Biology Department, University of Arkansas at Little Rock, 2801 South University Dr., Little Rock, AR 72204-1099, USA.
Exp Parasitol. 2013 Mar;133(3):314-26. doi: 10.1016/j.exppara.2012.12.001. Epub 2012 Dec 27.
Acanthamoeba castellanii is a free-living protozoan. Some strains are opportunistic pathogens. A type-I metacaspase was identified in A. castellanii (Acmcp) and was shown to be expressed through the encystation process. The model organism, Dictyostelium discoideum, has been used here as a model for studying these caspase-like proteins. Separate cell lines expressing a GFP-tagged version of the full length Acmcp protein, as well as a deletion proline region mutant of Acmcp protein (GFP-Acmcp-dpr), have been introduced into D. discoideum. Both mutants affect the cellular metabolism, characterized by an increase in the growth rate. Microscopic imaging revealed an association between Acmcp and the contractile vacuole system in D. discoideum. The treatment of cells with selected inhibitors in different environments added additional support to these findings. This evidence shows that Acmcp plays an important role in contractile vacuole regulation and mediated membrane trafficking in D. discoideum. Additionally, the severe defect in contractile vacuole function in GFP-Acmcp-dpr mutant cells suggests that the proline-rich region in Acmcp has an essential role in binding this protein with other partners to maintain this process. Furthermore, Yeast two-hybrid system identified there are weak interactions of the Dictyostelium contractile vacuolar proteins, including Calmodulin, RabD, Rab11 and vacuolar proton ATPase, with Acmcp protein. Taken together, our findings suggest that A. castellanii metacaspase associate with the contractile vacuole and have an essential role in cell osmoregulation, which contributes to its attractiveness as a possible target for treatment therapies against A. castellanii infection.
棘阿米巴是一种自由生活的原生动物。一些菌株是机会性病原体。在棘阿米巴（Acanthamoeba castellanii）中鉴定出一种 I 型胱天蛋白酶（Acmcp），并通过囊包形成过程显示其表达。这里使用模式生物粘菌（Dictyostelium discoideum）作为研究这些半胱天冬酶样蛋白的模型。分别引入了表达全长 Acmcp 蛋白的 GFP 标记细胞系以及 Acmcp 蛋白缺失脯氨酸区域突变体（GFP-Acmcp-dpr）的细胞系进入粘菌。这两种突变体都影响细胞代谢，其特征是生长速度增加。显微镜成像显示 Acmcp 与粘菌的收缩泡系统之间存在关联。在不同环境中用选定的抑制剂处理细胞为这些发现提供了更多支持。这些证据表明 Acmcp 在粘菌的收缩泡调节和介导膜运输中起重要作用。此外，GFP-Acmcp-dpr 突变体细胞中收缩泡功能严重缺陷表明 Acmcp 中的脯氨酸丰富区域在将该蛋白与其他伴侣结合以维持该过程中具有重要作用。此外，酵母双杂交系统鉴定出粘菌的收缩泡蛋白，包括钙调蛋白、RabD、Rab11 和液泡质子 ATP 酶与 Acmcp 蛋白之间存在弱相互作用。总之，我们的研究结果表明，棘阿米巴的胱天蛋白酶与收缩泡相关，在细胞渗透压调节中起关键作用，这使其成为治疗棘阿米巴感染的潜在治疗靶点的吸引力。
