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Pseudomonas aeruginosa breaches respiratory epithelia through goblet cell invasion in a microtissue model

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Abstract

Pseudomonas aeruginosa, a leading cause of severe hospital-acquired pneumonia, causes infections with up to 50% mortality rates in mechanically ventilated patients. Despite some knowledge of virulence factors involved, it remains unclear how P. aeruginosa disseminates on mucosal surfaces and invades the tissue barrier. Using infection of human respiratory epithelium organoids, here we observed that P. aeruginosa colonization of apical surfaces is promoted by cyclic di-GMP-dependent asymmetric division. Infection with mutant strains revealed that Type 6 Secretion System activities promote preferential invasion of goblet cells. Type 3 Secretion System activity by intracellular bacteria induced goblet cell death and expulsion, leading to epithelial rupture which increased bacterial translocation and dissemination to the basolateral epithelium. These findings show that under physiological conditions, P. aeruginosa uses coordinated activity of a specific combination of virulence factors and behaviours to invade goblet cells and breach the epithelial barrier from within, revealing mechanistic insight into lung infection dynamics.

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Fig. 1: Human airway model exposes individual steps of P. aeruginosa lung infection.
Fig. 2: Specific virulence factors promote P. aeruginosa lung tissue colonization and breaching.
Fig. 3: Goblet cells serve as entry gates for P. aeruginosa invasion of lung epithelia.
Fig. 4: Pathogen-mediated local tissue destruction provides access to the basolateral tissue side.

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Data availability

The microscopy data that support the findings of this study are openly available in the BioImage Archive (https://doi.org/10.6019/S-BIAD1083)93, and all other data types are in Zenodo (https://zenodo.org/records/10650981)94. Unique biological materials are available from the corresponding author on reasonable request.

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Acknowledgements

We thank J. Sollier for critical reading and editing of the paper and L. Boeck for expert input; S. Roig, K. Schleicher, A. Loynton-Ferrand and L. Guerard (Biozentrum - Imaging core facility), J. Bögli (Biozentrum - FACS core facility), C. Alampi, C. Tiberi Schmidt and M. Chami (Biozentrum – BioEM facility) for technical support; A. Kaczmarczyk and Fabienne Hamburger for help with complementation constructs; M. Basler (Biozentrum – University of Basel) for strains; and I. Schuler for help with histology preparations. Schematics were created with BioRender.com. This study was supported by the Swiss National Science Foundation NCCR AntiResist (51NF40_180541 to U.J.) and financial support from the Roche Institute of Human Biology (IHB).

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  1. These authors contributed equally: A. Leoni Swart, Benoît-Joseph Laventie.

Authors and Affiliations

  1. Biozentrum, University of Basel, Basel, Switzerland

    A. Leoni Swart, Benoît-Joseph Laventie, Rosmarie Sütterlin, Tina Junne, Luisa Lauer, Pablo Manfredi, Sandro Jakonia & Urs Jenal

  2. Cardiovascular, Metabolism, Immunology, Infectious Diseases and Ophthalmology (CMI2O), Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland

    Xiao Yu, Evdoxia Karagkiozi & Rusudan Okujava

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Contributions

A.L.S., B.-J.L., R.O. and U.J. conceptualized the project. A.L.S., B.-J.L., R.S., T.J., L.L., P.M., S.J., X.Y., E.K., R.O. and U.J. developed the methodology. A.L.S., B.-J.L., P.M., R.O. and U.J. conducted formal analysis. A.L.S., B.-J.L., R.S., T.J., L.L., S.J., X.Y. and E.K. conducted investigations. A.L.S., B.-J.L. and U.J. wrote the original paper draft. R.O. and U.J. acquired funding and supervised the project.

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Correspondence to Urs Jenal.

