-terminal in the nuclear transport of ME. As anticipated, the comp-ME strain displayed a fully restored lytic cycle (plaque formation, Fig.

），这表明C端在ME的核运输中起作用。正如预期的那样，comp-ME菌株表现出完全恢复的裂解周期（斑块形成，图。

9天

) and replication (Fig.

`) 和复制 (图`

) in the IAA-treated/ME-depleted mutant. A recovered phenotype of the comp-ME-N/C strain (+IAA) suggested a nonessential nature of the Tudor domain. Unlike the nuclear-localized constructs, the IAA-exposed comp-ME-N strain could not grow (Fig.

）在IAA处理/ME耗尽的突变体中。comp-ME-N/C菌株（+IAA）的恢复表型表明Tudor结构域并非必需。与核定位构建体不同，暴露于IAA的comp-ME-N菌株无法生长（图。

9d, e

9天，e

), highlighting the importance of ME expression in the parasite nucleus.

），强调了ME表达在寄生虫细胞核中的重要性。

Fig. 9: Functional analysis of the ME domain.

图 9：ME 结构域的功能分析。

The ME comprises the N-terminal, Tudor1, Tudor2, and C-terminal domains. The nuclear localization signal (NLS) of ME was analyzed using

ME 包含 N 端、Tudor1、Tudor2 和 C 端结构域。ME 的核定位信号 (NLS) 已通过分析得到。

https://www.novoprolabs.com/tools/nls-signal-prediction

。

The domains were expressed in the

这些结构域被表达在

UPRT

locus of miniAID-ME to create the comp-ME, comp-ME-N/C, and comp-ME-N mutants.

miniAID-ME 的位点用于创建 comp-ME、comp-ME-N/C 和 comp-ME-N 突变体。

The expression of ME-Ty, ME-N/C-Ty and ME-N-Ty were confirmed by IFA using an anti-Ty antibody.

通过使用抗Ty抗体的IFA确认了ME-Ty、ME-N/C-Ty和ME-N-Ty的表达。

Plaque assays were carried out under −/+500 μM IAA for 7 days.

在−/+500 μM IAA条件下进行了7天的斑块测定。

The parasites were subjected to intracellular replication assays under specified conditions. (

寄生虫在特定条件下进行了细胞内复制实验。

= 3 independent assays, means ± SEM; ****

= 3 次独立测定，平均值 ± 标准误；****

≤ 0.0001, two-way ANOVA).

≤ 0.0001，双因素方差分析）。

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Discussion

讨论

Herein, we demonstrate the network design of carbon metabolism in the mitochondrion and nucleus of a globally prevalent and clinically relevant human and animal pathogen,

在此，我们展示了全球普遍存在且与临床相关的人类和动物病原体的线粒体和细胞核中碳代谢的网络设计，

Toxoplasma gondii

弓形虫

. Our integrative approach discloses previously unknown plasticity, inter-organelle cooperativity, vulnerability and therapeutic potential of major metabolic enzymes in the parasite mitochondrion, cytosol and nucleus (Fig.

我们的综合方法揭示了寄生虫线粒体、细胞质和细胞核中主要代谢酶以前未知的可塑性、细胞器间协同性、脆弱性以及治疗潜力（图。

）。

Fig. 10: Mito-nuclear carbon metabolism in

图10：线粒体-核碳代谢在

Toxoplasma.

弓形虫。

Toxoplasma

弓形虫

actively catabolizes host glucose

积极分解宿主葡萄糖

via

通过

a canonical TCA cycle and also catabolizes glutamine

一个典型的TCA循环，同时也分解谷氨酰胺

via

通过

the GABA shunt and TCA cycle. Mitochondrial-localized MDH-FAD may participate in the TCA cycle, whereas cytoplasmic-localized MDH mediates the interconversion of oxaloacetate and malate. Malate is utilized by ME in the nucleus to produce pyruvate, a step that is essential for the growth and survival of .

GABA旁路和TCA循环。线粒体定位的MDH-FAD可能参与TCA循环，而细胞质定位的MDH则介导草酰乙酸和苹果酸的相互转化。苹果酸被细胞核中的ME用来生成丙酮酸，这一步骤对生长和存活至关重要。

Toxoplasma

弓形虫

. The question mark denotes the to-be-identified transporters in the mitochondrion, apicoplast or plasma membranes. Glc glucose, GT1 glucose transporter, PEP phosphoenolpyruvate, Pyr pyruvate, PYK1 pyruvate kinase 1, PYK2 pyruvate kinase 2, APT apicoplast phosphate translocator, APC apicoplast pyruvate carrier, MEP methylerythritol 4-phosphate pathway, FAS II type II fatty acid synthesis pathway, MPC mitochondrial pyruvate carrier, BCKDH branched-chain α-ketoacid dehydrogenase, PyC pyruvate carboxylase, Ac-CoA acetyl-CoA, Cit citrate, iCit isocitrate, α-KG α-ketoglutarate, CS1 citrate synthase 1, CS2 citrate synthase 2, PrpC 2-methylcitrate synthase, ACO aconitase, Iso isocitric acid, IDH1 isocitrate dehydrogenase 1, IDH2 isocitrate dehydrogenase 2, KDH α-ketoglutarate dehydrogenase, SCS succinyl-CoA synthetase, SSADH succinic semialdehyde dehydrogenase, Suc succinate, Fum fumarate, SDH succinate dehydrogenase, FH fumarase, Mal malate, MDH-FAD FAD malate-dehydrogenase, MDH malate dehydrogenase, Oxa oxaloacetate, ME malic enzyme, PEPCK phosphoenolpyruvate carboxykinase, AST aspartate aminotransferase, Asp aspartate, Gln glutamine, Glu glutamate, GluS glutaminase, GDH glutamate dehydrogenase, GAD glutamate decarboxylase, GABA γ-aminobutyric acid, GABA-AT gamma-aminobutyric acid-aminotransferase, SSA succinic semialdehyde, SP sodium propionate, Ac acetate, ACS Acetyl-CoA synthetase, NADH nicotinamide adenine dinucleotide, NADPH nicotinamide adenine dinucleotide phosphate, ETC electron transport chain, ATP adenosine triphosphate, AAC1 mitochondrial ADP/ATP Carrier 1, TCA cycle tricarboxylic acid cycle..

