Expression profiling by high throughput sequencing
Summary
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).
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