Sijwali Puran S, Koo Jamie, Singh Naresh, Rosenthal Philip J
Department of Medicine, San Francisco General Hospital, University of California San Francisco, CA 94143-0811, USA.
Mol Biochem Parasitol. 2006 Nov;150(1):96-106. doi: 10.1016/j.molbiopara.2006.06.013. Epub 2006 Jul 20.
Erythrocytic stages of the malaria parasite Plasmodium falciparum express four related papain-family cysteine proteases, termed falcipains. Falcipain-2 and falcipain-3 are food vacuole hemoglobinases, but determination of the specific roles of these and other falcipains has been incomplete. To better characterize biological roles, we attempted disruption of each falcipain gene in the same strain (3D7) of P. falciparum. Disruption of falcipain-1, falcipain-2, and falcipain-2' was achieved. In each case knockouts multiplied at the same rate as wild-type parasites. The morphologies of erythrocytic falcipain-1 and falcipain-2' knockout parasites were indistinguishable from those of wild-type parasites. In contrast, consistent with previous results, falcipain-2 knockout trophozoites developed swollen, hemoglobin-filled food vacuoles, indicative of a block in hemoglobin hydrolysis and were, compared to wild-type parasites, twice as sensitive to cysteine protease inhibitors and over 1000 times more sensitive to an aspartic protease inhibitor. The falcipain-3 gene could not be disrupted, but replacement with a tagged functional copy was readily achieved, strongly suggesting that falcipain-3 is essential to erythrocytic parasites. Our data suggest key roles for falcipain-2 and falcipain-3 in the development of erythrocytic malaria parasites and a complex interplay between P. falciparum cysteine and aspartic proteases.
恶性疟原虫的红细胞阶段表达四种相关的木瓜蛋白酶家族半胱氨酸蛋白酶，称为恶性疟原虫蛋白酶。恶性疟原虫蛋白酶-2和恶性疟原虫蛋白酶-3是食物泡血红蛋白酶，但对这些以及其他恶性疟原虫蛋白酶具体作用的确定并不完整。为了更好地描述其生物学作用，我们尝试在恶性疟原虫的同一菌株（3D7）中破坏每个恶性疟原虫蛋白酶基因。恶性疟原虫蛋白酶-1、恶性疟原虫蛋白酶-2和恶性疟原虫蛋白酶-2'的破坏已实现。在每种情况下，基因敲除体的增殖速度与野生型寄生虫相同。红细胞阶段的恶性疟原虫蛋白酶-1和恶性疟原虫蛋白酶-2'基因敲除寄生虫的形态与野生型寄生虫无法区分。相比之下，与先前的结果一致，恶性疟原虫蛋白酶-2基因敲除的滋养体形成了肿胀的、充满血红蛋白的食物泡，这表明血红蛋白水解受阻，并且与野生型寄生虫相比，对半胱氨酸蛋白酶抑制剂的敏感性高出两倍，对天冬氨酸蛋白酶抑制剂的敏感性高出1000倍以上。恶性疟原虫蛋白酶-3基因无法被破坏，但用一个带有标签的功能拷贝进行替换很容易实现，这强烈表明恶性疟原虫蛋白酶-3对红细胞寄生虫至关重要。我们的数据表明恶性疟原虫蛋白酶-2和恶性疟原虫蛋白酶-3在红细胞疟原虫发育中起关键作用，并且恶性疟原虫半胱氨酸蛋白酶和天冬氨酸蛋白酶之间存在复杂的相互作用。
