Cameron Pamela, McGachy Adrienne, Anderson Mary, Paul Andrew, Coombs Graham H, Mottram Jeremy C, Alexander James, Plevin Robin
Department of Immunology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom.
J Immunol. 2004 Sep 1;173(5):3297-304. doi: 10.4049/jimmunol.173.5.3297.
Infection with lesion-derived Leishmania mexicana amastigotes inhibited LPS-induced IL-12 production by mouse bone marrow-derived macrophages. This effect was associated with expression of cysteine peptidase B (CPB) because amastigotes of CPB deletion mutants had limited ability to inhibit IL-12 production, whereas preincubation of cells with a CPB inhibitor, cathepsin inhibitor IV, was able to suppress the effect of wild-type amastigotes. Infection with wild-type amastigotes resulted in a time-dependent proteolytic degradation of IkappaBalpha and IkappaBbeta and the related protein NF-kappaB. This effect did not occur with amastigotes of CPB deletion mutants or wild-type promastigotes, which do not express detectable CPB. NF-kappaB DNA binding was also inhibited by amastigote infection, although nuclear translocation of cleaved fragments of p65 NF-kappaB was still observed. Cysteine peptidase inhibitors prevented IkappaBalpha, IkappaBbeta, and NF-kappaB degradation induced by amastigotes, and recombinant CPB2.8, an amastigote-specific isoenzyme of CPB, was shown to degrade GST-IkappaBalpha in vitro. LPS-mediated IkappaBalpha and IkappaBbeta degradation was not affected by these inhibitors, confirming that the site of degradation of IkappaBalpha, IkappaBbeta, and NF-kappaB by the amastigotes was not receptor-driven, proteosomal-mediated cleavage. Infection of bone marrow macrophages with amastigotes resulted in cleavage of JNK and ERK, but not p38 MAPK, whereas preincubation with a cysteine peptidase inhibitor prevented degradation of these proteins, but did not result in enhanced protein kinase activation. Collectively, our results suggest that the amastigote-specific cysteine peptidases of L. mexicana are central to the ability of the parasite to modulate signaling via NF-kappaB and consequently inhibit IL-12 production.
用病变来源的墨西哥利什曼原虫无鞭毛体感染，可抑制脂多糖诱导的小鼠骨髓来源巨噬细胞产生白细胞介素-12。这种效应与半胱氨酸蛋白酶B（CPB）的表达有关，因为CPB缺失突变体的无鞭毛体抑制白细胞介素-12产生的能力有限，而用CPB抑制剂组织蛋白酶抑制剂IV对细胞进行预孵育，能够抑制野生型无鞭毛体的作用。用野生型无鞭毛体感染导致IκBα和IκBβ以及相关蛋白核因子κB（NF-κB）出现时间依赖性的蛋白水解降解。CPB缺失突变体的无鞭毛体或不表达可检测到的CPB的野生型前鞭毛体感染则不会出现这种效应。无鞭毛体感染也抑制NF-κB与DNA的结合，尽管仍观察到p65 NF-κB裂解片段的核转位。半胱氨酸蛋白酶抑制剂可阻止无鞭毛体诱导的IκBα、IκBβ和NF-κB降解，并且已证明重组CPB2.8（一种无鞭毛体特异性的CPB同工酶）在体外可降解谷胱甘肽S-转移酶-IκBα。脂多糖介导的IκBα和IκBβ降解不受这些抑制剂的影响，证实无鞭毛体对IκBα、IκBβ和NF-κB的降解位点不是受体驱动的、蛋白酶体介导的裂解。用无鞭毛体感染骨髓巨噬细胞导致JNK和ERK裂解，但p38丝裂原活化蛋白激酶（p38 MAPK）未裂解，而用半胱氨酸蛋白酶抑制剂预孵育可阻止这些蛋白的降解，但不会导致蛋白激酶激活增强。总体而言，我们的结果表明，墨西哥利什曼原虫的无鞭毛体特异性半胱氨酸蛋白酶对于该寄生虫通过NF-κB调节信号传导并因此抑制白细胞介素-12产生的能力至关重要。
