Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland.
Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
PLoS Biol. 2024 Apr 29;22(4):e3002597. doi: 10.1371/journal.pbio.3002597. eCollection 2024 Apr.
Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.
肠上皮细胞 (IECs) 在营养物质吸收和抵御肠道微生物方面发挥着关键作用。然而,某些肠道病原体,如鼠伤寒沙门氏菌 (S. Tm),可以通过鞭毛和 III 型分泌系统 (T3SS) 及其同源效应蛋白入侵 IECs,并利用 IECs 作为复制栖所。IECs 检测到鞭毛或 T3SS 蛋白会导致宿主细胞迅速作出反应,即激活炎症小体。在这里,我们引入了一种基于流体力显微镜 (FluidFM) 的单细胞操纵技术,该技术可将细菌直接递送到单层鼠类类器官内的单个 IEC 细胞的细胞质中。这种方法允许在与之前的事件(如对接、摄取起始或空泡逃逸)脱钩的情况下,特异性地研究细胞质中的病原体-宿主细胞相互作用。与现有认识一致,我们使用活细胞炎症小体报告基因表明,IEC 细胞质中 S. Tm 的暴露会通过其已知配体鞭毛蛋白和 T3SS 杆和针引发 NAIP/NLRC4 炎症小体。尽管缺乏这些入侵相关配体,但注射的缺乏这些入侵相关配体的 S. Tm 突变体仍能够在 IEC 的细胞质中生长,尽管缺乏 T3SS 功能,这表明,在没有 NAIP/NLRC4 炎症小体激活和随之而来的细胞死亡的情况下,不需要效应子介导的宿主细胞操作即可使上皮细胞质对 S. Tm 的生长变得许可。总体而言,将 S. Tm 引入单个类肠细胞的实验系统能够以高时空分辨率研究支配细胞质中宿主-病原体相互作用的分子基础。