Direct Salmonella injection into enteroid cells allows the study of host-pathogen interactions in the cytosol with high spatiotemporal resolution

• Published in: PLoS biology 2024-04, Vol.22 (4), p.e3002597-e3002597
• Main Authors: Ernst, Chantal, Andreassen, Patrick R, Giger, Gabriel H, Nguyen, Bidong D, Gäbelein, Christoph G, Guillaume-Gentil, Orane, Fattinger, Stefan A, Sellin, Mikael E, Hardt, Wolf-Dietrich, Vorholt, Julia A
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis; Apoptosis Regulatory Proteins - genetics; Apoptosis Regulatory Proteins - metabolism; Bacteria; Biology and Life Sciences; Calcium-Binding Proteins; CARD Signaling Adaptor Proteins - genetics; CARD Signaling Adaptor Proteins - metabolism; Cell activation; Cell death; Cell interactions; Cytosol; Cytosol - metabolism; Cytosol - microbiology; Epithelial cells; Epithelial Cells - metabolism; Epithelial Cells - microbiology; Epithelium; Flagella; Flagellin; Flagellin - metabolism; Host-parasite relationships; Host-Pathogen Interactions; Inflammasomes; Inflammasomes - metabolism; Intestinal Mucosa - metabolism; Intestinal Mucosa - microbiology; Ligands; Medicine and Health Sciences; Methods; Mice; Mice, Inbred C57BL; Microorganisms; Microscope and microscopy; Microscopy; Neuronal Apoptosis-Inhibitory Protein - genetics; Neuronal Apoptosis-Inhibitory Protein - metabolism; Nutrient uptake; Pathogens; Physiological aspects; Proteins; Research and Analysis Methods; Salmonella; Salmonella Infections - immunology; Salmonella Infections - metabolism; Salmonella Infections - microbiology; Salmonella typhimurium - metabolism; Salmonella typhimurium - pathogenicity; Short Reports; Single-Cell Analysis - methods; Type III Secretion Systems - metabolism
• ISSN: 1545-7885; 1544-9173; 1545-7885
• DOI: 10.1371/journal.pbio.3002597

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.