Part 5: Kiss and spit metabolomics highlight the role of host purine metabolism during pathogen infection

Intracellular bacteria and protists rely on the host cell to supply many metabolites, but the mechanisms through which pathogens manipulate host metabolism to their benefit are not understood. Here, we demonstrate that when the obligate intracellular parasite Toxoplasma gondii secretes its rhoptry organelle contents into the host cytoplasm before invasion—a process called “kiss and spit”—host cell metabolite abundance is altered in nucleotide synthesis, the pentose phosphate pathway, glycolysis, and amino acid synthesis. U-13C6 labeling metabolomics confirmed that kiss and spit increased the flow of carbon through the pentose phosphate pathway and nucleotide synthesis. An increase in 2,3-bisphosphoglycerate abundance led us to investigate the activation of host cytosolic nucleosidase II (cN-II) to provide purines for the parasite. We found that T. gondii manipulates the host cN-II enzyme to dephosphorylate GMP and IMP that it needs for replication. Further, we found that the approved anti-cancer drug fludarabine, which inhibits cN-II, also inhibits Toxoplasma replication. These results reveal Toxoplasma host cell manipulation and highlight potential therapies for toxoplasmosis.

There are several datasets related to T. gondii kiss and spit

Part 1: Kiss and spit metabolomics highlight the role of host purine metabolism during pathogen infection: 10.5061/dryad.b2rbnzsjd : Time course of T. gondii kiss and spit-HFF cells metabolomics

Part 2: Kiss and spit metabolomics highlight the role of host purine metabolism during pathogen infection: 10.5061/dryad.69p8cz9b5:  U-13C6 labeling of ME49 T. gondii  kiss and spit and full infection in HFF cells

Part 3: Kiss and spit metabolomics highlight the role of host purine metabolism during pathogen infection: 10.5061/dryad.9p8cz8wrn:  Effect of fludarabine on purine metabolism in T. gondii infected HFF host cells

Part 4: Kiss and spit metabolomics highlight the role of host purine metabolism during pathogen infection: 10.5061/dryad.7d7wm383s: ME49T. gondii infected MDAMB231 cells Metabolomics at 24 and 48 HPI

Part 5: Kiss and spit metabolomics highlight the role of host purine metabolism during pathogen infection: 10.5061/dryad.ghx3ffbxx: Effect of AMP addition on purine metabolism in T. gondii infected host cells at 48 HPI

Part 6: Kiss and spit metabolomics highlight the role of host purine metabolism during pathogen infection: 10.5061/dryad.zkh1893jn: ME49 T. gondii Kiss and spit negative controls

Methods
T. gondii preferentially uses AMP salvage pathway to obtain purines. So, it is reasonable to think that the parasites treated with fludarabine or with genetic deletion of cN-II, use AMP to compensate for inosine and guanosine reduction. Thus, we performed target metabolomics in uninfected and infected MDAMB231 parental and cN-II KO cells, compensated with different concentrations of AMP (0, 1, 2, 4 µM) during 48 HPI.

MDAMB231 cells dishes in triplicate were treated with 2 x 106 ME49 tachyzoites and supplemented with AMP.  At 48 HPI, dishes were washed three times with ice cold PBS, then quenched with 80:20 HPLC grade Methanol: Water (Sigma-Aldrich). Dishes were incubated on dry ice at -80°C for 15 minutes. Plates were scraped, the solution removed, and spun at 2500 x g for 5 minutes at 4°C. The supernatant was removed and stored on ice, then the pellet was washed again in quenching solution and re-spun. Supernatants were combined, dried down under N2, and stored at -80°C.

Samples were resuspended in 100 µL HPLC grade water (Fisher Optima) for analysis on a Thermo-Fisher Vanquish Horizon UHPLC coupled to an electrospray ionization source (HESI) part of a hybrid quadrupole-Orbitrap high resolution mass spectrometer (Q Exactive Orbitrap; Thermo Scientific). Chromatography was performed using a 100 mm x 2.1 mm x 1.7 µm BEH C18 column (Acquity) at 30°C. 20 µL of the sample was injected via an autosampler at 4°C and flow rate was 200 µL/min. Solvent A was 97:3 water/methanol with 10 mM tributylamine (TBA) (Sigma-Aldrich) adjusted to a pH of 8.2 using approximately 9 mM Acetate (final concentration, Sigma-Aldrich). Solvent B was 100% methanol with no TBA (Sigma- Aldrich). Products were eluted in 95% A / 5% B for 2.5 minutes, then a gradient of 95% A / 5% B to 5% A / 95% B over 14.5 minutes, then held for an additional 2.5 minutes at 5%A / 95%B. Finally, the gradient was returned to 95% A / 5% B over 0.5 minutes and held for 5 minutes to re-equilibrate the column. MS parameters included: scan in negative mode; scan range = 70 - 1000 m/z; Automatic Gain control (AGC) = 1e6, spray voltage = 3.0 kV, maximum ion collection time = 40 ms, and capillary temperature = 350C. Peaks were matched to known standards for identification. Data analysis was performed using the Metabolomics Analysis and Visualization Engine (MAVEN) software