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Extended data

Extended Data Fig. 1 Development of in vitro lung tissues.

a. Schematics of the cell culture process to develop fully differentiated (+21 days post airlift) airway epithelium on Transwell inserts and subsequent apical infection. b, c. Development of the lung tissue post airlift (day 0 to 60) visualized by histology (b) or ICC (c). d, e. Quantification of the thickness (d) and total cell numbers (e) of tissues at day 0, 30 or 60 post airlift, inferred from ICC images. For (d) and (e): Statistical significance was calculated using Kruskal–Wallis H-test with Dunnett’s multiple comparison test. ns; not significant. For panels a-c: n = 3 independent experiments (same donor). For d and e.: n = 15 3D images (30 regions/images for d) examined over 3 independent experiments. Data are presented as mean values +/− SD. f, g. Comparison of airway epithelium on Transwell inserts from 3 donors visualized by histology (f) or ICC (g). n = 3 independent experiments (different donors). Histology: hematoxylin and eosin staining. ICC staining: nuclei: DAPI, mucus: Muc5AC, actin: phalloidin-647, cilia: acetylated-α-tubulin.

Extended Data Fig. 2 Cellular composition and functional properties of the bronchial lung epithelium model.

a, b. Visualization (a) and quantification (b) of the major cell types present in the bronchial epithelium by ICC or FACS, respectively. n = 2 independent experiments (same donor). c. Measurements of cilia beating frequencies by microscopy at day 30 or 60 post airlift. n = 36 inserts over 3 independent experiments (same donor); Box plot: center line, median; box limits, upper and lower quartiles; whiskers, min-max. d, e. Assessment of epithelial barrier integrity (TEER) of lung epithelium at day 0, 30 or 60 post airlift (d, n = 3 independent experiments, same donor) and epithelia from 3 different donors (e, n = 5 independent experiments). For (c), (d) and (e): statistical significance was calculated using Kruskal–Wallis H-test with Dunnett’s multiple comparison test. ns; not significant. f. Visualization of tight junctions by ICC of lung epithelium at day 0 or 30 post airlift. g. Assessment of epithelial barrier integrity (Lucifer yellow permeability) of lung epithelium from 3 different donors (n = 6 inserts over 3 independent experiments). h. Wound healing after physical tissue damage (indicated by the white arrow) of the lung epithelium was followed by time-lapse live imaging for 36 hours (see: Supplementary Information Movie 2). For b, c and f: data are presented as mean values +/− SD. For d, e and g: data are presented as mean values +/− SD. Staining: nuclei: DAPI, cycling basal cells: mucus: Muc5AC, cycling basal cells: α-Ki67, basal cells: α-p63, club cells: α-SCG1A1, cilia: acetylated-α-tubulin, actin: phalloidin-647, tight junctions: ZO-1 Alexa Fluor 555, membrane: CellMask.

Extended Data Fig. 3 Taxonomic distribution of virulence factors among 285 Pseudomonas aeruginosa complete genomes.

a. The phylogenetic tree depicts the isolation source of the PAO1 (blue arc) and PA14 (orange arc) clades. Purple: clinical isolates, black dots indicate clinical strains isolated from human airways, teal: environmental isolates, grey: undetermined or other sources. b. The phylogenetic trees depict the presence of an orthologue of the virulence factors (indicated in the center, using PAO1 nomenclature) among these strains. Presence: purple or pink leaflet, absence: uncolored leaflet. For pilA, two types of homologs were identified within the considered taxonomic range. Branches length relate to genetic distance with an arbitrary scale unit.

Extended Data Fig. 4 Human airway model exposes distinct steps of P. aeruginosa tissue colonization.

a. Images of the lung tissue surface before and after washing inserts with HBSS shows efficient mucus removal. Staining: mucus: Jacalin-fluorescein (green), membrane: CellMask DeepRed (purple). b. P. aeruginosa growth on the apical side of the lung tissues (continuous line) and spread to the basal compartment after tissue breaching (dotted line) with (black) and without (blue) apical mucus upon infection. c. Epithelial barrier integrity (TEER, plain lines) and tissue viability (LDH release, dashed lines) during infection in absence (blue) or presence (black) of mucus. d. Stills of live imaging (25×) of apoptotic events upon infection with P. aeruginosa. Staining: apoptotic cells: NucView (caspase-3/7 activity), membrane: CellMask DeepRed. e. Quantification of dead epithelial cells by FACS, using a viability marker (Zombie NIR), upon infection with P. aeruginosa. Unpaired t test (two-tailed) compared to uninfected; ** P = 0.0011. f. P. aeruginosa population on the airway epithelium (continuous line) and in the basal compartment (dashed line) with PneumaCult-ALI culture medium (black) or HBSS buffer (green) in the basolateral compartment. g, h, i. P. aeruginosa population on the apical (g) and basal side (h) of the lung tissues as well as tissue viability (LDH release) (i) upon infection with different inoculum sizes as indicated. For panels a-i: same donor. j, k. Comparison for 3 different donors of P. aeruginosa growth on the apical side of the lung tissues (continuous line) and spread to the basal compartment after tissue breaching (dotted line) with (black) (j) as well as tissue viability (LDH release) (k). For panels a-f, j, k: inoculum = 103. For all panels n = 3 independent experiments. For j and k: data are presented as mean values +/− SD.

