Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay.
Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay.
Proc Natl Acad Sci U S A. 2019 Apr 30;116(18):8879-8888. doi: 10.1073/pnas.1821487116. Epub 2019 Apr 12.
, the causative agent of Chagas disease (CD), contains exclusively Fe-dependent superoxide dismutases (Fe-SODs). During invasion to macrophages, superoxide radical (O) is produced at the phagosomal compartment toward the internalized parasite via NOX-2 (gp91-) activation. In this work, cytosolic Fe-SODB overexpressers (pRIBOTEX-Fe-SODB) exhibited higher resistance to macrophage-dependent killing and enhanced intracellular proliferation compared with wild-type (WT) parasites. The higher infectivity of Fe-SODB overexpressers compared with WT parasites was lost in gp91- macrophages, underscoring the role of O in parasite killing. Herein, we studied the entrance of O and its protonated form, perhydroxyl radical [(HO); pK = 4.8], to at the phagosome compartment. At the acidic pH values of the phagosome lumen (pH 5.3 ± 0.1), high steady-state concentrations of O and HO were estimated (∼28 and 8 µM, respectively). Phagosomal acidification was crucial for O permeation, because inhibition of the macrophage H-ATPase proton pump significantly decreased O detection in the internalized parasite. Importantly, O detection, aconitase inactivation, and peroxynitrite generation were lower in Fe-SODB than in WT parasites exposed to external fluxes of O or during macrophage infections. Other mechanisms of O entrance participate at neutral pH values, because the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid decreased O detection. Finally, parasitemia and tissue parasite burden in mice were higher in Fe-SODB-overexpressing parasites, supporting the role of the cytosolic O-catabolizing enzyme as a virulence factor for CD.
克氏锥虫（Trypanosoma cruzi），恰加斯病（Chagas disease，CD）的病原体，仅含有铁依赖性超氧化物歧化酶（Fe-SODs）。在巨噬细胞内入侵期间，通过 NOX-2（gp91-）激活，在吞噬体隔室中产生超氧自由基（O ）。在这项工作中，与野生型（WT）寄生虫相比，胞质 Fe-SODB 过表达（pRIBOTEX-Fe-SODB）的寄生虫表现出更高的抵抗巨噬细胞依赖性杀伤和增强的细胞内增殖能力。与 WT 寄生虫相比，Fe-SODB 过表达寄生虫的更高感染力在 gp91-巨噬细胞中丧失，这突显了 O 在寄生虫杀伤中的作用。在此，我们研究了 O 和其质子化形式过氧羟自由基 [(HO)；pK = 4.8] 在吞噬体隔室中进入 的情况。在吞噬体腔的酸性 pH 值（pH 5.3 ± 0.1）下，估计存在高稳态浓度的 O 和 HO（分别约为 28 和 8 µM）。吞噬体酸化对于 O 的渗透至关重要，因为巨噬细胞 H-ATP 酶质子泵的抑制显著降低了内化寄生虫中 O 的检测。重要的是，在暴露于外部 O 通量或在巨噬细胞感染期间，与 WT 寄生虫相比，Fe-SODB 中的 O 检测、乌头酸酶失活和过氧亚硝酸盐生成均较低。O 进入的其他机制在中性 pH 值下起作用，因为阴离子通道抑制剂 5-硝基-2-（3-苯基丙基氨基）苯甲酸降低了 O 的检测。最后，在过表达 Fe-SODB 的寄生虫中，小鼠中的寄生虫血症和组织寄生虫负担更高，这支持了胞质 O 分解代谢酶作为 CD 毒力因子的作用。
