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However, certain enteric pathogens, such as <jats:italic>Salmonella enterica<\/jats:italic> serovar Typhimurium (<jats:italic>S<\/jats:italic>. 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\u2013host 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 <jats:italic>S.<\/jats:italic> Tm induces NAIP\/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected <jats:italic>S.<\/jats:italic> 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 <jats:italic>S<\/jats:italic>. Tm. Overall, the experimental system to introduce <jats:italic>S<\/jats:italic>. Tm into single enteroid cells enables investigations into the molecular basis governing host\u2013pathogen interactions in the cytosol with high spatiotemporal resolution.<\/jats:p>","DOI":"10.1371\/journal.pbio.3002597","type":"journal-article","created":{"date-parts":[[2024,4,29]],"date-time":"2024-04-29T20:00:53Z","timestamp":1714420853000},"page":"e3002597","update-policy":"https:\/\/doi.org\/10.1371\/journal.pbio.corrections_policy","source":"Crossref","is-referenced-by-count":16,"title":["Direct Salmonella injection into enteroid cells allows the study of host\u2013pathogen interactions in the cytosol with high spatiotemporal resolution"],"prefix":"10.1371","volume":"22","author":[{"given":"Chantal","family":"Ernst","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick R.","family":"Andreassen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriel 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