Extended Data Fig. 5 Virulence factors promoting P. aeruginosa lung tissue colonization and breaching.

a. Growth of P. aeruginosa wild type and mutant strains in SCFM medium. b, e, f. Complementation of the PAO1 ΔpilA and ΔpscC mutants. Apical replication (b), basolateral invasion (e) and tissue viability (LDH release) (f) of the mutants and respective complemented strains were assessed. c. P. aeruginosa population in the basolateral compartment up to 48 h p.i. Dunnett’s One-Way ANOVA compared to wild type; P = 0.0274 (ΔfliC), <0.0001 (ΔpilA), 0.0003 (ΔpscC), 0.1585 (Δpch) or <0.0001 (ΔcyaAB); ns: not significant. d. tissue viability (LDH release) during infection with P. aeruginosa. g. Percentage actively beating cilia (>3.5Hz) of the airway epithelium during P. aeruginosa infection measured by live microscopy. h. Stills of live imaging (10x) of the tissue integrity during with P. aeruginosa wild type and Δpch mutant over time. Cell membrane staining: CellMask DeepRed. Inoculum = 101, 103 or 106 c.f.u. as indicated on the left. Representative images, quantified in Fig. 2f. i. P. aeruginosa ΔfliC colonization of the airway epithelium. 10x overview of the entire Transwell (upper panel) and 100× details (middle and lower panels) (ICC). Related to Fig. 2c, g. Staining: nuclei: DAPI, actin: phalloidin-647. j. Quantification of the airway epithelium attachment by P. aeruginosa mutants from ICC images. Related to Fig. 2h. Dunnett’s One-Way ANOVA compared to wild type; ** P < 0.01; ns: not significant. For all panels: Inoculum = 103, n = 3 independent experiments (same donor). For j: n = 51 images examined over 3 independent experiments. For b-f: data are presented as mean values +/− SD.

Extended Data Fig. 6 Selective internalization into goblet cells allows P. aeruginosa to invade lung epithelia.

a-d. Representative examples of 100× ICC images of P. aeruginosa attaching to and invading lung epithelial cells at 6 h and 12 h p.i. Attached (a), internalized (b) and replicating bacteria (c) are shown. Goblet cell invasion is illustrated in (d) using an additional stain for mucus, and was consistently observed in all three donors tested (e). For all panels: Inoculum = 103. n = 3 independent experiments. For a-d: same donor, for e: different donors. Staining: bacteria: constitutive chromosomal expression of mNeonGreen (yellow) or mCherry (pink), nuclei: DAPI, mucus: Muc5AC, membrane: CellMask, actin: phalloidin-647, tight junctions: ZO-1 Alexa Fluor 555.

Extended Data Fig. 7 Internalization of P. aeruginosa and sloughing of infected epithelial cells.

a. Quantification of the complemented T6SS mutants ΔH2 (ΔtssL2) and ΔH3 (ΔtssM3) internalized in epithelial cells 12 hours p.i. Unpaired t test (two-tailed) compared to wild type; P = <0.0001 (ΔtssL2), 0.0321 (ΔtssL2+tssL2), 0.0003 (ΔtssM3) or 0.0444 (Δ tssM3+tssM3); n = 51 images examined over 3 independent experiments. Data are presented as mean values +/− SD. b, c. Representative ICC images (100×) depicting sloughing of epithelial cells infected with P. aeruginosa (b) and colonization of sloughed cells by extracellular bacteria (c). For all panels: inoculum = 103. n = 3 independent experiments (same donor). Staining: bacteria: constitutive chromosomal expression of mNeonGreen (yellow) or mCherry (pink), nuclei: DAPI, actin: phalloidin-647.

