Rey-Ladino J A, Reiner N E
Department of Medicine, Faculty of Medicine, University of British Columbia, Vancouver, Canada.
Infect Immun. 1993 Aug;61(8):3265-72. doi: 10.1128/iai.61.8.3265-3272.1993.
Heat shock protein (HSP) expression was examined in murine bone marrow-derived macrophages infected with stationary-phase promastigotes of Leishmania donovani. Immunoblotting performed with a rabbit polyclonal antiserum raised against HSP60 from Heliothis virescens (moth) revealed the de novo appearance of 65- and 67-kDa proteins in leishmania-infected macrophages. A third protein of 60 kDa, which represented murine HSP60, was also detected, and its expression did not change in response to infection. In contrast, expression of the novel 65- and 67-kDa proteins in infected cells was coordinately regulated and, at 24 h of infection, reached maximal levels of 52 to 100% increases above initial levels determined at 3 h. Proteins which had identical electrophoretic mobilities and were similarly regulated in response to heat were also detected in promastigotes. The appearance of these proteins in macrophages was specific to leishmania infection in that neither protein was detected in noninfected cells either in the basal state or following several treatments, including (i) infection with Yersinia pseudotuberculosis, (ii) phagocytosis of Staphylococcus aureus, (iii) NaAsO2 treatment, and (iv) heat shock. Expression of the 65- and 67-kDa heat-regulated Leishmania proteins was also observed to be selective, in that as their concentration was increasing, the abundance of the Leishmania surface protease gp63 in infected cells was noted to decrease. Murine HSP60 but not the Leishmania heat-regulated proteins was also recognized by a distinct rabbit antiserum raised against human HSP60, suggesting the presence of specific determinants within these Leishmania proteins. A monoclonal antibody that recognizes both mammalian HSP70 and HSP70 from plasmodia detected single isoforms of both Leishmania and murine HSP70 in infected cells, and the level of neither protein changed during infection. Moreover, although a murine HSP of 73 kDa was induced in response to both heat shock and NaAsO2 treatment, it was not induced to detectable levels by infection. The rapid and relatively high level of expression of inducible HSP60-related proteins of L. donovani and Leishmania HSP70 in infected macrophages suggests that these proteins are involved in pathogenesis and may be important targets of the immune response.
研究了感染杜氏利什曼原虫静止期前鞭毛体的小鼠骨髓源性巨噬细胞中热休克蛋白（HSP）的表达。用针对烟草天蛾HSP60制备的兔多克隆抗血清进行免疫印迹分析，结果显示在利什曼原虫感染的巨噬细胞中出现了65 kDa和67 kDa的新生蛋白。还检测到了代表小鼠HSP60的第三种60 kDa蛋白，其表达在感染后没有变化。相比之下，感染细胞中新型65 kDa和67 kDa蛋白的表达受到协同调节，在感染24小时时，其水平比3小时时的初始水平最高增加了52%至100%。在前鞭毛体中也检测到了具有相同电泳迁移率且对热反应类似调节的蛋白。这些蛋白在巨噬细胞中的出现是利什曼原虫感染特有的，因为在基础状态下或经过几种处理后，包括（i）感染假结核耶尔森菌、（ii）吞噬金黄色葡萄球菌、（iii）亚砷酸钠处理和（iv）热休克，未感染细胞中均未检测到这两种蛋白。还观察到65 kDa和67 kDa热调节利什曼原虫蛋白的表达具有选择性，即随着它们浓度的增加，感染细胞中利什曼原虫表面蛋白酶gp63的丰度会降低。针对人HSP60制备的另一种兔抗血清可识别小鼠HSP60，但不能识别利什曼原虫热调节蛋白，这表明这些利什曼原虫蛋白中存在特定的决定簇。一种既能识别哺乳动物HSP70又能识别疟原虫HSP70的单克隆抗体，在感染细胞中检测到了利什曼原虫和小鼠HSP70的单一异构体，且两种蛋白的水平在感染过程中均未改变。此外，尽管73 kDa的小鼠HSP在热休克和亚砷酸钠处理后均被诱导，但感染并未将其诱导至可检测水平。杜氏利什曼原虫诱导型HSP60相关蛋白和利什曼原虫HSP70在感染巨噬细胞中快速且相对高水平的表达表明，这些蛋白参与了发病机制，可能是免疫反应的重要靶点。
