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First published online 26 October 2004
doi: 10.1242/jcs.01500

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Salmonella typhimurium transcytoses flagellin via an SPI2-mediated vesicular transport pathway

¹ Epithelial Pathobiology Division, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, USA
² Division of Digestive Diseases, Department of Medicine, Emory University, Atlanta, GA 30322, USA
³ Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322, USA
⁴ Department of Anatomy, University of Virginia, Charlottesville, Virginia 22908-0732, USA

View larger version (35K): [in a new window]   Fig. 1. S. typhimurium, but not E. coli transcytoses flagellin across polarized epithelia. (A) T84 model epithelia were apically colonized with 5x108 CFU S. typhimurium (SL3201) or a flagellated commensal E. coli strain as described in Materials and Methods. At the indicated times, the apical or basolateral media was isolated and assayed for flagellin by immunoblotting. Apical supernatants were diluted 100-fold and basolateral supernatants were concentrated 20-fold. (B) MDCK model epithelia were colonized as in panel A. Basolateral supernatants were assayed as above for flagellin at the indicated time points. (C) Known concentrations of flagellin were subjected to a similar analysis to provide quantitative data. All data are representative of several experiments.

View larger version (11K): [in a new window]   Fig. 2. S. typhimurium does not alter epithelial permeability to macromolecules. T84 model epithelia were colonized as in Fig. 1 with S. typhimurium and/or E. coli in the presence of 10 mg/ml FITC or 40 kDa FITC-dextran. (A) Transepithelial electrical resistance (TEER), (B) FITC or (C) FITC-dextran concentrations in the basolateral media were measured as described in Materials and Methods. , no bacteria; , S. typhimurium; , E. coli. Data are the means±s.e.m. of three parallel experiments.

View larger version (22K): [in a new window]   Fig. 3. Transcytosis of flagellin does not correlate with bacterial movement. (A,B) T84 model epithelia were colonized apically with 5x108 wild-type S. typhimurium or isogenic fliD mutant. At indicated time, basolateral media was assayed for CFU (A) and western blots were probed for flagellin (B). (C) IL-8 secretion was measured after 5 hours.

View larger version (31K): [in a new window]   Fig. 4. Transcytosis of flagellin is blocked at 20°C. T84 model epithelia were treated in a 37°C incubator or at ambient room temperature (observed to vary between 18.7 and 20.4°C). (A,B) Epithelia were colonized by 5x108 CFU S. typhimurium. Flagellin transcytosis (A) and bacterial invasion (B) were measured as described in Fig. 1 and Materials and Methods. (C) 10 mg/ml FITC-dextran (40 kDa) was added to the apical surface and its appearance was measured in the basolateral reservoir. , 37°C; , room temperature.

View larger version (33K): [in a new window]   Fig. 5. Cellular fractionation reveals the presence of vesicular flagellin not associated with bacteria. T84 model epithelia were colonized with S. typhimurium for 1 hour after which epithelia were disrupted and fractionated by centrifugation as described in Materials and Methods. Fractions were normalized for protein content and then analyzed by SDS-PAGE and immunoblotting for bacterial GroEl and flagellin. This data is representative of several experiments.

View larger version (34K): [in a new window]   Fig. 6. S. typhimurium transcytosis of flagellin is dependent on SPI-2, but not SPI-1. T84 model epithelia were apically colonized with 5x108 CFU wild-type S. typhimurium strains (SL1344 or 12023) or corresponding isogenic mutants (SPI-1 and SPI-2 mutants of SL1344 are invA and ssaR, SPI-2 mutants of 12023 are ssaV and sseB). (A,B) 2 hours after addition of bacteria, flagellin in the apical and basolateral supernatants was measured by SDS-PAGE and immunoblotting as described in Materials and Methods. Results in A are from three parallel experiments whereas those in B are representative of several experiments. (C) Supernatants, in the absence of host cells, of the wild type and SPI-2 mutants were diluted by indicated dilution factor (DF) and subjected to SDS-PAGE immunoblot analysis for flagellin. (D) Internalized bacteria were quantified 1 hour after colonization as described in Materials and Methods. (E) TEER was measured at indicated times in model epithelia colonized by the wild type or SPI mutants. , no bacteria; , SL1344; , SPI-1 mutant; , SPI-2 mutant. Results in D and E are the means±s.e.m. of three parallel experiments. (F,G) At 1 hour or indicated time point, epithelial cells were fractionated as in Fig. 5 and flagellin was assayed in small vesicle isolation (F) or initial lysate (G). Results are representative of several experiments.

View larger version (132K): [in a new window]   Fig. 7. Altered levels/localization of flagellin in epithelia infected with SPI-2 deficient S. typhimurium. MDCK model epithelia were colonized with wild-type S. typhimurium (SL1344) or isogenic mutants for 1 hour, after which epithelia were paraformaldehyde-fixed, immunostained and analyzed by confocal microscopy. Actin is shown in purple. Salmonella complete surface antigens (CSA) are shown in red. Flagellin is shown in green. Flagellae stain with both CSA and flagellin antibodies and thus appear yellow. The sub-apical planes 1.5 µm and 3 µm below the apical surface are shown. Magnification x1000.

View larger version (72K): [in a new window]   Fig. 8. S. typhimurium SPI-2 mutant localizes to the cytoplasm in polarized epithelia. T84 Model epithelia were colonized with wild-type S. typhimurium or isogenic SPI-2 mutant. Two hours following colonization, cells were fixed and analyzed by electron microscopy as described in Methods. Arrows indicate bacteria. Bar, 2.5 µm.

View larger version (16K): [in a new window]   Fig. 9. S. typhimurium SPI-2 mutant exhibits reduced ability to activate IL-8 expression when colonizing the physiologically relevant apical surface of model intestinal epithelia. The indicated bacterial strains were applied to polarized model epithelia and IL-8 secretion measured as described. (A) Bacteria were applied directly to the basolateral reservoir. (B) Bacteria were applied to the apical reservoir; i.e. the physiological site of colonization. IL-8 in the basolateral compartment was assayed 5 hours later by ELISA. Data are the means±s.e.m. of three parallel experiments.

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