问号表示线粒体、顶质体或质膜中待鉴定的转运蛋白。Glc 葡萄糖，GT1 葡萄糖转运蛋白，PEP 磷酸烯醇式丙酮酸，Pyr 丙酮酸，PYK1 丙酮酸激酶1，PYK2 丙酮酸激酶2，APT 顶质体磷酸转运蛋白，APC 顶质体丙酮酸载体，MEP 甲基赤藓糖醇-4-磷酸途径，FAS II II型脂肪酸合成途径，MPC 线粒体丙酮酸载体，BCKDH 支链α-酮酸脱氢酶，PyC 丙酮酸羧化酶，Ac-CoA 乙酰辅酶A，Cit 柠檬酸，iCit 异柠檬酸，α-KG α-酮戊二酸，CS1 柠檬酸合酶1，CS2 柠檬酸合酶2，PrpC 2-甲基柠檬酸合酶，ACO 乌头酸酶，Iso 异柠檬酸，IDH1 异柠檬酸脱氢酶1，IDH2 异柠檬酸脱氢酶2，KDH α-酮戊二酸脱氢酶，SCS 琥珀酰辅酶A合成酶，SSADH 琥珀酸半醛脱氢酶，Suc 琥珀酸，Fum 延胡索酸，SDH 琥珀酸脱氢酶，FH 延胡索酸酶，Mal 苹果酸，MDH-FAD FAD苹果酸脱氢酶，MDH 苹果酸脱氢酶，Oxa 草酰乙酸，ME 苹果酸酶，PEPCK 磷酸烯醇式丙酮酸羧激酶，AST 天冬氨酸氨基转移酶，Asp 天冬氨酸，Gln 谷氨酰胺，Glu 谷氨酸，GluS 谷氨酰胺酶，GDH 谷氨酸脱氢酶，GAD 谷氨酸脱羧酶，GABA γ-氨基丁酸，GABA-AT γ-氨基丁酸转氨酶，SSA 琥珀酸半醛，SP 丙酸钠，Ac 乙酸，ACS 乙酰辅酶A合成酶，NADH 烟酰胺腺嘌呤二核苷酸，NADPH 烟酰胺腺嘌呤二核苷酸磷酸，ETC 电子传递链，ATP 三磷酸腺苷，AAC1 线粒体ADP/ATP载体1，TCA cycle 三羧酸循环。

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We show a significant role of the mitochondrial isoform of citrate synthase (CS1) for in vitro growth of tachyzoites and in vivo virulence. A similar outcome was reported recently

我们展示了柠檬酸合酶的线粒体异构体（CS1）在速殖子体外生长和体内毒力方面的重要作用。最近也有类似的报道。

. Our study revealed a more pronounced reduction in virulence of the

。我们的研究揭示了更为显著的毒力降低

cs1

计算机科学1

mutants, which may be attributed to the disparate genetic backgrounds of the parental strains. The depletion of

突变体，这可能归因于亲本菌株的不同遗传背景。消耗

CS1

计算机科学1

partly phenocopies the mutants of MPC1 and MPC2 transporting glycolysis-derived pyruvate into the mitochondrion and BCKDH-E1α subunit facilitating acetyl-CoA synthesis from MPC-imported pyruvate. These knockout strains are not lethal to the parasite survival in vitro, but exhibit varying degrees of attenuation in their virulence in mice.

部分表型模拟了将糖酵解衍生的丙酮酸转运至线粒体的MPC1和MPC2突变体，以及促进从MPC导入的丙酮酸合成乙酰辅酶A的BCKDH-E1α亚基。这些敲除菌株对寄生虫在体外生存并非致命，但在小鼠中表现出不同程度的毒力减弱。

，

. It has been reported that the mitochondria-localized 2-methylcitrate synthase (PrpC) has citrate synthase activity, although the catalytic efficiency of CS1 is notably higher than that of PrpC

据报道，线粒体定位的2-甲基柠檬酸合酶（PrpC）具有柠檬酸合酶活性，尽管CS1的催化效率明显高于PrpC。

. Consequently, when

。因此，当

CS1

计算机科学1

is knocked out, PrpC may facilitate citrate formation from acetyl-CoA, thereby maintaining the functionality of the TCA cycle (Fig.

被敲除时，PrpC可能促进乙酰辅酶A生成柠檬酸，从而维持TCA循环的功能（图。

）。

T. gondii

弓形虫

also expresses a CS2 in the cytoplasm, producing citrate

同样在细胞质中表达 CS2，产生柠檬酸

, which is hypothesized to be transported into the apicoplast and subsequently serve as a substrate for ACO to generate isocitric acid. The latter is metabolized by IDH2 to produce α-ketoglutarate and NADPH, contributing to FASII pathways in the organelle (Fig.

，该物质被假设会运输到顶质体中，随后作为ACO的底物生成异柠檬酸。后者被IDH2代谢以产生α-酮戊二酸和NADPH，为该细胞器中的FASII途径提供支持（图。

). This premise is supported by the evidence that the parasite encodes an aconitase (ACO) and two isocitrate dehydrogenases (IDH1 and IDH2). The ACO is dual-localized in the mitochondrion and the apicoplast

). 这一前提得到了以下证据的支持：该寄生虫编码了一种乌头酸酶（ACO）和两种异柠檬酸脱氢酶（IDH1 和 IDH2）。ACO 在线粒体和顶质体中具有双重定位。

, whereas The IDH1 and IDH2 are present in the mitochondrion and apicoplast, respectively

，而IDH1和IDH2分别存在于线粒体和顶质体中。

. Whether CS2-derived citrate can traffic to the apicoplast and mitochondrion to support biosynthetic pathways in both organelles requires additional research.

是否 CS2 衍生的柠檬酸可以运输到顶质体和线粒体以支持这两个细胞器中的生物合成途径，还需要进一步研究。

SCSα and SSADH may synergize to supply succinate to the cycle (Fig.

SCSα 和 SSADH 可能协同作用，为循环提供琥珀酸（图。

). A notably mild phenotype of the

). 显著温和的表型

Δssadh

mutant suggests that the GABA-derived succinic semialdehyde is not a significant contributor of succinate. Similarly, loss of GABA-AT, which converts GABA to succinic semialdehyde, does not affect parasite growth

突变体表明，GABA衍生的琥珀酸半醛并不是琥珀酸的重要来源。同样，将GABA转化为琥珀酸半醛的GABA-AT的缺失也不会影响寄生虫的生长。

. By contrast, the

相比之下，

Δscsα

strain exhibited a more severe phenotype which indicates its primary function in supplying succinate. However, the

该菌株表现出更严重的表型，这表明其主要功能是提供琥珀酸。然而，

Δscsα

mutant could be maintained in culture, suggesting that SSADH may compensate for succinate, albeit not sufficiently to ensure normal growth without

突变体可以在培养基中维持，这表明SSADH可能补偿了琥珀酸，但不足以确保在没有的情况下正常生长。

SCSα

. A double deletion of

. 一个双重删除

SSADH and SCSα

SSADH 和 SCSα

further detrimented parasite growth but the mutant was viable in culture. We postulate that propionate-derived succinate

进一步损害了寄生虫的生长，但该突变体在培养中是可存活的。我们推测丙酸衍生的琥珀酸

via

通过

the 2-methylcitrate cycle

2-甲基柠檬酸循环

may partly fuel the TCA cycle in tachyzoites (Fig.

可能部分地在速殖子中为TCA循环提供燃料（图。

）。

We also found that

我们还发现

SDHA

琥珀酸脱氢酶复合体黄素蛋白亚基A

is vital for parasite growth, and the

对寄生虫的生长至关重要，而且

Δsdha

Δsdha （注：该文本为字母和符号组合，无明确语义，无法翻译为中文。）

mutant is avirulent in mice. Our attempts to delete the FH protein, converting SDHA-derived fumarate to malate, were unsuccessful, indicating its crucial role for the growth of

突变体在小鼠中无毒力。我们尝试删除FH蛋白（将SDHA衍生的延胡索酸转化为苹果酸）未能成功，表明其对生长至关重要。

T. gondii

弓形虫

. Interestingly, FH-deficient

有趣的是，FH缺陷型

Plasmodium berghei

伯氏疟原虫

is viable

是可行的

. A conditional mutant of FH in tachyzoites of

. 速殖子中FH的条件突变体

T. gondii

弓形虫

is required to define its role better. Besides PEPCK

需要更好地定义其作用。除了PEPCK之外

, MDH-FAD likely enables the parasite to reprogram its oxaloacetate supply in varying milieus (Fig.

