State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei Province, PR China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Wuhan, Hubei Province, PR China.
PLoS Pathog. 2022 Nov 30;18(11):e1011009. doi: 10.1371/journal.ppat.1011009. eCollection 2022 Nov.
Many apicomplexan parasites harbor a non-photosynthetic plastid called the apicoplast, which hosts important metabolic pathways like the methylerythritol 4-phosphate (MEP) pathway that synthesizes isoprenoid precursors. Yet many details in apicoplast metabolism are not well understood. In this study, we examined the physiological roles of four glycolytic enzymes in the apicoplast of Toxoplasma gondii. Many glycolytic enzymes in T. gondii have two or more isoforms. Endogenous tagging each of these enzymes found that four of them were localized to the apicoplast, including pyruvate kinase2 (PYK2), phosphoglycerate kinase 2 (PGK2), triosephosphate isomerase 2 (TPI2) and phosphoglyceraldehyde dehydrogenase 2 (GAPDH2). The ATP generating enzymes PYK2 and PGK2 were thought to be the main energy source of the apicoplast. Surprisingly, deleting PYK2 and PGK2 individually or simultaneously did not cause major defects on parasite growth or virulence. In contrast, TPI2 and GAPDH2 are critical for tachyzoite proliferation. Conditional depletion of TPI2 caused significant reduction in the levels of MEP pathway intermediates and led to parasite growth arrest. Reconstitution of another isoprenoid precursor synthesis pathway called the mevalonate pathway in the TPI2 depletion mutant partially rescued its growth defects. Similarly, knocking down the GAPDH2 enzyme that produces NADPH also reduced isoprenoid precursor synthesis through the MEP pathway and inhibited parasite proliferation. In addition, it reduced de novo fatty acid synthesis in the apicoplast. Together, these data suggest a model that the apicoplast dwelling TPI2 provides carbon source for the synthesis of isoprenoid precursor, whereas GAPDH2 supplies reducing power for pathways like MEP, fatty acid synthesis and ferredoxin redox system in T. gondii. As such, both enzymes are critical for parasite growth and serve as potential targets for anti-toxoplasmic intervention designs. On the other hand, the dispensability of PYK2 and PGK2 suggest additional sources for energy in the apicoplast, which deserves further investigation.
许多顶复门寄生虫都含有一个非光合质体，称为顶质体，它承载着重要的代谢途径，如甲基赤藓醇 4-磷酸（MEP）途径，该途径合成异戊烯前体。然而，顶质体代谢的许多细节还不是很清楚。在这项研究中，我们研究了四个糖酵解酶在刚地弓形虫顶质体中的生理作用。刚地弓形虫中的许多糖酵解酶有两个或更多的同工酶。内源性标记这些酶中的每一种都发现，其中四种定位于顶质体，包括丙酮酸激酶 2（PYK2）、磷酸甘油酸激酶 2（PGK2）、磷酸丙糖异构酶 2（TPI2）和磷酸甘油醛脱氢酶 2（GAPDH2）。产生 ATP 的酶 PYK2 和 PGK2 被认为是顶质体的主要能量来源。令人惊讶的是，单独或同时删除 PYK2 和 PGK2 并没有导致寄生虫生长或毒力的主要缺陷。相比之下，TPI2 和 GAPDH2 对速殖子增殖至关重要。TPI2 缺失导致 MEP 途径中间产物水平显著降低，并导致寄生虫生长停滞。在 TPI2 缺失突变体中重建另一种异戊烯前体合成途径，称为甲羟戊酸途径，部分挽救了其生长缺陷。同样，敲低产生 NADPH 的 GAPDH2 酶也通过 MEP 途径减少异戊烯前体合成并抑制寄生虫增殖。此外，它还减少了顶质体中新脂肪酸的合成。总之，这些数据表明，定位于顶质体的 TPI2 为异戊烯前体的合成提供碳源，而 GAPDH2 为 MEP、脂肪酸合成和铁氧还蛋白氧化还原系统等途径提供还原力。因此，这两种酶对寄生虫的生长都至关重要，是抗弓形虫干预设计的潜在靶点。另一方面，PYK2 和 PGK2 的非必需性表明顶质体中有额外的能量来源，值得进一步研究。
