Spaceflight analogue culture enhances the host-pathogen interaction between Salmonella and a 3-D biomimetic intestinal co-culture model

Physical forces associated with spaceflight and spaceflight analogue culture regulate a wide range of physiological responses by both bacterial and mammalian cells that can impact infection. However, our mechanistic understanding of how these environments regulate host-pathogen interactions in humans is poorly understood. Using a spaceflight analogue low fluid shear culture system, we investigated the effect of Low Shear Modeled Microgravity (LSMMG) culture on the colonization of Salmonella Typhimurium in a 3-D biomimetic model of human colonic epithelium containing macrophages. RNA-seq profiling of stationary phase wild type and hfq mutant bacteria alone indicated that LSMMG culture induced global changes in gene expression in both strains and that the RNA-binding protein Hfq played a significant role in regulating the transcriptional response of the pathogen to LSMMG culture. However, a core set of genes important for adhesion, invasion, and motility were commonly induced in both strains. LSMMG culture enhanced the colonization (adherence, invasion and intracellular survival) of Salmonella in this advanced model of intestinal epithelium using a mechanism that was independent of Hfq. Although S. Typhimurium hfq mutants are normally defective for invasion when grown as conventional shaking cultures, LSMMG conditions unexpectedly enabled high levels of colonization by an isogenic hfq mutant. In response to infection with either the wild type or mutant, host cells upregulated transcripts involved in inflammation, tissue remodeling, and wound healing during intracellular survival. Interestingly, infection by the hfq mutant led to fewer transcriptional differences between LSMMG- and control-infected host cells relative to infection with the wild type strain. This is the first study to investigate the effect of LSMMG culture on the interaction between S. Typhimurium and a 3-D model of human intestinal tissue. These findings advance our understanding of how physical forces can impact the early stages of human enteric salmonellosis. Overall design: Wild type (WT) Salmonella enterica serovar Typhimurium (S. Typhimurium) and an isogenic hfq deletion mutant (hfq) were grown to stationary phase in Rotating Wall Vessel (RWV) bioreactors positioned in the Low Shear Modeled Microgravity (LSMMG - sample names contain the term LFS) or control orientation. A subset of the samples analyzed in this dataset are triplicate samples of these bacteria alone (no host cells) that were fixed just prior to infection and analyzed using the Illumina NexSeq platform. Comparisons were made between the LSMMG cultures of each strain to the same strain grown as a re-oreinted control. Comparisons were also made between wild type and mutant bacteria under the LSMMG culture condition only or the control culture condition only. The second set of samples in the dataset are the host transcriptional responses to infection with these RWV-cultured strains during adherence (30 minutes post-infection), invasion (3 hours post-infection) and survival (24 hours post-infection). The model host in this study was an RWV-derived, three-dimensional (3-D) intestinal co-culture model comprised of colonic epithelial cells (HT-29) and macrophages (U937).

与太空飞行及太空飞行模拟培养体系相关的物理作用力，可调控细菌与哺乳动物细胞的多种生理应答，进而影响感染过程。然而，目前学界对这类物理环境如何调控人类宿主-病原体互作的机制认知仍十分有限。本研究采用太空飞行模拟低流体剪切培养体系，探究了低剪切模拟微重力（Low Shear Modeled Microgravity, LSMMG）培养对鼠伤寒沙门氏菌（Salmonella Typhimurium）在含巨噬细胞的人类结肠上皮三维仿生模型中定植能力的影响。
仅对处于稳定期的野生型与hfq突变体细菌进行RNA测序（RNA-seq）转录组分析后发现，LSMMG培养可诱导两种菌株的全基因组基因表达发生全局性改变，且RNA结合蛋白Hfq在调控病原体对LSMMG培养的转录应答中发挥了重要作用。不过，两类菌株中均共同诱导了一批与黏附、侵袭及运动相关的核心基因。LSMMG培养可通过不依赖Hfq的机制，增强鼠伤寒沙门氏菌在该先进肠道上皮模型中的定植能力（包括黏附、侵袭与胞内存活）。尽管常规振荡培养条件下，鼠伤寒沙门氏菌hfq突变体通常存在侵袭缺陷，但LSMMG培养条件却意外地让同基因hfq突变体实现了高水平定植。无论是野生型还是突变体菌株感染宿主细胞后，胞内存活阶段的宿主细胞均会上调与炎症、组织重塑及伤口愈合相关的转录本表达。值得注意的是，与野生型菌株感染相比，hfq突变体感染后，LSMMG处理组与对照组感染宿主细胞之间的转录组差异更少。本研究是首个探究LSMMG培养对鼠伤寒沙门氏菌与人类肠道组织三维模型之间互作影响的研究，这些发现加深了我们对物理作用力如何影响人类肠道沙门氏菌病早期进程的认知。
研究整体设计：将野生型（WT）肠炎沙门氏菌鼠伤寒血清型（Salmonella enterica serovar Typhimurium, S. Typhimurium）及其同基因hfq缺失突变体（hfq）置于旋转壁容器（Rotating Wall Vessel, RWV）生物反应器中，分别置于低剪切模拟微重力（LSMMG，样本名称含LFS字样）或对照取向进行培养，至稳定期。本数据集分析的部分样本为仅含细菌（无宿主细胞）的三份重复样本，于感染前固定，并采用Illumina NexSeq平台进行测序分析。比较内容包括：各菌株的LSMMG培养组与同菌株的重新定向对照组；以及仅在LSMMG培养条件下或仅在对照培养条件下的野生型与突变体细菌之间的表达差异。数据集的第二组样本为宿主细胞对上述RWV培养菌株的转录应答，分别采集感染黏附阶段（感染后30分钟）、侵袭阶段（感染后3小时）与胞内存活阶段（感染后24小时）的样本。本研究使用的宿主模型为RWV培养获得的三维（3-D）肠道共培养模型，由结肠上皮细胞（HT-29）与巨噬细胞（U937）组成。