，MDH-FAD 可能使寄生虫在不同环境中重新规划其草酰乙酸供应（图。

). It can complement a

）。它可以补充一个

Δmdh1

yeast mutant impaired in growth on glycerol (non-fermentable carbon source), and the

在甘油（非发酵碳源）上生长受损的酵母突变体，以及

Δmdh-fad

tachyzoite shows a mild phenotype. The mutant accumulates malate, its NADH pool is severely compromised and the mitochondrial membrane potential collapses. Despite such metabolic anomalies, the mere survival and virulence of the

速殖子表现出轻微的表型。该突变体积累了苹果酸，其NADH池严重受损，线粒体膜电位崩溃。尽管存在这样的代谢异常，但仅仅是生存和毒力

Δmdh-fad

mutant is unprecedented. We believe PEPCK

突变体是前所未有的。我们相信PEPCK

may offset the deficiency of oxaloacetate

可能抵消草酰乙酸的不足

, and ATP can be supplied through glycolysis and salvage pathways. Indeed, tachyzoites express an ATP carrier (AAC1) in the mitochondrion

，并且可以通过糖酵解和补救途径提供ATP。事实上，速殖子在线粒体中表达一种ATP载体（AAC1）。

，

and can acquire host-derived ATP

并且可以获取宿主来源的ATP

. However, there appears to be no substitute mechanism for maintaining the NADH and membrane potential in the

但是，似乎没有任何替代机制来维持NADH和膜电位在

Δmdh-fad

mutant. The adaptive reprogramming in the

变异体。其中的适应性重编程

Δmdh-fad

strain warrants further investigation.

菌株需要进一步调查。

A second isoform of MDH is expressed in parasite cytosol, indicating the presence of a malate-aspartate shuttle for the transfer of NADH/NAD

第二种MDH异构体在寄生虫细胞质中表达，表明存在一个苹果酸-天冬氨酸穿梭系统用于NADH/NAD的转运。

to the mitochondrion (Fig.

到线粒体（图。

). Previous work showed the myc-tagged MDH localizing to the mitochondrion of

）。之前的研究显示，带有myc标签的MDH定位于线粒体中。

T. gondii

弓形虫

. However, our findings indicate that MDH-HA co-localizes with the cytoplasmic protein ALD. Consistent with our finding, the location of MDH was predicted to be in the cytoplasm by HyperLOPIT

然而，我们的研究结果表明，MDH-HA 与细胞质蛋白 ALD 共定位。与我们的发现一致，HyperLOPIT 预测 MDH 的位置在细胞质中。

. This discrepancy in the localization of MDH can be attributed to the use of different transgenic approaches. The

这种MDH定位的差异可以归因于使用了不同的转基因方法。

Δmdh

mutant shows no growth phenotype, and therefore seems dispensable for malate synthesis. Tachyzoites encode a 2-oxoglutarate/malate translocase (OMT, TGGT1_274060) located in the mitochondrion-membranes

突变体未显示生长表型，因此似乎对于苹果酸合成是可有可无的。速殖子编码一种位于线粒体膜上的2-氧戊二酸/苹果酸转位酶（OMT，TGGT1_274060）。

, which could potentially compensate for the loss of cytosolic MDH. On the other hand, the malic enzyme, producing NADPH and pyruvate in the nucleus, is essential. NADPH is required for cellular processes, including nucleotide biosynthesis and redox balance. Likewise, acetyl-CoA synthesis from pyruvate in the nucleus is vital for histone acetylation and chromatin remodeling.

，这可能在一定程度上弥补胞质MDH的缺失。另一方面，生成NADPH和丙酮酸的苹果酸酶在细胞核中至关重要。NADPH是包括核苷酸生物合成和氧化还原平衡在内的细胞过程所必需的。同样，由丙酮酸在细胞核中合成乙酰辅酶A对组蛋白乙酰化和染色质重塑也非常重要。

，

. Our transcriptomics data displayed that conditional knockdown of ME is associated with the modulation of replication, mismatch repair, amino sugar and nucleotide sugar metabolism. Loss of ME did not impact the total NADPH, likely due to other NADPH-generating pathways, including oxidative pentose phosphate pathway (oxPPP) and folate metabolism.

我们的转录组学数据显示，ME的条件性敲低与复制、错配修复、氨基糖和核苷酸糖代谢的调节有关。ME的缺失并未影响总的NADPH，可能是由于其他产生NADPH的途径，包括氧化戊糖磷酸途径（oxPPP）和叶酸代谢。

，

。

Previous research indicated that Tudor domain proteins are molecular adaptors involved in RNA metabolism, DNA damage response, and chromatin modification

以往的研究表明，Tudor结构域蛋白是参与RNA代谢、DNA损伤反应和染色质修饰的分子适配器。

，

. However, it is noteworthy that we could rescue the ME-depleted strain by complementing the C-terminus and N-terminus, excluding the Tudor domain. Our data also show that the ME expression in the nucleus is regulated

然而，值得注意的是，我们可以通过补充C端和N端来挽救ME缺失的菌株，但不包括Tudor结构域。我们的数据还显示，细胞核中的ME表达受到调控。

via

通过

the C-terminal, and its nuclear expression is essential for the lytic cycle. In mammalian cells, increasing evidence shows nuclear-mitochondrial crosstalk involving the translocation of metabolic enzymes

C端，并且其核表达对裂解周期至关重要。在哺乳动物细胞中，越来越多的证据表明存在涉及代谢酶易位的核-线粒体串扰。

. There is even relocation of pyruvate dehydrogenase complex to the nucleus and the occurrence of a nuclear TCA cycle regulating the metabolic-epigenetic circuitry

甚至有丙酮酸脱氢酶复合体向细胞核的迁移，以及调控代谢-表观遗传网络的核TCA循环的发生。

，

. It is, therefore, tempting to surmise a role of ME-derived pyruvate in acetyl-CoA synthesis for epigenetic regulation in tachyzoites – a premise supported by our initial studies of histone acetylation. Future work shall focus on yet-uncharacterized isoforms of metabolic enzymes to study the nuclear metabolism and mito-nuclear crosstalk in .

因此，很容易推测 ME 衍生的丙酮酸在速殖子乙酰辅酶 A 合成中对表观遗传调控的作用——这一前提得到了我们关于组蛋白乙酰化初步研究的支持。未来的工作将集中于尚未特征化的代谢酶异构体，以研究核代谢和线粒体-核交叉对话。

T. gondii

弓形虫

. Lastly, ME, SCSα and SDHA emerge as potential therapeutic targets against acute toxoplasmosis.

最后，ME、SCSα 和 SDHA 成为针对急性弓形虫病的潜在治疗靶点。

Materials and methods

材料与方法

Biological resources and ethics statement

生物资源与伦理声明

The RH

Δku80

, RH

，RH

Δku80

-TiR1, RH DiCre_T2A

-TiR1，RH DiCre_T2A

Δku80Δhxgprt

(

DiCre

迪克雷

)

，

, RH_

，RH_

Luc

吕克

and ME49 strains were provided by Bang Shen (Huazhong Agricultural University, Wuhan), respectively. Indole-3-acetic acid (IAA) was purchased from Sigma-Aldrich. Anti-Ty (mouse), anti-HSP60 (mouse), and anti-ALD (rabbit) antibodies were provided by Bang Shen (Huazhong Agricultural University, Wuhan).