胞内细菌与原生生物依赖宿主细胞提供多种代谢物，但病原体调控宿主代谢以获益的具体机制仍未阐明。本研究证实，当专性胞内寄生虫刚地弓形虫（Toxoplasma gondii）在入侵宿主细胞前将棒状体（rhoptry organelle）内容物分泌至宿主细胞质中——这一过程被称为“吻-吐”（kiss and spit）——宿主细胞的核苷酸合成、戊糖磷酸途径、糖酵解及氨基酸合成通路中的代谢物丰度均发生改变。U-13C6标记代谢组学（U-13C6 labeling metabolomics）验证表明，“吻-吐”过程可增强碳流通过戊糖磷酸途径与核苷酸合成通路。2,3-双磷酸甘油酸（2,3-bisphosphoglycerate）丰度的提升促使我们探究宿主胞质核苷酶II（cytosolic nucleosidase II，cN-II）的激活以满足寄生虫对嘌呤的需求。研究发现，刚地弓形虫可调控宿主cN-II酶，使其脱磷酸化自身复制所需的鸟苷单磷酸（GMP）与次黄苷单磷酸（IMP）。此外，本研究证实获批抗癌药物氟达拉滨（fludarabine）可通过抑制cN-II进而抑制刚地弓形虫的增殖。上述结果揭示了刚地弓形虫对宿主细胞的调控机制，并为弓形虫病（toxoplasmosis）的潜在治疗方案提供了新思路。

与刚地弓形虫“吻-吐”相关的数据集共6项：
1. 《“吻-吐”代谢组学揭示病原体感染过程中宿主嘌呤代谢的作用》，DOI：10.5061/dryad.b2rbnzsjd：刚地弓形虫“吻-吐”-人包皮成纤维（HFF）细胞代谢组学时间序列
2. 《“吻-吐”代谢组学揭示病原体感染过程中宿主嘌呤代谢的作用》，DOI：10.5061/dryad.69p8cz9b5：ME49株刚地弓形虫“吻-吐”及完整感染HFF细胞的U-13C6标记实验
3. 《“吻-吐”代谢组学揭示病原体感染过程中宿主嘌呤代谢的作用》，DOI：10.5061/dryad.9p8cz8wrn：氟达拉滨对刚地弓形虫感染HFF宿主细胞嘌呤代谢的影响
4. 《“吻-吐”代谢组学揭示病原体感染过程中宿主嘌呤代谢的作用》，DOI：10.5061/dryad.7d7wm383s：ME49株刚地弓形虫感染MDAMB231细胞24小时与48小时的代谢组学分析
5. 《“吻-吐”代谢组学揭示病原体感染过程中宿主嘌呤代谢的作用》，DOI：10.5061/dryad.ghx3ffbxx：感染后48小时添加AMP对刚地弓形虫感染宿主细胞嘌呤代谢的影响
6. 《“吻-吐”代谢组学揭示病原体感染过程中宿主嘌呤代谢的作用》，DOI：10.5061/dryad.zkh1893jn：ME49株刚地弓形虫“吻-吐”阴性对照实验

研究方法
刚地弓形虫优先利用AMP补救途径获取嘌呤，因此推测经氟达拉滨处理或cN-II基因敲除（KO）的寄生虫可通过AMP补偿肌苷与鸟苷的减少。为此，本研究对未感染及感染的MDAMB231亲本细胞与cN-II敲除（KO）细胞进行靶向代谢组学分析，感染后48小时内分别添加不同浓度的AMP（0、1、2、4 µM）。
将接种有2×10^6个ME49株速殖子的MDAMB231细胞培养皿设置3个生物学重复，并添加AMP处理。感染后48小时，用冰预冷PBS洗涤培养皿3次，随后用80:20比例的HPLC级甲醇:水溶液（Sigma-Aldrich）终止代谢反应。将培养皿置于干冰上，-80℃孵育15分钟。刮取培养皿内细胞，收集混合液，4℃下2500×g离心5分钟。收集上清液并置于冰上，沉淀用淬灭液再次洗涤后重悬离心，合并两次上清液，经氮气吹干后于-80℃保存。
将样品重悬于100 µL HPLC级纯水（Fisher Optima）中，采用Thermo-Fisher Vanquish Horizon超高效液相色谱（UHPLC）结合电喷雾电离源（HESI）进行分析，该系统搭载四极杆-轨道阱混合高分辨质谱仪（Q Exactive Orbitrap; Thermo Scientific）。色谱分离采用100 mm × 2.1 mm × 1.7 µm的BEH C18色谱柱（Acquity），柱温维持30℃。自动进样器于4℃下进样20 µL，流速设为200 µL/min。流动相A为含10 mM三丁胺（TBA，Sigma-Aldrich）的97:3水/甲醇混合液，用约9 mM乙酸调节pH至8.2（终浓度，Sigma-Aldrich）；流动相B为不含三丁胺的100%甲醇（Sigma-Aldrich）。洗脱程序为：初始以95% A / 5% B洗脱2.5分钟，随后在14.5分钟内梯度洗脱至5% A / 95% B，维持该比例2.5分钟，再在0.5分钟内恢复至95% A / 5% B并维持5分钟以平衡色谱柱。质谱参数设置如下：负离子扫描模式，扫描范围70–1000 m/z，自动增益控制（AGC）=1e6，喷雾电压3.0 kV，最大离子收集时间40 ms，毛细管温度350℃。通过与已知标准品比对对色谱峰进行定性。数据分析采用代谢组学分析与可视化引擎（MAVEN）软件完成。