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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.
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