ME49株系由华中农业大学的沈邦提供。吲哚-3-乙酸 (IAA) 购自Sigma-Aldrich公司。抗Ty（小鼠）、抗HSP60（小鼠）和抗ALD（兔）抗体也由华中农业大学的沈邦提供。

Anti-HA (mouse, Cat#M180-3) was purchased from MBL (Medical & Biological Laboratories Co., Japan). Rabbit anti-H3K9ac antibodies (Cat#9649) were purchased from CST (Cell Signaling Technology, USA). Alexa Fluor 488 (Cat#4408) and Alexa Fluor 594 (Cat#8889) antibodies were purchased from CST (Cell Signaling Technology, USA).

抗HA（小鼠，货号M180-3）购自MBL（医学与生物实验室公司，日本）。兔抗H3K9ac抗体（货号9649）购自CST（细胞信号技术公司，美国）。Alexa Fluor 488（货号4408）和Alexa Fluor 594（货号8889）抗体购自CST（细胞信号技术公司，美国）。

Hoechst dye (Cat#94403) were purchased from Sigma-Aldrich. The female ICR mice (six-week-old) were obtained from the Guangdong Medical Experimental Animal Center (Guangdong, China). The Balb/c-nu mice were acquired from Guangzhou Ruige Biotechnology Co., LTD. We have complied with all relevant ethical regulations for animal use.

Hoechst染料（货号94403）购自Sigma-Aldrich。雌性ICR小鼠（六周龄）购自广东省医学实验动物中心（中国广东）。Balb/c-nu小鼠购自广州瑞格生物科技有限公司。我们已遵守所有相关的动物使用伦理规定。

Animals were raised under standard conditions approved by the Ethics Committee of South China Agricultural University (Permit 2023f266)..

动物在华南农业大学伦理委员会批准的标准条件下饲养（许可号2023f266）。

Cell culture

细胞培养

Genetically modified strains were generated using the RH

使用RH生成了转基因菌株。

Δku80

, RH

，RH

Δku80

-TiR1 and

-TiR1 和

DiCre

迪克莱

as the parental strains

作为亲本菌株

，

. Parasites were propagated in human foreskin fibroblasts (HFF) (ATCC, USA) using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2% fetal bovine serum, 4500 mg/L glucose, 2 mM glutamine, 10 U/mL penicillin and 100 μg/mL streptomycin.

寄生虫在人包皮成纤维细胞（HFF）（ATCC，美国）中培养，使用添加了2%胎牛血清、4500毫克/升葡萄糖、2毫摩尔谷氨酰胺、10单位/毫升青霉素和100微克/毫升链霉素的Dulbecco改良Eagle培养基（DMEM）。

Making of transgenic parasites

制作转基因寄生虫

Supplementary Tables

补充表格

(Supplementary Data

（补充数据

) and S

) 和 S

(Supplementary Data

（补充数据

) describe primers and plasmids deployed in this study. The CRISPR/Cas9 plasmid was constructed using pEASY-Basic Seamless Cloning and Assembly Kit (TransGen Biotech, China). Other plasmids were constructed using the ClonExpress II one-step cloning kit (Vazyme Biotechnology, China). For 3’-genomic tagging, we amplified the 10×HA sequence flanked by homologous arms of the target gene and co-transfected it with the matching CRISPR/Cas9 constructs into the RH.

）描述了本研究中使用的引物和质粒。CRISPR/Cas9质粒是使用pEASY-Basic无缝克隆与组装试剂盒（TransGen Biotech，中国）构建的。其他质粒则使用ClonExpress II一步克隆试剂盒（Vazyme Biotechnology，中国）构建。对于3’-基因组标记，我们扩增了带有目标基因同源臂的10×HA序列，并将其与匹配的CRISPR/Cas9构建体共转染到RH中。

Δku80

或

DiCre

迪克里

strain. Transgenic strains were selected with 1 μM pyrimethamine (Sigma Aldrich, USA) and identified through diagnostic PCR and indirect immunofluorescence (IFA) assays. The

菌株。转基因菌株通过1 μM乙胺嘧啶（Sigma Aldrich，美国）筛选，并通过诊断性PCR和间接免疫荧光（IFA）分析进行鉴定。

CS1

计算机科学1

(TGGT1_268890),

SSADH

(TGGT1_257480),

SCSα

(TGGT1_290600),

SDHA

琥珀酸脱氢酶复合体黄素蛋白亚基A

(TGGT1_215590),

MDH

(TGGT1_318430),

OMT

(TGGT1_274060),

AST

抽象语法树

(TGGT1_248600) and

(TGGT1_248600) 和

MDH-FAD

(TGGT1_288500) knockout strains (Supplementary Data

(TGGT1_288500) 敲除菌株（补充数据）

) were generated by co-transfecting YFP

）是通过共转染YFP生成的

-DHFR

* or

* 或

DHFR

二氢叶酸还原酶

* cassettes flanked by homologous arms of the corresponding target genes and CRISPR/Cas9 constructs into the

* 由对应目标基因的同源臂和CRISPR/Cas9构建体侧翼的盒插入

DiCre

双Cre体系

strain. The strains were selected with 1 μM pyrimethamine, cloned by limiting dilution and clonal strains were screened by diagnostic PCRs and the YFP

菌株。这些菌株用1微摩尔的乙胺嘧啶进行选择，通过限制稀释法克隆，并通过诊断性PCR和YFP筛选克隆菌株。

加号

signal. The

信号。这个

ΔscsαΔssadh

strain was generated by replacing

菌株是通过替换生成的

SSADH

with Tub-CAT-mCherry

带有Tub-CAT-mCherry

via

通过

the CRISPR/Cas9 method and chloramphenicol selection (30 μM, Sigma-Aldrich, USA). The double mutant was isolated by diagnostic PCR screening and the mCherry

CRISPR/Cas9 方法和氯霉素筛选（30 μM，Sigma-Aldrich，美国）。通过诊断性 PCR 筛选分离出双突变体，并使用 mCherry。

signal.

信号。

The conditional knockdown strain for ME (TGGT1_286440) was generated by co-transfecting the locus-specific CRISPR/Cas9 construct and homology donor template into the RH

ME（TGGT1_286440）的条件性敲低菌株是通过将位点特异性的CRISPR/Cas9构建体和同源供体模板共转染到RH中生成的。

Δku80

-TiR1 strain (refer Supplementary Data

-TiR1菌株（参见补充数据

), followed by selection with 25 μg/mL mycophenolic acid and 50 μg/mL xanthine. Degradation of ME in the eventual mutant (miniAID-ME) was induced by 500 μM IAA. The miniAID-ME strain was used further to generate the comp-ME, comp-ME-N/C and comp-ME-N strains by inserting the specific expression cassette into the .

