Yahiro Kinnosuke, Ogura Kohei, Tsutsuki Hiroyasu, Iyoda Sunao, Ohnishi Makoto, Moss Joel
Department of Microbiology and Infection Control Sciences, Division of Biological Sciences, Kyoto Pharmaceutical University, Kyoto, Japan.
Advanced Health Care Science Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Kumamoto, Kumamoto, Japan.
Cell Death Discov. 2021 Nov 19;7(1):360. doi: 10.1038/s41420-021-00753-0.
Locus for Enterocyte Effacement (LEE)-positive Shiga-toxigenic Escherichia coli (STEC) contributes to many global foodborne diseases, with infection characterized by severe gastrointestinal symptoms, including bloody diarrhea. The incidence of LEE-negative STEC-mediated disease is also increasing globally. Subtilase cytotoxin (SubAB) is released by some LEE-negative STEC strains. It cleaves BiP, which is a chaperone protein located in the endoplasmic reticulum (ER), thereby causing apoptosis induced by ER stress. To date, the apoptotic signaling pathway mediated by SubAB has not been identified. In the current study, RNA-seq analysis showed that SubAB significantly induced the expression of Kelch domain containing 7B (KLHDC7B). We explored the role of KLHDC7B in the SubAB-induced apoptotic pathway. SubAB-induced KLHDC7B mRNA expression was increased after 12 h of incubation of toxin with HeLa cells. KLHDC7B expression was downregulated by knockdown of PKR-like endoplasmic reticulum kinase (PERK), CEBP homologous protein (CHOP), activating transcription factor 4 (ATF4), and CEBP β (CEBPB). KLHDC7B knockdown suppressed SubAB-stimulated CHOP expression, poly(ADP-ribose) polymerase (PARP) cleavage, and cytotoxicity. The over-expressed KLHDC7B was localized to the nucleus and cytosolic fractions. Next, we used RNA-seq to analyze the effect of KLHDC7B knockdown on apoptosis induced by SubAB, and found that the gene encoding for the pro-apoptotic Bcl-2 family protein, Harakiri (HRK), was upregulated in SubAB-treated control cells. However, this effect was not observed in SubAB-treated KLHDC7B-knockdown cells. Therefore, we identified the pathway through which SubAB-induced KLHDC7B regulates HRK expression, which is essential for apoptosis in toxin-mediated ER stress.
肠上皮细胞脱落位点（LEE）阳性的产志贺毒素大肠杆菌（STEC）导致了许多全球食源性疾病，其感染的特征是严重的胃肠道症状，包括血性腹泻。LEE阴性的STEC介导的疾病在全球的发病率也在上升。枯草杆菌蛋白酶细胞毒素（SubAB）由一些LEE阴性的STEC菌株释放。它能切割BiP，BiP是一种位于内质网（ER）的伴侣蛋白，从而导致内质网应激诱导的细胞凋亡。迄今为止，尚未确定SubAB介导的凋亡信号通路。在本研究中，RNA测序分析表明SubAB显著诱导含 Kelch 结构域 7B（KLHDC7B）的表达。我们探讨了KLHDC7B在SubAB诱导的凋亡途径中的作用。毒素与HeLa细胞孵育12小时后，SubAB诱导的KLHDC7B mRNA表达增加。通过敲低蛋白激酶R样内质网激酶（PERK）、CEBP同源蛋白（CHOP）、激活转录因子4（ATF4）和CEBPβ（CEBPB），KLHDC7B的表达下调。敲低KLHDC7B可抑制SubAB刺激的CHOP表达、聚（ADP-核糖）聚合酶（PARP）切割和细胞毒性。过表达的KLHDC7B定位于细胞核和细胞质部分。接下来，我们使用RNA测序分析敲低KLHDC7B对SubAB诱导的细胞凋亡的影响，发现促凋亡Bcl-2家族蛋白Harakiri（HRK）的编码基因在SubAB处理的对照细胞中上调。然而，在SubAB处理的敲低KLHDC7B的细胞中未观察到这种效应。因此，我们确定了SubAB诱导的KLHDC7B调节HRK表达的途径，这对于毒素介导的内质网应激中的细胞凋亡至关重要。
