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

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101183755 Publication Model: eCollection Cited Medium: Internet ISSN: 1545-7885 (Electronic) Linking ISSN: 15449173 NLM ISO Abbreviation: PLoS Biol Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science, [2003]-

Cytosol*/metabolism ; Cytosol*/microbiology ; Salmonella typhimurium*/pathogenicity ; Salmonella typhimurium*/metabolism ; Host-Pathogen Interactions* ; Type III Secretion Systems*/metabolism ; Inflammasomes*/metabolism ; Flagellin*/metabolism ; Calcium-Binding Proteins*; Animals ; Mice ; Neuronal Apoptosis-Inhibitory Protein/metabolism ; Neuronal Apoptosis-Inhibitory Protein/genetics ; Epithelial Cells/microbiology ; Epithelial Cells/metabolism ; Apoptosis Regulatory Proteins/metabolism ; Apoptosis Regulatory Proteins/genetics ; Mice, Inbred C57BL ; CARD Signaling Adaptor Proteins/metabolism ; CARD Signaling Adaptor Proteins/genetics ; Single-Cell Analysis/methods ; Salmonella Infections/microbiology ; Salmonella Infections/metabolism ; Salmonella Infections/immunology ; Intestinal Mucosa/microbiology ; Intestinal Mucosa/metabolism

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.
Competing Interests: The authors have declared that no competing interests exist.
(Copyright: © 2024 Ernst et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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0 (Type III Secretion Systems)
0 (Inflammasomes)
12777-81-0 (Flagellin)
0 (Neuronal Apoptosis-Inhibitory Protein)
0 (Apoptosis Regulatory Proteins)
0 (Ipaf protein, mouse)
0 (CARD Signaling Adaptor Proteins)
0 (Calcium-Binding Proteins)

Date Created: 20240429 Date Completed: 20240429 Latest Revision: 20240502

20240502

PMC11057982

10.1371/journal.pbio.3002597

38684033