），随后使用25 μg/mL霉酚酸和50 μg/mL黄嘌呤进行筛选。最终突变体（miniAID-ME）中ME的降解由500 μM IAA诱导。通过将特定表达盒插入到miniAID-ME菌株中，进一步生成了comp-ME、comp-ME-N/C和comp-ME-N菌株。

UPRT

locus (selection with 10 μM 5-fluorodeoxyuracil). Clonal strains were identified by diagnostic PCRs and IFA.

位点（用10 μM 5-氟脱氧尿嘧啶进行选择）。通过诊断性PCR和IFA鉴定克隆菌株。

TgMDH-FAD complementation in

TgMDH-FAD互补性在

S. cerevisiae

酿酒酵母

The empty vector (

空向量 (

pRS416

) and the indicated

）以及所指示的

TgMDH-FAD

construct (

构造（

pRS416

MDH-FAD

) were transformed into the

) 被转化为

ScΔmdh1

mutant. Briefly, the

突变体。简而言之，

ScΔmdh1

strain was grown in 2% yeast extract, 1% tryptone, and 2% glucose. Following transformation with the plasmids described above, all yeast strains were cultured in synthetic dropout (uracil-free) minimal medium (0.67% yeast nitrogen base) supplemented with appropriate amino acids and 2% glucose. The transfectants were cloned on selective plates and tested for growth complementation on a medium with glucose or glycerin as a sole carbon source by serial dilution assay (30 °C for 3 days).

菌株在2%酵母提取物、1%胰蛋白胨和2%葡萄糖中生长。用上述质粒转化后，所有酵母菌株均在合成脱落（无尿嘧啶）最小培养基（0.67%酵母氮源碱基）中培养，并添加适当的氨基酸和2%葡萄糖。通过系列稀释实验（30°C下培养3天），将转染子克隆在选择性平板上，并在以葡萄糖或甘油为唯一碳源的培养基中测试其生长互补性。

The .

。

电报

MDH-FAD expression cassette was introduced into chromosome XI of the

MDH-FAD表达盒被引入到第XI号染色体中

ScΔmdh1

. Clonal strains were selected on plates and tested for growth complementation on a medium with glucose or glycerin as a sole carbon source by serial dilution assay (30 °C for 3 days).

通过连续稀释实验，在葡萄糖或甘油作为唯一碳源的培养基上，选择克隆菌株并测试其生长互补性（30°C培养3天）。

Production of polyclonal antibodies

多克隆抗体的生产

The open reading frames of ME were amplified from the RH

ME的开放阅读框从RH中扩增得到。

Δku80

strain and cloned into the

菌株并克隆到

pCold

vector containing a 6×His tag. The

包含6×His标签的载体。

pCold-ME

construct was transformed into the BL21(DE3) strain of

构建体被转化到BL21(DE3)菌株中

E. coli

大肠杆菌

, and protein expression was induced by IPTG. The recombinant proteins were purified

，然后用IPTG诱导蛋白质表达。重组蛋白被纯化

via

通过

Ni-NTA affinity resin and used to immunize female mice (six-week-old). The purified protein preparation and positive sera were aliquoted and stored at −80 °C.

Ni-NTA亲和树脂，并用于免疫雌性小鼠（六周龄）。纯化的蛋白制剂和阳性血清被分装并储存在-80°C。

Immunofluorescence assays

免疫荧光测定

Based on a protocol described previously

基于先前描述的协议

, HFF cells were infected by the parental and transgenic strains and then fixed with 4% paraformaldehyde (15 min), permeabilized with 0.1% Triton X-100 (20 min) and blocked with 10% FBS (2 hours). Samples were treated with mouse anti-HA (1:1000), rabbit anti-ALD (1:1000) and rabbit anti-H3K9ac antibodies (30 min, 1:400) and stained with Alexa Fluor488 (goat anti-mouse, 1:1000), Alexa Fluor594 (goat anti-rabbit, 1:1000) IgG and Hoechst dye (30 min, 1:1000).

,HFF细胞被亲本和转基因菌株感染，然后用4%多聚甲醛固定（15分钟），用0.1% Triton X-100透化（20分钟），并用10%胎牛血清封闭（2小时）。样品用小鼠抗-HA（1:1000）、兔抗-ALD（1:1000）和兔抗-H3K9ac抗体处理（30分钟，1:400），并用Alexa Fluor488（山羊抗小鼠，1:1000）、Alexa Fluor594（山羊抗兔，1:1000）IgG和Hoechst染料染色（30分钟，1:1000）。

Parasitized cells were imaged using a BX53 fluorescence microscope (Olympus, Japan) or ZEISS LSM 980 with Airyscan 2 (Carl Zeiss, Germany) and refined using ZEN software (Carl Zeiss, Germany). Image J software was used to process the fluorescent images..

被寄生的细胞使用BX53荧光显微镜（奥林巴斯，日本）或配备Airyscan 2的ZEISS LSM 980（卡尔蔡司，德国）进行成像，并使用ZEN软件（卡尔蔡司，德国）进行优化。荧光图像使用Image J软件进行处理。

Parasite phenotyping

寄生虫表型分析

Plaque assay were set up in 6-well plates seeded with HFF cells (100 tachyzoites/well, 3 wells per strain), essentially as reported earlier

斑块测定是在接种了HFF细胞的6孔板中进行的（每孔100个速殖子，每株3个孔），基本如前所述。

四十三

. Cultures were incubated unperturbed at 37 °C and 5% CO

培养物在37°C和5% CO₂条件下未受干扰地孵育。

for 6–10 days. Samples were fixed with 4% paraformaldehyde (20 min), washed by PBS, stained with 0.1% crystal violet (20 min) and scanned (Microtek Scan Marker i600, Microtek, China) to analyze the plaque size. To assess the replication efficiency, HFFs were infected with parental (RH

持续6-10天。样品用4%多聚甲醛固定（20分钟），用PBS洗涤，用0.1%结晶紫染色（20分钟），并扫描（Microtek Scan Marker i600，Microtek，中国）以分析斑块大小。为了评估复制效率，HFFs被亲本（RH）感染。

Δku80

-TiR1) or miniAID-ME strains preincubated for 2 h with or without 500 μM IAA. Cells were fixed using 4% paraformaldehyde and then incubated with mouse anti-

-TiR1) 或 miniAID-ME 菌株预孵育 2 小时，加入或不加入 500 μM IAA。细胞用 4% 多聚甲醛固定，然后与小鼠抗-

电报

IgG (30 min). Samples were permeabilized by 0.1% Triton X-100 (20 min) and blocked with 10% FBS (2 h). Subsequently, they were incubated with rabbit anti-ALD (30 min, 1:1000), followed by Alexa Fluor 488 (goat anti-mouse, 1:1000) and Alexa Fluor 594 (goat anti-rabbit, 1:1000), and Hoechst (1:1000) was used for 30 min.

IgG（30分钟）。样品用0.1% Triton X-100透化处理（20分钟），并用10% FBS封闭（2小时）。随后，与兔抗ALD（30分钟，1:1000）孵育，接着使用Alexa Fluor 488（山羊抗小鼠，1:1000）和Alexa Fluor 594（山羊抗兔，1:1000），并用Hoechst（1:1000）染色30分钟。

The .