Extended Data Fig. 8 Intracellular replication and localization of P. aeruginosa T3SS mutants.

a, b. Schematic (a) and 100× ICC images (b) of the intracellular replication of a P. aeruginosa T3SS mutant (ΔpscC) resulting in mechanical cell rupture and release of intracellular bacteria. The progression shown in (b) is a compilation of different stages of breaching sites observed in samples 24 h p.i. Staining: bacteria: constitutive chromosomal expression of mNeonGreen, nuclei: DAPI, membrane: CellMask, tight junctions: ZO-1 Alexa Fluor 555. Inoculum = 103. c. Subcellular localization in lung epithelial cells of P. aeruginosa T3SS mutant (ΔpscC) compared to wild type PAO1 using Transmission Electron Microscopy. The tissue sections were performed on the epithelium apical surface, and the cilia are visible on the left of overview images. Intracellular bacteria are marked with white asterisks. Inoculum = 106 (WT) or 103 (ΔpscC). For all panels: n = 3 independent experiments (same donor).

Extended Data Fig. 9 P. aeruginosa breaching and basolateral invasion of the lower airway epithelium.

a, b. Schematic (a) and 100× ICC images (b) of P. aeruginosa invasion of the basolateral side of the lung epithelium after successful establishment of a breaching site. c. Progression of lower airway infection by P. aeruginosa PAO1 wild type recorded by SEM. d, e. Immunocytochemistry images of P. aeruginosa breaching sites seen for all three donors tested (d) and for the PAO1 ΔpscC complemented mutants (e). For all panels: Inoculum = 103. n = 3 independent experiments (same donor [a-c and e] or different donors [d]). ICC staining: bacteria: constitutive chromosomal expression of mNeonGreen, nuclei: DAPI, tight junctions: ZO-1 Alexa Fluor 555.

Extended Data Fig. 10 Rapid colonization of breaching sites is driven by chemotaxis.

Breaching site colonization in samples infected with a 1:1 mixture of differentially labeled (pink or yellow) wild type and ΔcheA1/2 mutant strains. a. Wild type + wild type example of co-colonized breach. b. Wild type + ΔcheA1/2 example of co-colonized breach. c. Wild type + ΔcheA1/2 example of a breach solely colonize by wild type. d. ΔcheA1/2 + ΔcheA1/2 example of breaches colonized by only one of the two ΔcheA1/2 strains. For all panels: inoculum = 103, n = 3 independent experiments (same donor). Staining: bacteria: constitutive chromosomal expression of mNeonGreen or mCherry, nuclei: DAPI, actin: phalloidin-647.

Supplementary information

Supplementary Information

Suplementary Fig. 1.

Reporting Summary

Supplementary Video 1

Cilia beating and mucus flow. Real-time recording of beating cilia and mucus flow on the apical surface of the lower airway model. Staining: membrane, CellMask.

Supplementary Video 2

Tissue regeneration and P. aeruginosa infection of injured lung epithelium. Wound healing after physical tissue damage of uninfected lower airway epithelium (top panels) and selective P. aeruginosa invasion of the insured site upon infection (bottom panels). Staining: bacteria, constitutive chromosomal expression of mNeonGreen; membrane, CellMask. Inoculum = 103, n = 3 (same donor).

Supplementary Video 3

Increased frequency of apoptotic lung cell events upon infection with P. aeruginosa. Live imaging (25×) of apoptotic events upon infection with P. aeruginosa. Staining: apoptotic cells, NucView (caspase-3/7 activity); membrane, CellMask. Inoculum = 103, n = 3 (same donor).

Supplementary Video 4

Pseudomonas aeruginosa invades lung cells. Example of immunocytochemistry image (100×) where bacteria are visible both on the lung epithelium surface and internalized in a lung cell (12 h p.i.). P. aeruginosa, mNeonGreen (yellow); actin, phalloidin-647 (purple); nuclei, DAPI (blue). To facilitate the visualization, cells where segmented and rendered in 3D using IMARIS.

Supplementary Tables

Supplementary Table 1. Bacterial strains used in this study. Related to key resources table. Table 2. Key resources used in this study.

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Swart, A.L., Laventie, BJ., Sütterlin, R. et al. Pseudomonas aeruginosa breaches respiratory epithelia through goblet cell invasion in a microtissue model. Nat Microbiol 9, 1725–1737 (2024). https://doi.org/10.1038/s41564-024-01718-6

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