TMT-Based Quantitative Proteomic Analysis of Intestinal Organoids Infected byListeria monocytogeneswith Different Virulence

Abstract

13Listeria monocytogenes (Lm) is an opportunistic food-borne pathogen that cause 14 listeriosis. L. monocytogenes belonged to different serovars presents with different 15 virulence in the host and caused different host reactions. To investigate the remodeling 16 of host proteome by differently toxic strains, the cellular protein responses of intestinal 17 organoids were analyzed using TMT labeling and high performance liquid 18 chromatography-mass spectrometry. Quantitative proteomic analysis revealed 6564 19 differentially expressed proteins, of which 5591 proteins were quantified. The fold-20 change cutoff was set at 1.3 (Lm vs control), the virulent strain caused 102 up-regulated 21 proteins and 52 down-regulated proteins, while the low virulent strain caused 188 up-22 regulated proteins and 25 down-regulated proteins. These identified proteins were 23 involved in the regulation of essential processes such as biological metabolism, energy 24 metabolism, and immune system process. Some selected proteins were screened by 25 Real-time PCR and Western blotting. These results revealed that differently toxic L. 26 monocytogenes induced similar biological functions and immune responses while had 27 different regulation on differential proteins in the pathway.28 Keywords: Listeria monocytogenes; virulence; proteomic; host response; intestinal 29 organoid.30to evade or modulate immune defences. For example, L. monocytogenes modified 87 bacterial ligands to avoid detection, modulated host signalling pathways to alter host 88 innate defences [32] . The detailed understanding of L. monocytogenes-host interactions 89 and infection and immunity is essential for elucidating these mechanisms. Therefore, 90 investigating the changes in the overall protein abundance of host cells by L. 91 monocytogenes can help to understand the relationship between the pathogenicity and 92 toxicity of the bacteria. 93The majority of studies on L. monocytogenes infection have been conducted in animals 94 such as mice, and single cell line models such as colon adenocarcinoma cells or 95 macrophage cells [25,33] . Most knowledge of the innate and adaptive immune responses 96 has been learned from experimental L. monocytogenes infections of mice [34] . In 97 addition to immune cells, intestinal epithelial cells were also involved in the defense 98 against L. monocytogenes infections. Studies explored that host responses caused by L. 99 monocytogenes included innate immune responses first activated by PRRs of intestinal 100 epithelial cells, immune cells recruited by downstream cytokines and adaptive immune 101 responses subsequently stimulated [32, 34] . However, a single immune model or intestinal 102 cell model both cannot completely reflect the damage of L. monocytogenes to the entire 103 intestinal epithelium. Recently, intestinal organoid was emerging as a more effective 104 infection model that reproduced the differentiation of intestinal epithelial cells, and 105showed the greatest similarity to the intestinal epithelium with respect to...

The application of intestinal organoids and their co-culture systems in the study of gastrointestinal diseases

“…Once the fragments were reassembled into organoids, the introduced gut microbiota or bacterial products were entrapped within the lumen. This model allows the bacteria to encounter the apical and basolateral sides of the IECs [52]. The infection of intestinal organoids with Listeria monocytogenes by the fragmentation method was used for proteomic analysis [52].…”

“…This model allows the bacteria to encounter the apical and basolateral sides of the IECs [52]. The infection of intestinal organoids with Listeria monocytogenes by the fragmentation method was used for proteomic analysis [52]. Muramyl dipeptide, a component of the bacterial cell wall, was introduced into intestinal organoids via fragmentation to study the effect of this bacterial molecule on IECs [53].…”