。

DiCre

迪克雷

and

和

Δmdh

parasites were immunostained by mouse anti-

寄生虫通过小鼠抗-免疫染色

电报

IgG (before permeabilization, 1:1000) and rabbit anti-ALD (after permeabilization, 1:1000) to visualize non-invaded and total parasites, respectively. The

IgG（渗透前，1:1000）和兔抗ALD（渗透后，1:1000）分别用于可视化未侵入和总寄生虫。The

cs1

计算机科学1

，

Δssadh, Δscsα

，

ΔscsαΔssadh, Δsdha

and

和

Δmdh-fad

parasites were visualized by YFP

寄生虫通过YFP可视化

加号

signal and mouse anti-

信号和鼠标抗

电报

IgG (1:1000), followed by Alexa Fluor 594 (goat anti-mouse, 1:1000). At least 100 vacuoles per sample were scored for the number of parasites developing within them.

IgG（1:1000），随后使用Alexa Fluor 594（山羊抗小鼠，1:1000）。每个样本至少计数100个液泡中发育的寄生虫数量。

NAD

烟酰胺腺嘌呤二核苷酸

/NADH measurement

/NADH 测量

Freshly isolated tachyzoites of the

freshly isolated tachyzoites of the

DiCre

迪克莱

and

和

Δmdh-fad

strains were lysed in 200 μL of NAD

菌株在200 μL的NAD中裂解

/NADH extraction buffer on ice (30 min) and centrifuged at 12,000

/NADH 提取缓冲液置于冰上（30分钟），并在12,000转速下离心

(10 min). The total NAD

（10分钟）。总NAD

加号

and NADH levels in the supernatant were detected using an NAD

使用NAD检测上清液中的NADH水平

加号

/NADH assay kit with WST-8 (Beyotime, Shanghai, China)

/NADH检测试剂盒与WST-8（碧云天，上海，中国）

. Briefly, a 20 μL sample or NADH standards (0–20 μM) was added to a 96-well plate. Subsequently, 90 μL of alcohol dehydrogenase was added and incubated at 37 °C (10 min), followed by the addition of 10 μL chromogenic solution (37 °C, 45 min). The absorbance values were determined at 450 nm using a plate reader (Synergy, BioTek Instruments, USA)..

简而言之，将20 μL样品或NADH标准品（0–20 μM）加入96孔板中。随后加入90 μL醇脱氢酶，并在37°C下孵育10分钟，然后加入10 μL显色溶液（37°C，45分钟）。使用酶标仪（Synergy，BioTek Instruments，美国）在450 nm处测定吸光度值。

Parasite virulence and burden

寄生虫毒力与负担

The female ICR mice were infected with extracellular parasites of the specified strains by intraperitoneal injection (100 tachyzoites/mouse, 10 mice/strain). Infection by the

雌性ICR小鼠通过腹腔注射感染指定菌株的细胞外寄生虫（每只小鼠100个速殖子，每种菌株10只小鼠）。感染通过

Δscsα

mutant was performed at doses of 10

mutant 在 10 剂量下进行

, 10

，10

parasites (8 mice/group) and 10

寄生虫（每组8只小鼠）和10

, 10

，10

parasites (5 mice/group). The clinical signs and survival were monitored daily, and blood samples were collected from the surviving mice. Seronegative animals were not included in the data analysis

寄生虫（每组5只小鼠）。每天监测临床症状和存活情况，并从存活的小鼠中采集血样。血清阴性动物未纳入数据分析。

，

. To determine the parasite proliferation, we infected female ICR or Balb/c-nu mice by intraperitoneal injection (10

为了确定寄生虫的增殖情况，我们通过腹腔注射感染了雌性ICR或Balb/c-nu小鼠（10

parasites, 5–6 mice/strain). Animals infected with the

寄生虫，5-6只小鼠/品系）。感染的动物

cs1

计算机科学1

，

Δssadh, Δscsα

，

Δsdha

Δsdha （注：该文本为非标准字符组合，无法直接翻译成中文，保留原文。）

，

Δmdh-fad

, and

，以及

DiCre

迪克雷

strains were provided with normal purified water, while those harboring the RH

菌株提供了正常的纯净水，而那些携带RH的

Δku80

-TiR1 and miniAID-ME strains were given water with or without 500 μM IAA. Peritoneal fluids were collected 5 days post-infection, and genomic DNA was extracted using the TIANamp Blood DNA Kit from Tiangen Biotechnology Co., Ltd. (China). Parasites in the peritoneal fluids were detected by amplifying the non-coding fragment (529 bp, primers in Supplementary Data .

-TiR1 和 miniAID-ME 菌株分别给予含或不含 500 μM IAA 的水。感染后 5 天收集腹腔液，并使用天根生化科技有限公司（中国）的 TIANamp 血液 DNA 试剂盒提取基因组 DNA。通过扩增非编码片段（529 bp，引物见补充数据）检测腹腔液中的寄生虫。

) using Power SYBR Green PCR Master Mix from Toyobo Co., Ltd. (Japan)

) 使用来自日本东洋纺公司 (Toyobo Co., Ltd.) 的 Power SYBR Green PCR Master Mix。

，

。

Bioluminescence imaging

生物发光成像

ICR mice were immunized with the

ICR小鼠被免疫接种了

Δscsα

或

Δsdha

mutant (10

突变体 (10

)

via

通过

intraperitoneal injection. 34–39 days post-immunization, mice were infected with the luciferase-expressing RH-

腹腔注射。免疫后34-39天，小鼠感染表达荧光素酶的RH-

Luc

卢克

strain (10

菌株 (10

tachyzoites/animal, 3 or 4 mice/group, i.p.). Naïve mice were inoculated with the same dose of the indicated strains as control groups (3 or 4 mice/group, i.p.). Animals were anesthetized (1.25% tribromoethanol, MeilunBio Co., Ltd, China) five days after infection and 300 μL of 15 mg/mL D-luciferin was injected intraperitoneally (Yeasen Biotechnology Co., Ltd, Shanghai, China).

速殖子/动物，每组3或4只小鼠，腹腔注射）。未感染的小鼠作为对照组，接种相同剂量的指定菌株（每组3或4只小鼠，腹腔注射）。感染后五天，动物被麻醉（1.25%三溴乙醇，美仑生物公司，中国），并腹腔注射300 μL 15 mg/mL的D-荧光素（翌圣生物科技有限公司，上海，中国）。

The IVIS spectrum imaging (PerkinElmer, USA) was performed to detect the parasite load.

使用IVIS光谱成像系统（PerkinElmer，美国）检测寄生虫负荷。

。

Genomics and transcriptomics

基因组学与转录组学

The

cs1

计算机科学1

，

Δmdh

and

和

Δmdh-fad

tachyzoites were purified, and genomic DNA was extracted using the TIANamp Blood DNA Kit (Tiangen Biotechnology Co., Ltd., China). The genome was then sequenced as reported previously

速殖子被纯化，并使用天根生化科技有限公司（中国）的TIANamp血液DNA试剂盒提取基因组DNA。随后，按照先前报道的方法对基因组进行测序。

. The curated datasets were compared to the reference genome of the GT1 strain (ToxoDB), and the results were visualized using the integrated genome viewer. For transcriptomics, the miniAID-ME strain was pre-treated with −/+500 μM IAA for 12 h, and parasites were purified. Total RNA was extracted using Transzol UP reagent (TransGen Biotech Co., Ltd., China).

