Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan.
Faculty of Pharmacy, Yokohama University of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama-shi, Kanagawa 245-0066, Japan.
Toxins (Basel). 2021 Apr 9;13(4):272. doi: 10.3390/toxins13040272.
C2 toxin is a clostridial binary toxin consisting of actin ADP-ribosyltransferase (C2I) and C2II binding components. Activated C2II (C2IIa) binds to cellular receptors and forms oligomer in membrane rafts. C2IIa oligomer assembles with C2I and contributes to the transport of C2I into the cytoplasm of host cells. C2IIa induces Ca-induced lysosomal exocytosis, extracellular release of the acid sphingomyelinase (ASMase), and membrane invagination and endocytosis through generating ceramides in the membrane by ASMase. Here, we reveal that C2 toxin requires the lysosomal enzyme cathepsin B (CTSB) during endocytosis. Lysosomes are a rich source of proteases, containing cysteine protease CTSB and cathepsin L (CTSL), and aspartyl protease cathepsin D (CTSD). Cysteine protease inhibitor E64 blocked C2 toxin-induced cell rounding, but aspartyl protease inhibitor pepstatin-A did not. E64 inhibited the C2IIa-promoted extracellular ASMase activity, indicating that the protease contributes to the activation of ASMase. C2IIa induced the extracellular release of CTSB and CTSL, but not CTSD. CTSB knockdown by siRNA suppressed C2 toxin-caused cytotoxicity, but not siCTSL. These findings demonstrate that CTSB is important for effective cellular entry of C2 toxin into cells through increasing ASMase activity.
C2 毒素是一种梭菌双效毒素，由肌动蛋白 ADP-核糖基转移酶（C2I）和 C2II 结合组成部分组成。活化的 C2II（C2IIa）与细胞受体结合，并在膜筏中形成寡聚体。C2IIa 寡聚体与 C2I 组装，并有助于 C2I 向宿主细胞的细胞质中转运。C2IIa 通过 ASMase 在膜中产生神经酰胺，诱导 Ca2+诱导的溶酶体胞吐、细胞外酸性鞘磷脂酶（ASMase）的释放以及膜内陷和内吞作用。在这里，我们揭示 C2 毒素在胞吞作用过程中需要溶酶体酶组织蛋白酶 B（CTSB）。溶酶体是蛋白酶的丰富来源，包含半胱氨酸蛋白酶 CTSB 和组织蛋白酶 L（CTSL）以及天冬氨酸蛋白酶组织蛋白酶 D（CTSD）。半胱氨酸蛋白酶抑制剂 E64 阻断了 C2 毒素诱导的细胞圆化，但天冬氨酸蛋白酶抑制剂 pepstatin-A 没有。E64 抑制了 C2IIa 促进的细胞外 ASMase 活性，表明该蛋白酶有助于 ASMase 的激活。C2IIa 诱导细胞外释放 CTSB 和 CTSL，但不释放 CTSD。siRNA 敲低 CTSB 抑制了 C2 毒素引起的细胞毒性，但不抑制 siCTSL。这些发现表明，CTSB 通过增加 ASMase 活性，对于 C2 毒素有效进入细胞至关重要。
