Department of Immunology, Max Rady College of Medicine, University of Manitoba, Winnipeg, Manitoba R3T 0T5, Canada.
Department of Pathology, Max Rady College of Medicine, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
J Immunol. 2021 Mar 1;206(5):1013-1026. doi: 10.4049/jimmunol.2000517. Epub 2021 Jan 18.
There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of We show that targeted loss of PEPCK results in impaired proliferation of in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4 T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for , and its deletion results in altered metabolic activity and attenuation of virulence.
目前，由于缺乏足够的知识来刺激宿主保护性和持久的 T 细胞介导的免疫应答，针对利什曼病还没有有效的疫苗。我们之前已经鉴定出磷酸烯醇丙酮酸羧激酶（PEPCK，一种糖异生酶）是一种免疫优势抗原，在寄生虫的昆虫（前鞭毛体）和哺乳动物（无鞭毛体）阶段都有表达。在这项研究中，我们研究了 PEPCK 在代谢、毒力和免疫发病机制中的作用。我们发现，PEPCK 的靶向缺失导致寄生虫在无细胞培养和骨髓来源的巨噬细胞中的增殖受损。此外，PEPCK 的缺乏导致体内病理学高度减弱。感染 PEPCK 缺陷寄生虫的 BALB/c 小鼠尽管在感染部位携带寄生虫，但仍未能发展出任何皮肤损伤。这与脾脏和感染部位引流淋巴结中产生细胞因子（IFN-γ、IL-4 和 IL-10）的 CD4 T 细胞的频率显著减少有关。用可溶性 Ag 体外再刺激后，来自感染 PEPCK 缺陷寄生虫的小鼠的细胞也会产生明显较低水平的这些细胞因子进入培养上清液。与野生型和补加型相比，PEPCK 缺陷寄生虫表现出显著更高的细胞外酸化率、增加的质子泄漏、降低的 ATP 偶联效率和耗氧率。总之，这些结果表明 PEPCK 是一种关键的代谢酶，其缺失导致代谢活性改变和毒力减弱。