精心整理的数据集与GT1菌株（ToxoDB）的参考基因组进行了比较，并使用集成基因组查看器对结果进行了可视化。对于转录组学，miniAID-ME菌株预先用−/+500 μM IAA处理12小时，并纯化了寄生虫。使用Transzol UP试剂（中国TransGen生物技术有限公司）提取总RNA。

RNA sequencing was performed as described before.

RNA测序按照之前描述的方法进行。

. The transcriptome datasets were analyzed using the Majorbio Cloud Platform (

转录组数据集使用 Majorbio 云平台进行分析 (

www.majorbio.com

）。

Metabolomics

代谢组学

Metabolic labeling of extracellular tachyzoites with stable isotopes of glucose or glutamine was carried out following a modified method described previously

使用葡萄糖或谷氨酰胺的稳定同位素对细胞外速殖子进行代谢标记，按照先前描述的改良方法进行。

，

. Tachyzoites (5 × 10

速殖子（5×10

) of the

) 的

DiCre

迪克雷

and

和

Δmdh-fad

strains were cultured for 4 hours in a glucose-free medium containing 8 mM

菌株在含有8 mM葡萄糖的无葡萄糖培养基中培养了4小时

C语言

-glucose or

-葡萄糖或

-glutamine. Subsequently, parasites were washed with PBS and treated with 1 mL of ice-cold methanol (80%)

-谷氨酰胺。随后，用PBS洗涤寄生虫，并用1毫升冰冷的甲醇（80%）处理。

. Samples were sonicated (5 cycles, 1 min each with intermittent ice cooling) and stored at −20 °C for 30 min.

样品经超声处理（5个循环，每次1分钟，期间间断冰浴冷却），然后在-20°C下保存30分钟。

Metabolites were extracted by sample centrifugation, and the supernatant was evaporated to dryness. The residue was reconstituted in 100 μL of water and then mixed with 50 μL of 175 mM 3-NPH (Sigma Aldrich, USA) in 75% methanol, 50 μL of 105 mM EDC (Sigma Aldrich, USA) in methanol and 50 μL of 2.5% pyridine (Sigma Aldrich, USA) in methanol.

通过样品离心提取代谢物，上清液蒸发至干。残渣用100 μL水重新溶解，然后与50 μL 175 mM 3-NPH（Sigma Aldrich，美国）的75%甲醇溶液、50 μL 105 mM EDC（Sigma Aldrich，美国）的甲醇溶液和50 μL 2.5%吡啶（Sigma Aldrich，美国）的甲醇溶液混合。

. After reaction at 4 °C for 30 min, the mixtures were evaporated to dryness and re-dissolved in 50 µL of 50% methanol for the UHPLC-HRMS assay

在4°C下反应30分钟后，将混合物蒸发至干，并重新溶解于50 µL 50%甲醇中用于UHPLC-HRMS分析。

. The chromatographic separation was performed on a Thermo Fisher Ultimate 3000 UHPLC system using a Waters HSS T3 column (2.1 mm × 100 mm, 1.7 μm) (Waters, USA). The mobile phases comprised water with 0.1% formate (phase A) and acetonitrile with 0.1% formate (phase B). Metabolites were separated by linear gradient mode (0–0.5 min, 5% B; 2.5 min, 30% B; 5.5 min, 60% B; 9–13 min, 100% B; 13.1–15 min, 5% B) and mass spectrometry data were collected using Thermo Fisher Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometry (QE) in Heated Electrospray Ionization Negative (HESI-) mode.

色谱分离在Thermo Fisher Ultimate 3000 UHPLC系统上进行，使用Waters HSS T3柱（2.1 mm × 100 mm，1.7 μm）（Waters，美国）。流动相包括含0.1%甲酸的水（A相）和含0.1%甲酸的乙腈（B相）。代谢物通过线性梯度模式分离（0–0.5 min，5% B；2.5 min，30% B；5.5 min，60% B；9–13 min，100% B；13.1–15 min，5% B），质谱数据通过Thermo Fisher Q Exactive混合四极杆-轨道阱质谱仪（QE）在加热电喷雾电离负离子（HESI-）模式下采集。

The spray voltage was set to 2800 V. The capillary and probe heater temperatures were adjusted to 320 °C and 350 °C, respectively. The sheath gas flow rate was 50 Arb (arbitrary unit), the auxiliary gas flow rate was tuned to 15 Arb, and the S-Lens RF level was 50 Arb. The full scan was operated at a high-resolution 7 × 10.

喷雾电压设置为2800伏。毛细管和探针加热器温度分别调整为320°C和350°C。鞘气流速为50 Arb（任意单位），辅助气流速调整为15 Arb，S-Lens射频电平为50 Arb。全扫描以7×10的高分辨率运行。

FWHM (

m/z

质荷比

, 200) at 100–1000

，200）在 100–1000

m/z

质荷比

with an AGC target setting of 1 × 10

AGC目标设置为1 × 10

. Data were analyzed using Xcalibur software, and IsoCor v2 was used to correct the original mass spectra

数据使用Xcalibur软件进行分析，并用IsoCor v2对原始质谱进行校正。

。

Reporting summary

报告摘要

Further information on research design is available in the

有关研究设计的更多信息，请参见

Nature Portfolio Reporting Summary

《自然》系列报告摘要

linked to this article.

与本文相关联。

Data availability

数据可用性

Numerical data were plotted using GraphPad Prism 8 (GraphPad Software Inc., USA). Statistical analysis was performed using the Student’s

数值数据使用GraphPad Prism 8（GraphPad Software Inc.，美国）进行绘图。统计分析采用Student's方法进行。

test, two-way ANOVA and log-rank Mantel–Cox test. The genome sequencing data are deposited and released with the accession numbers PRJNA1108415, PRJNA1109294 and PRJNA1108442. Transcriptomics sequencing data are available in the short read archive (SRA) of the National Center for Biotechnology Information database (accession number PRJNA943448).

测试、双向ANOVA和log-rank Mantel-Cox检验。基因组测序数据已存入并发布，登录号为PRJNA1108415、PRJNA1109294和PRJNA1108442。转录组学测序数据可在国家生物技术信息中心数据库的短读取档案（SRA）中获取（登录号PRJNA943448）。

Reference genome of the .

参考基因组。

Toxoplasma

弓形虫

GT1 strain:

GT1菌株：

https://toxodb.org/toxo/app/record/dataset/NCBITAXON_507601

. All data are presented in the article or the supplementary information. The source data for the graphs and images can be found in the Supplementary Data 4. All resources described herein are available upon reasonable request.

所有数据均在文章或补充信息中提供。图表和图像的源数据可在补充数据4中找到。本文所述所有资源均可在合理要求下获取。

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Acknowledgements

致谢

We thank Na Li, Yaqiong Guo, Dongjuan Yuan and Rui Xu (College of Veterinary Medicine, South China Agricultural University, Guangzhou) for the discussion. We thank Xianfu Gao (Shanghai Profleader Biotech Co., Ltd.) and Wenchao Wang (Phenions Biotech Co., Ltd.) for LC-MS/MS. The authors also thank the Institute of Hematology, Jinan University, for providing flow cytometric sorting.

