Evaluating the effect of spaceflight on the host–pathogen interaction between human intestinal epithelial cells and Salmonella Typhimurium

Spaceflight uniquely alters the physiology of both human cells and microbial pathogens, stimulating cellular and molecular changes directly relevant to infectious disease. However, the influence of this environment on host–pathogen interactions remains poorly understood. Here we report our results from the STL-IMMUNE study flown aboard Space Shuttle mission STS-131, which investigated multi-omic responses (transcriptomic, proteomic) of human intestinal epithelial cells to infection with Salmonella Typhimurium when both host and pathogen were simultaneously exposed to spaceflight. To our knowledge, this was the first in-flight infection and dual RNA-seq analysis using human cells.

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A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

Software and code
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Data analysis
Protein identification and quantification was performed with ProteinPilot 4.0 software using Paragon algorithm.
RNA integrity was analyzed using Agilent 2100 Bioanalyzer Expert Software.
RNA-seq reads for each sample were quality checked using FastQC v0.10.1 and aligned to Human GRCh38.p7 primary assembly and S. Typhimurium LT2 genome using STAR v2.5.1b. A series of quality control metrics were generated on the STAR outputs. Cufflinks v2.2.1 was used to report FPKM values (Fragments Per Kilobase of transcript per Million mapped reads) and read counts. TPM (Transcripts Per Million) was calculated by an in-house R script. Differential expression (DE) analysis was performed with EdgeR package from Bioconductor v3.2 in R 3.2.3. DAVID 6.8 was used to perform enrichment analysis of RNA-seq data.
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April 2020
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability All data generated during this study are either included in the manuscript and its Supplementary files (Supplementary Table 12) or are available from the corresponding author upon reasonable request. RNA-Seq data are available at the Gene Expression Omnibus (GEO) database (GSE156066; BioProject PRJNA656571) and NASA GeneLab (https://genelab-data.ndc.nasa.gov/genelab/accession/GLDS-323).

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Sample size
Proteomics: Proteomic analysis using iTRAQ was performed on total cellular protein from HT-29 cells from triplicate flight and ground bioreactors (N=3 for flight and ground infected and uninfected cultures).
RNA seq: RNA was extracted from flight and ground cultures using remaining samples following the proteomics processing. From the remaining samples, RNA-seq analyses were performed using duplicate flight and ground bioreactors for all conditions (N=2 biological replicates) except for the infected flight samples, which only had sufficient sample remaining to evaluate duplicate technical aliquots from a single bioreactor (i.e., N=1 biological; N=2 technical replicates).
Data exclusions The presence of FBS in the culture media resulted in strong interference from bovine proteins; a subset of which could not be distinguished from human proteins due to their identical sequence homologies and thus were excluded from our analysis.

Replication
The study was performed in spaceflight and thus all replicates were performed during that single spaceflight. It is not easy to obtain funding for or access to the microgravity platform to replicate spaceflight experiments.