Xi De, Hofmann Lukas, Alter Thomas, Einspanier Ralf, Bereswill Stefan, Heimesaat Markus M, Gölz Greta, Sharbati Soroush
Institute of Veterinary Biochemistry, Freie Universität Berlin, Berlin, Germany.
Institute of Food Safety and Food Hygiene, Freie Universität Berlin, Berlin, Germany.
Gut Pathog. 2021 Jun 28;13(1):42. doi: 10.1186/s13099-021-00437-1.
Campylobacter jejuni (C. jejuni) infections are of increasing importance worldwide. As a typical mucosal pathogen, the interaction of C. jejuni with mucins is a prominent step in the colonisation of mucosal surfaces. Despite recent advances in understanding the interaction between bacterial pathogens and host mucins, the mechanisms of mucin glycosylation during intestinal C. jejuni infection remain largely unclear. This prompted us to identify relevant regulatory networks that are concerted by miRNAs and could play a role in the mucin modification and interaction.
We firstly used a human intestinal in vitro model, in which we observed altered transcription of MUC2 and TFF3 upon C. jejuni NCTC 11168 infection. Using a combined approach consisting of in silico analysis together with in vitro expression analysis, we identified the conserved miRNAs miR-125a-5p and miR-615-3p associated with MUC2 and TFF3. Further pathway analyses showed that both miRNAs appear to regulate glycosyltransferases, which are related to the KEGG pathway 'Mucin type O-glycan biosynthesis'. To validate the proposed interactions, we applied an in vivo approach utilising a well-established secondary abiotic IL-10 mouse model for infection with C. jejuni 81-176. In colonic tissue samples, we confirmed infection-dependent aberrant transcription of MUC2 and TFF3. Moreover, two predicted glycosyltransferases, the sialyltransferases ST3GAL1 and ST3GAL2, exhibited inversely correlated transcriptional levels compared to the expression of the identified miRNAs miR-125a-5p and miR-615-3p, respectively. In this study, we mainly focused on the interaction between miR-615-3p and ST3GAL2 and were able to demonstrate their molecular interaction using luciferase reporter assays and RNAi. Detection of ST3GAL2 in murine colonic tissue by immunofluorescence demonstrated reduced intensity after C. jejuni 81-176 infection and was thus consistent with the observations made above.
We report here for the first time the regulation of glycosyltransferases by miRNAs during murine infection with C. jejuni 81-176. Our data suggest that mucin type O-glycan biosynthesis is concerted by the interplay of miRNAs and glycosyltransferases, which could determine the shape of intestinal glycosylated proteins during infection.
空肠弯曲菌（C. jejuni）感染在全球范围内的重要性日益增加。作为一种典型的黏膜病原体，空肠弯曲菌与黏蛋白的相互作用是其在黏膜表面定植的关键步骤。尽管在理解细菌病原体与宿主黏蛋白之间的相互作用方面取得了最新进展，但空肠弯曲菌肠道感染期间黏蛋白糖基化的机制仍基本不清楚。这促使我们确定由微小RNA（miRNA）协同作用且可能在黏蛋白修饰和相互作用中发挥作用的相关调控网络。
我们首先使用了一种人类肠道体外模型，在此模型中，我们观察到空肠弯曲菌NCTC 11168感染后MUC2和TFF3的转录发生改变。通过将计算机分析与体外表达分析相结合的方法，我们鉴定出与MUC2和TFF3相关的保守miRNA miR-125a-5p和miR-615-3p。进一步的通路分析表明，这两种miRNA似乎都调控糖基转移酶，这些酶与KEGG通路“黏蛋白型O-聚糖生物合成”相关。为了验证所提出的相互作用，我们采用了一种体内方法，利用一个成熟的非生物性白细胞介素-10（IL-10）小鼠模型来感染空肠弯曲菌81-176。在结肠组织样本中，我们证实了MUC2和TFF3存在依赖于感染的异常转录。此外，两种预测的糖基转移酶，即唾液酸转移酶ST3GAL1和ST3GAL2，与所鉴定的miRNA miR-125a-5p和miR-615-3p的表达相比，转录水平呈现负相关。在本研究中，我们主要关注miR-615-3p与ST3GAL2之间的相互作用，并能够通过荧光素酶报告基因检测和RNA干扰来证明它们的分子相互作用。通过免疫荧光检测小鼠结肠组织中的ST3GAL2，发现在空肠弯曲菌81-176感染后强度降低，这与上述观察结果一致。
我们首次报道了在小鼠感染空肠弯曲菌81-176期间miRNA对糖基转移酶的调控。我们的数据表明，黏蛋白型O-聚糖生物合成是由miRNA和糖基转移酶的相互作用协同进行的，这可能决定感染期间肠道糖基化蛋白的形态。
