Salmonella enterica Serovar Typhimurium Exploits Cycling through Epithelial Cells To Colonize Human and Murine Enteroids

Abstract

Enterobacterial pathogens infect the gut by a multistep process, resulting in colonization of both the lumen and the mucosal epithelium. Due to experimental constraints, it remains challenging to address how luminal and epithelium-lodged pathogen populations cross-feed each other in vivo. Enteroids are cultured three-dimensional miniature intestinal organs with a single layer of primary intestinal epithelial cells (IECs) surrounding a central lumen. They offer new opportunities to study enterobacterial infection under near-physiological conditions, at a temporal and spatial resolution not attainable in animal models, but remain poorly explored in this context. We employed microinjection, time-lapse microscopy, bacterial genetics, and barcoded consortium infections to describe the complete infection cycle of Salmonella enterica serovar Typhimurium in both human and murine enteroids. Flagellar motility and type III secretion system 1 (TTSS-1) promoted Salmonella Typhimurium targeting of the intraepithelial compartment and breaching of the epithelial barrier. Strikingly, however, TTSS-1 also potently boosted colonization of the enteroid lumen. By tracing the infection over time, we identified a cycle(s) of TTSS-1-driven IEC invasion, intraepithelial replication, and reemergence through infected IEC expulsion as a key mechanism for Salmonella Typhimurium luminal colonization. These findings suggest a positive feed-forward loop, through which IEC invasion by planktonic bacteria fuels further luminal population expansion, thereby ensuring efficient colonization of both the intraepithelial and luminal niches. IMPORTANCE Pathogenic gut bacteria are common causes of intestinal disease. Enteroids—cultured three-dimensional replicas of the mammalian gut—offer an emerging model system to study disease mechanisms under conditions that recapitulate key features of the intestinal tract. In this study, we describe the full life cycle of the prototype gut pathogen Salmonella enterica serovar Typhimurium within human and mouse enteroids. We map the consecutive steps and define the bacterial virulence factors that drive colonization of luminal and epithelial compartments, as well as breaching of the epithelial barrier. Strikingly, our work reveals how bacterial colonization of the epithelium potently fuels expansion also in the luminal compartment, through a mechanism involving the death and expulsion of bacterium-infected epithelial cells. These findings have repercussions for our understanding of the Salmonella infection cycle. Moreover, our work provides a comprehensive foundation for the use of microinjected enteroids to model gut bacterial diseases.

High definition DIC imaging uncovers transient stages of pathogen infection cycles on the surface of human adult stem cell-derived intestinal epithelium

“…Successful entry of the bacterium was occasionally followed by prompt neighbor-coordinated extrusion of the targeted IEC from the monolayer (Fig 3C). The extrusion phenotype and timeframe corresponded well with the extrusions we previously reported in Salmonella -infected 3D enteroids (15), and which have also been observed in vivo (49–51). Again, using DIC imaging alone, the morphological changes in both the extruding IEC and the neighboring cells could be traced over time (Fig 3C).…”

“…Furthermore, 2D organoid cultures offer an attractive alternative to 3D culture specifically for studies of microbe interactions with the apical cell surface. The latter can be microinjected (15, 54, 55), fragmented (56), or inverted (57), but because of the 3D-topology these structures are better suited for population-scale dynamics than single-microbe behavior in a stable apical plane. While live-cell imaging of microbe dynamics has been published on monolayers of continuous cell lines (3, 47, 58) and to an extent in microinjected 3D enteroids (15), dynamic behaviors of microbes at the surface of a physiologically arranged human intestinal epithelium remain poorly investigated.…”

“…The latter can be microinjected (15, 54, 55), fragmented (56), or inverted (57), but because of the 3D-topology these structures are better suited for population-scale dynamics than single-microbe behavior in a stable apical plane. While live-cell imaging of microbe dynamics has been published on monolayers of continuous cell lines (3, 47, 58) and to an extent in microinjected 3D enteroids (15), dynamic behaviors of microbes at the surface of a physiologically arranged human intestinal epithelium remain poorly investigated. We show that this short-coming can be explained by the inherent conflict between the technical prerequisites for high-definition light microscopy (e.g.…”

“…Human jejunal enteroids were established and cultured as described previously (15, 17). Briefly, pieces of intestinal resections were washed thoroughly in ice-cold PBS and epithelial crypts were subsequently dissociated using Gentle Cell Dissociation Reagent (STEMCELL Technologies, Vancouver, BC, Canada) by nutating at 4°C for 30 minutes followed by trituration.…”

New Insights on the Early Interaction Between Typhoid and Non-typhoid Salmonella Serovars and the Host Cells

“…Several reports have evaluated the functionality of this system during S. Typhimurium infection, showing that the model is suitable. A recent study using both human and mouse enteroids has mapped the full cell cycle of S. Typhimurium, revealing that the invasion and colonization of the epithelial barrier and luminal compartment is boosted by TSS-1 and flagellar motility ( Geiser et al, 2021 ). In this case, the use of enteroids has allowed the identification of each consecutive step leading to effective infection of the epithelial barrier and the contribution of particular virulence factors.…”