miR-215 Modulates Ubiquitination to Impair Inflammasome Activation and Autophagy During Salmonella Typhimurium Infection in Porcine Intestinal Cells

This article belongs to the Special Issue Salmonella and Salmonellosis: Implications in Public Health.

Sequencing data have been deposited at National Institutes of Health Sequencing Reading Archive (NCBI SRA) database under Bioproject accession ID PRJNA1160452. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the Proteomics Identification Database (PRIDE) (81) partner repository with the dataset identifier PXD054446.

The host response to S. Typhimurium infection can be post-transcriptionally regulated by miRNAs. In this study, we investigated the role of miR-215 using both in vivo porcine infection models and in vitro intestinal epithelial cell lines. Several miRNAs were found to be dysregulated in the porcine ileum during infection with wild-type and SPI2-defective mutant strains of S. Typhimurium, with some changes being SPI2-dependent. Notably, miR-215 was significantly downregulated during infection. To explore its functional role, gain-of-function experiments were performed by transfecting porcine intestinal epithelial cells (IPEC-J2) with a miR-215-5p mimic, followed by label-free quantitative (LFQ) proteomic analysis. This analysis identified 157 proteins, of which 35 were downregulated in response to miR-215 overexpression, suggesting they are potential targets of this miRNA. Among these, E2 small ubiquitin-like modifier (SUMO)-conjugating enzyme UBC9 and E3 ubiquitin-ligase HUWE1 were identified as key targets, both of which are upregulated during S. Typhimurium infection. The miR-215-mediated downregulation of these proteins resulted in a significant decrease in overall ubiquitination, a process crucial for regulating inflammasome activation and autophagy. Consistently, inflammasome markers caspase 1 (CASP1) and apoptosis-associated speck-like protein containing a CARD (ASC), as well as autophagy markers microtubule-associated protein 1A/1B-light chain 3 (LC3B) and Ras-related protein Rab-11 (RAB11A), showed decreased expression in miR-215 mimic-transfected and infected IPEC-J2 cells. To further validate these findings, human intestinal epithelial cells (HT29) were used as a complementary model, providing additional insights into conserved immune pathways and extending the observations made in the porcine system. Overall, our findings demonstrate that miR-215 plays a significant role in modulating host inflammasome activation and autophagy by targeting proteins involved in ubiquitination during S. Typhimurium infection.

This work was supported by the Spanish Ministry of Economy and Competitiveness (AGL2017-87415-R), the Spanish Ministry of Science, Innovation and Universities (PID2022-142887OB-I00) and Plan Propio de Investigación 2020 from Universidad de Cordoba

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