Herdman Brock P, Paton James C, Wang Hui, Beddoe Travis, Paton Adrienne W
Research Centre for Infectious Diseases, Department of Molecular and Cellular Biology, University of Adelaide, Adelaide, SA, Australia.
Department of Animal, Plant and Soil Science, Centre for AgriBiosciences, La Trobe University, Bundoora, Victoria, Australia.
Infect Immun. 2017 Jul 19;85(8). doi: 10.1128/IAI.00214-17. Print 2017 Aug.
Various serovars, including serovar Typhi, encode an AB5 toxin (ArtAB), the A subunit of which is an ADP-ribosyltransferase related to the S1 subunit of pertussis toxin. However, although the A subunit is able to catalyze ADP-ribosylation of host G proteins, a cytotoxic phenotype has yet to be identified for the holotoxin. Here we show that its B subunit pentamer (ArtB) binds to receptors on the surface of Vero (African green monkey kidney) cell, CHO (Chinese hamster ovary) cell, U937 (human monocyte) cell, and HBMEC (human brain microvascular endothelial cell) lines. Moreover, ArtB induced marked vacuolation in all cell lines after 4 h of incubation. Further studies in Vero cells showed that vacuolation was inhibited by bafilomycin A1 and was dependent on the clathrin-mediated uptake of ArtB. Vacuolation was also inhibited by treatment of cells with neuraminidase, indicating that sialylated glycans are functional receptors for ArtB. Confocal colocalization studies indicated that after cell binding and internalization, ArtB undergoes retrograde transport via early endosomes, the -Golgi network, and the Golgi apparatus, reaching the endoplasmic reticulum (ER) after approximately 2 h. The onset of vacuolation also coincided with gross cytoskeletal reorganization. At later time points, ArtB colocalized with ER-Tracker Red in the vacuolar membrane, implying that vacuolation is a consequence of ER disorganization. Thus, the isolated B subunit of this cryptic AB5 toxin has significant effects on target cells with the potential to contribute directly to pathogenesis independently of the catalytic A subunit.
多种血清型，包括伤寒血清型，编码一种AB5毒素（ArtAB），其A亚基是一种与百日咳毒素S1亚基相关的ADP核糖基转移酶。然而，尽管A亚基能够催化宿主G蛋白的ADP核糖基化，但尚未确定该全毒素的细胞毒性表型。在这里，我们表明其B亚基五聚体（ArtB）与Vero（非洲绿猴肾）细胞、CHO（中国仓鼠卵巢）细胞、U937（人单核细胞）细胞和HBMEC（人脑微血管内皮细胞）系表面的受体结合。此外，孵育4小时后，ArtB在所有细胞系中均诱导出明显的空泡化。在Vero细胞中的进一步研究表明，巴弗洛霉素A1可抑制空泡化，且空泡化依赖于网格蛋白介导的ArtB摄取。用神经氨酸酶处理细胞也可抑制空泡化，表明唾液酸化聚糖是ArtB的功能性受体。共聚焦共定位研究表明，细胞结合和内化后，ArtB通过早期内体、反式高尔基体网络和高尔基体进行逆行转运，约2小时后到达内质网（ER）。空泡化的开始也与明显的细胞骨架重组同时发生。在后期时间点，ArtB与空泡膜中的ER-Tracker Red共定位，这意味着空泡化是内质网紊乱的结果。因此，这种隐性AB5毒素的分离B亚基对靶细胞有显著影响，有可能独立于催化性A亚基直接促进发病机制。