我们感谢华南农业大学兽医学院（广州）的李娜、郭亚琼、袁东娟和徐睿参与讨论。我们感谢上海普洛菲生物科技有限公司的高先富和菲尼昂生物科技有限公司的王文超进行LC-MS/MS分析。作者还感谢暨南大学血液学研究所提供流式细胞分选支持。

This research was supported by the National Key Research and Development Program of China (2023YFD1801000); Guangdong Major Project of Basic and Applied Basic Research (2020B0301030007); National Key Research and Development Program of China (2022YFD1800200; 2022YFD1801700); Natural Science Foundation of Guangdong Province (2022A1515011104; 2024A1515011346); 111 Project (D20008); Double first-class discipline promotion project (2023B10564003).

本研究得到了以下项目的资助：中国国家重点研发计划（2023YFD1801000）；广东省基础与应用基础研究重大项目（2020B0301030007）；中国国家重点研发计划（2022YFD1800200；2022YFD1801700）；广东省自然科学基金（2022A1515011104；2024A1515011346）；111计划（D20008）；双一流学科推进项目（2023B10564003）。

Supplementary funding was provided .

补充资金已提供。

via

通过

a Core Research Grant to Nishith Gupta (CRG/2021/000919) by the Department of Science and Technology – Science and Engineering Research Board (DST-SERB), India. Nishith Gupta also acknowledges the extended support of the Senior fellowship by the DBT–Wellcome Trust (India Alliance, IA/S/19/1/504263) and the Scholar Mobility Program from the Sino-German Science Center (M0074).

印度科学技术部-科学与工程研究委员会（DST-SERB）授予Nishith Gupta核心研究基金（CRG/2021/000919）。Nishith Gupta还感谢DBT-Wellcome Trust（印度联盟，IA/S/19/1/504263）提供的高级研究员奖学金的长期支持，以及中德科学中心（M0074）的学者交流计划的支持。

The funders had no role in the design, data collection, analysis, preparation or decision to publish this work..

资助者在本工作的设计、数据收集、分析、准备或决定发表方面没有任何作用。

Author information

作者信息

Author notes

作者笔记

These authors contributed equally: Hongxi Zhang, Nuo Ji, Shuxin Su.

这些作者贡献相同：张宏曦、季诺、苏淑欣。

Authors and Affiliations

作者与所属机构

State Key Laboratory for Animal Disease Control and Prevention, South China Agricultural University, Guangzhou, China

华南农业大学动物疾病防控国家重点实验室，广州，中国

Hongxi Zhang, Nuo Ji, Meng Zhao, Huiyu Du, Yaoyu Feng, Lihua Xiao & Ningbo Xia

洪熙张，诺记，梦赵，惠玉杜，瑶玉冯，李华肖，宁波夏

Frontiers Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China

合成生物学前沿科学中心，系统生物工程教育部重点实验室，天津大学化工学院，中国天津

Shuxin Su & Yi Wu

苏淑欣和吴毅

Intracellular Parasite Education And Research Labs (iPEARL), Department of Biological Sciences, Birla Institute of Technology and Science, Pilani (BITS Pilani), Hyderabad, India

印度海得拉巴比尔拉理工学院（BITS Pilani）生物科学系细胞内寄生虫教育与研究实验室（iPEARL）

Lakesh Kumar Sahoo & Nishith Gupta

拉凯什·库马尔·萨胡和尼希特·古普塔

Guangdong Laboratory for Lingnan Modern Agriculture, Center for Emerging and Zoonotic Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

岭南现代农业科学与技术广东省实验室，华南农业大学兽医学院，新兴与人畜共患病研究中心，广州，中国

Yaoyu Feng, Lihua Xiao & Ningbo Xia

冯耀宇，肖丽华，夏宁波

Department of Molecular Parasitology, Institute of Biology, Faculty of Life Sciences, Humboldt University, Berlin, Germany

德国柏林洪堡大学生命科学学院生物学研究所分子寄生虫学系

Nishith Gupta

尼希特·古普塔

Authors

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Hongxi Zhang

洪熙张

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Nuo Ji

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Contributions

贡献

Hongxi Zhang: Resources; Data curation; Validation; Investigation; Methodology; Writing–original draft. Nuo Ji: Resources; Data curation; Validation; Investigation; Methodology; Writing–original draft. Shuxin Su: Resources; Data curation; Validation; Investigation; Methodology; Writing–original draft.

洪熙张：资源；数据管理；验证；调查；方法论；撰写原始稿件。诺吉：资源；数据管理；验证；调查；方法论；撰写原始稿件。舒欣苏：资源；数据管理；验证；调查；方法论；撰写原始稿件。

Meng Zhao: Resources; Data curation; Validation; Investigation; Methodology. Huiyu Du: Resources; Data curation; Validation; Investigation; Methodology. Lakesh Kumar Sahoo: Resources; Validation; Investigation; Methodology. Yi Wu: Resources; Data curation; Validation; Investigation; Methodology; Supervision; Writing–original draft.

孟昭：资源；数据管理；验证；调查；方法论。杜慧玉：资源；数据管理；验证；调查；方法论。Lakesh Kumar Sahoo：资源；验证；调查；方法论。吴毅：资源；数据管理；验证；调查；方法论；监督；写作–原始草稿。

Yaoyu Feng: Resources; Data curation; Formal analysis; Supervision; Funding acquisition; Project administration; Nishith Gupta: Resources; Data curation; Supervision; Funding acquisition; Project administration; Writing—review and editing. Lihua Xiao: Resources; Data curation; Formal analysis; Supervision; Funding acquisition; Project administration; Ningbo Xia: Conceptualization; Resources; Data curation; Software; Formal analysis; Validation; Investigation; Visualization; Methodology; Supervision; Funding acquisition; Project administration; Writing—original draft; Writing—review and editing..

冯耀宇：资源；数据管理；正式分析；监督；资金获取；项目管理；尼希特·古普塔：资源；数据管理；监督；资金获取；项目管理；写作—审阅与编辑。肖丽华：资源；数据管理；正式分析；监督；资金获取；项目管理；夏宁波：概念化；资源；数据管理；软件；正式分析；验证；调查；可视化；方法论；监督；资金获取；项目管理；写作—原始草稿；写作—审阅与编辑。

Corresponding authors

通讯作者

Correspondence to

致信给

Nishith Gupta

尼希特·古普塔

，

Lihua Xiao

肖力华

或

Ningbo Xia

宁波霞

。

Ethics declarations

伦理声明

Competing interests

竞争利益

The authors declare that they have no conflict of interest. N.G. is an Editorial Board Member for Communications Biology, but was not involved in the editorial review of, nor the decision to publish this article.

作者声明他们没有利益冲突。N.G.是《通讯生物学》的编辑委员会成员，但未参与本文的编辑审查，也未参与发表本文的决策。

Peer review

同行评审

Peer review information

同行评审信息

Communications Biology

通讯生物学

thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Karthika Rajeeve and Dario Ummarino.

感谢匿名评审员对该工作的同行评审所做出的贡献。主要处理编辑：Karthika Rajeeve 和 Dario Ummarino。

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Supplementary information

补充信息

Supplementary Information

补充信息

Description of Additional Supplementary Files

附加补充文件的描述

Supplementary Data 1

补充数据 1

Supplementary Data 2

补充数据 2

Supplementary Data 3

补充数据3

Supplementary Data 4

补充数据 4

Reporting Summary

报告摘要

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Zhang, H., Ji, N., Su, S.

张, H., 季, N., 苏, S.