Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
Population Health & Immunity Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.
Front Cell Infect Microbiol. 2018 Jul 16;8:244. doi: 10.3389/fcimb.2018.00244. eCollection 2018.
is a parasitic protist that causes diarrhea in humans, affecting mainly children of the developing world, elderly and immunocompromised individuals. Humans are infected by two major assemblages (i.e. genetic subtypes), A and B, with the latter being the most common. So far, there is little information on molecular or cellular changes during infections with assemblage B. Here, we used RNA sequencing to study transcriptional changes in Caco-2 intestinal epithelial cells (IECs) co-incubated with assemblage B (GS isolate) trophozoites for 1.5, 3, and 4.5 h. We aimed to identify early molecular events associated with the establishment of infection and followed cellular protein changes up to 10 h. IEC transcriptomes showed a dominance of immediate early response genes which was sustained across all time points. Transcription of inflammatory cytokines (e.g., , and ) peaked at 1.5 and 3 h of infection. Compared to co-incubation with assemblage A , we identified the induction of novel cytokines () and showed that inflammatory signaling is mediated by Erk1/2 phosphorylation (mitogen activated protein kinase, MAPK), nuclear factor kappa B (NFκB) and adaptor protein-1 (AP-1). We also showed that GS trophozoites attenuate P38 (MAPK) phosphorylation in IECs. Low amounts of IL-8, CXCL1 and CCL20 proteins were measured in the interaction medium, which was attributed to cytokine degradation by trophozoite secreted proteases. Based on the transcriptome, the decay of cytokines mRNA mediated by zinc finger protein 36 might be another mechanism controlling cytokine levels at later time points. IEC transcriptomes suggested homeostatic responses to counter oxidative stress, glucose starvation, and disturbances in amino acid and lipid metabolism. A large group of differentially transcribed genes were associated with cell cycle arrest and induction of apoptosis, which was validated at protein level. IEC transcriptomes also suggested changes in tight junction's integrity, microvilli structure and the extracellular mucin layer. This is the first study to illuminate transcriptional and protein regulatory events underlying IECs responses and pathogenesis during assemblage B infection. It highlights differences compared to assemblage A infections which might account for the differences observed in human infections with the two assemblages.
是一种寄生的原生动物，会导致人类腹泻，主要影响发展中国家的儿童、老年人和免疫功能低下者。人类感染两种主要的集合体（即遗传亚型），A 和 B，后者更为常见。到目前为止，关于 B 集合体感染期间分子或细胞变化的信息很少。在这里，我们使用 RNA 测序来研究共培养的 Caco-2 肠上皮细胞（IEC）与 B 集合体（GS 分离株）滋养体 1.5、3 和 4.5 h 时的转录变化。我们的目的是鉴定与感染建立相关的早期分子事件，并跟踪细胞蛋白变化，直至 10 h。IEC 转录组显示立即早期反应基因的优势，这种优势在所有时间点都持续存在。炎症细胞因子（如 、 和 ）的转录在感染后 1.5 和 3 h 达到峰值。与与 A 集合体共培养相比，我们鉴定了新型细胞因子的诱导（），并表明炎症信号通过 Erk1/2 磷酸化（丝裂原激活蛋白激酶，MAPK）、核因子 kappa B（NFκB）和衔接蛋白-1（AP-1）介导。我们还表明，GS 滋养体减弱了 IEC 中的 P38（MAPK）磷酸化。在相互作用介质中测量到少量的 IL-8、CXCL1 和 CCL20 蛋白，这归因于滋养体分泌的蛋白酶对细胞因子的降解。基于转录组，锌指蛋白 36 介导的细胞因子 mRNA 衰减可能是控制后期细胞因子水平的另一种机制。IEC 转录组提示稳态反应以对抗氧化应激、葡萄糖饥饿以及氨基酸和脂质代谢紊乱。一大组差异转录基因与细胞周期停滞和凋亡诱导有关，这在蛋白质水平上得到了验证。IEC 转录组还提示紧密连接完整性、微绒毛结构和细胞外粘蛋白层的变化。这是第一项阐明 B 集合体感染期间 IEC 反应和发病机制的转录和蛋白质调节事件的研究。它突出了与 A 集合体感染的差异，这可能解释了两种集合体在人类感染中的差异。
