Hodgson Andrea, Wier Eric M, Fu Kai, Sun Xin, Yu Hongbing, Zheng Wenxin, Sham Ho Pan, Johnson Kaitlin, Bailey Scott, Vallance Bruce A, Wan Fengyi
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America; Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.
PLoS Pathog. 2015 Mar 10;11(3):e1004705. doi: 10.1371/journal.ppat.1004705. eCollection 2015 Mar.
Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors) encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS) into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB) signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle) effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p65¹⁻³⁸ fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p65¹⁻³⁸ fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3), a 'specifier' subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.
包括肠致病性大肠杆菌（EPEC）、肠出血性大肠杆菌（EHEC）以及啮齿动物等效物鼠柠檬酸杆菌在内的黏附/抹除（A/E）病原体是食源性疾病的重要致病因子。感染后，A/E病原体编码的众多毒力蛋白（效应蛋白）通过其保守的III型分泌系统（T3SS）注入宿主细胞，在那里它们干扰细胞信号级联反应，特别是协调宿主防御的先天性和适应性免疫反应的核因子κB（NF-κB）信号通路。在T3SS分泌的非LEE编码（Nle）效应蛋白中，金属蛋白酶NleC最近被阐明可通过切割NF-κB Rel亚基来调节宿主NF-κB信号传导。然而，NleC如何识别NF-κB Rel亚基以及NleC介导的切割如何影响感染细胞和动物中的宿主免疫反应仍不清楚。在本研究中，我们表明NleC通过与p65中独特的N端序列相互作用特异性靶向p65/RelA。在培养细胞中鼠柠檬酸杆菌感染期间，NleC在肠上皮细胞中切割p65，尽管只有一小部分分子被切割，以产生p65¹⁻³⁸片段。此外，NleC介导的p65切割显著影响编码促炎细胞因子/趋化因子的NF-κB靶基因子集的表达、结肠中的免疫细胞浸润以及鼠柠檬酸杆菌感染小鼠的组织损伤。从机制上讲，NleC切割产生的p65¹⁻³⁸片段干扰了p65与核糖体蛋白S3（RPS3）之间的相互作用，RPS3是NF-κB的一个“特异性”亚基，可赋予促炎基因转录的一个子集，这放大了仅切割一小部分p65以调节NF-κB介导的基因表达的效果。因此，我们的结果揭示了A/E病原体通过切割一小部分p65并靶向宿主细胞中的p65/RPS3相互作用来特异性阻断NF-κB信号传导和炎症反应的新机制，从而为食源性疾病发病机制提供了新见解。
