Department of Molecular Biotechnology and Microbiology, Gdansk University of Technology, Gdansk, Poland
Published Date: 16/09/2021.
*Corresponding author: Roman Kotłowski, Department of Molecular Biotechnology and Microbiology, Gdansk University of Technology, Gdansk, Poland
The aim of this study was to identify genes of the HT29 cell line, whose transcription occurs in response to infection by pathogenic strains of E. coli UM146 and E. coli UM147 isolated from individuals with CD and UC, respectively and in response to the presence of non-pathogenic strain of E. coli Nissle 1917, for better understanding of the mechanism of IBD. General findings from our experiments are related to protective action of chemokine ligand 6 indicating very important role of IL6 in inducing inflammation processes driven by two pathogenic E. coli strains used in our study. We also determined important role of molecules participating in transport of ions, carbohydrates, mRNAs and proteins as well as higher expression levels for molecules involved in RNA modification processes. Those expression differences may somehow influence host-pathogen interaction differences for two pathogenic strains with different toxin types versus one non-pathogenic E. coli.
Keywords: Inflammatory bowel Disease; Microarray; HT-29 cell line; Escherichia coli
Abbreviations: CD: Crohn’s Disease; IBD: Inflammatory Bowel Disease; PCR: Polymerase chain; reaction; RMA: Robust MultiChip Average; RNA: Ribonucleic acid; SPATE: Serine Protease Autotransporters; t-RNA: Transporting ribonucleic acid; UC: Ulcerative Colitis
Crohn's disease (CD) and Ulcerative Colitis (UC) are most common diseases in the group of inflammatory bowel diseases (IBD). One of the features characteristics to both diseases is as yet undetermined etiology. Usually, patients with CD or UC have mutations in the genes engaged in the immune responses [1,2], and epithelial gaps shown by sophisticated microscopic examination . It has also been found that the levels of expression of Secretory Leukocyte Protease Inhibitor (SLPI) can contribute to healing of lesions . Likewise, protease inhibitors may also regulate the inflammation . Furthermore, point mutations in the nod2 gene particularly in patients with CD decrease expression of proinflammatory cytokines, antimicrobial peptides including defensins . In addition, studies on intestinal microflora composition [6,7] and pure Lipopolysaccharide (LPS) use  confirmed the involvement of microorganisms in IBD. Kind of treatments against excessive apoptosis postulated in IBD patients together with factors involved in IBD is presented in Table 1.
Table 1: IBD Factors and Suggested Treatments.
Material consisted of two pathogenic strains of E. coli, UM146 and UM147, isolated from patients with CD and UC, respectively, non-pathogenic strain of E. coli Nissle 1917, and human colon adenocarcinoma cell line HT-29. The common feature of both strains is the presence of S-fimbriae, a vacuolating autotransporter toxin belonging to Serine Protease Autotransporters (SPATE) and antigen 43. In addition, E. coli UM146 is P-fimbriae positive in the PCR test and E. coli UM147 has a sort of trypsin activity. Single colonies of the bacteria were cultured at 37° C for 18 h in 10 ml LB broth (Part No. 1427-500 BP) from Fisher Scientific. In order to determine the level of gene transcription of the HT29 line in response to bacterial infection, 1 ml of bacterial culture (ca. 107 CFU) was added to a flat-bottomed vessel with a bottom area equal to 75 cm2, in which HT-29 were cultured until cells became confluent and grew on the 70% area of the bottom. The samples after the addition of the bacteria were incubated for 3 hours at 37 °C in the presence of 5% CO2, after which a trypsin-containing solution was added in order to separate the cell line from the bottom surface - the resulting cell suspension was transferred into centrifuge tubes. Cells were centrifuged at 1.000 rpm for 10 min at 4°C. RNA isolation was performed by extraction with guanidine thiocyanate, phenol and chloroform (3). Hybridization of RNA to immobilized probes of single-stranded DNA on a GeneChip HG-U133A Plus 2.0 microarray and the detection of biotinylated RNA sequences were carried out according to the recommendations of Affymetrix®. Cultures of HT29 cells and E. coli as well as experiments involving microarrays were performed in a single experiment due to reproducibility difficulties. The results of the microarray were normalized using Robust MultiChip Average (RMA) using the Affymetrix® Expression Console™, and selection of line HT-29 genes differing from one another depending on the strain of E. coli, were determined using the equations from Table 2.
Table 2: Selection of most significant results for RNA transcripts of HT-29 cell line.
Only results with %-difference values higher than ±10 were collected in Table 3 as supplementary material, while additional information concerning ontology terms and involvement in Reactome pathways in Table 4 as supplementary material. Presented in Tables 3 results are divided into three groups of RNA transcripts grouped based on %-differences and skewness values in comparison to the reference results. In the first group characterized by negative %-difference and positive skewness values 4 out of 5 ID-probes were identified: (I.) Heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A) – HNRNPU mainly involved in mRNA spicing; (II.) Chemokine (C-X-C motif) ligand 6 - CXCL6 involved in neutrophil mediated immunity; (III.) Mir210 host gene - MIR210HG with unknown function and (IV.) Zinc finger protein 207 - ZNF207 participating in regulation of chromosome segregation in mitotic cell division were identified. To the second group characterized by positive %-difference and positive skewness only four out of nine ID-probes were identified. These are: (I.) Midasin AAAATP-ase 1 - MDN1 involved in ribosomal large subunit protein complex assembly; (II.) Small nuclear ribonucleoprotein polypeptide A' - SNRPA1 participating in mRNA splicing, via spliceosome; (III.) Nucleoporin 58 kDa - NUP58 involved mainly in mitotic cell cycle, t-RNA processing transmembrane transport of carbohydrates, mRNA and proteins as well as regulation of cellular response to heat and (IV.) Solute carrier family 20, SLC20A1 is responsible for phosphate-containing compound metabolic processes like phosphate and sodium ions transmembrane transport and positive regulation of I-kappaB kinase/NF-kappaB signaling. Third group we found constitute 46 ID Probes, and their characteristics are presented in Table 5. The most repeated function for this group is mediation of RNA processing connected with the gene expression of HT-29 cells in the response to pathogenic E. coli strains in terms of elevated level of RNA transcripts [9,10].
Table 5: Function of mRNA transcripts characterized by positive %-difference and negative skewness values.
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The authors declare that they do not have any competing interests.