Extracellular Vesicles from Probiotic and Beneficial Escherichia coli Strains Exert Multifaceted Protective Effects Against Rotavirus Infection in Intestinal Epithelial Cells

Abstract

Rotavirus remains a major cause of severe acute gastroenteritisin infants worldwide. The suboptimal efficacy of current vaccines underscores the needfor alternative microbiome-based interventions, including postbiotics. Extracellularvesicles (EVs) from probiotic and commensal <em>E. coli</em> strains have been shown to mitigatediarrhea and enhance immune responses in a suckling-rat model of rotavirus infection.Here, we investigate the regulatory mechanisms activated by EVs in rotavirus-infectedenterocytes. <strong>Methods: </strong>Polarized Caco-2 monolayers were used as a model of matureenterocytes. Cells were pre-incubated with EVs from the probiotic <em>E. coli</em> Nissle 1917 (EcN)or the commensal EcoR12 strain before rotavirus infection. Intracellular Ca²⁺concentration, ROS levels, and the expression of immune- and barrier-related genes andproteins were assessed at multiple time points post-infection. <strong>Results:</strong> EVs from bothstrains exerted broad protective effects against rotavirus-induced cellular dysregulation,with several responses being strain-specific. EVs interfered with viral replication bycounteracting host cellular processes essential for rotavirus propagation. Specifically, EVtreatment significantly reduced rotavirus-induced intracellular Ca²⁺ mobilization, ROSproduction, and COX-2 expression. In addition, both EV types reduced virus-inducedmucin secretion and preserved tight junction organization, thereby limiting viral accessto basolateral coreceptors. Additionally, EVs enhanced innate antiviral defenses viadistinct, strain-dependent pathways: EcN EVs amplified IL-8-mediated responses,whereas EcoR12 EVs preserved the expression of interferon-related signaling genes.<strong>Conclusions:</strong> EVs from EcN and EcoR12 act through multiple complementarymechanisms to restrict rotavirus replication, spread, and immune evasion. These findingssupport their potential as effective postbiotic candidates for preventing or treatingrotavirus infection.