Characterization of the micro-environment of Salmonella typhimurium-containing vacuoles within MDCK epithelial cells.
Salmonella typhimurium has the capacity to enter into and multiply within epithelial cells. During the entire intracellular stage, bacteria are enclosed within a vacuole. To characterize the micro-environment of the bacteria-containing vacuoles, we have used a new method to measure the expression levels of several S. typhimurium genes in intracellular bacteria within Madin-Darby canine kidney (MDCK) epithelial cells. Our study was based on the determination of beta-galactosidase activity derived from lacZ transcriptional fusions using the highly sensitive substrate fluorescein-di-beta-D-galactoside (FDG). Expression of the iroA and mgtB genes (induced by Fe2+ and Mg2+ limitation respectively), and cadA (induced by pH 6.0 in the presence of lysine, with enhanced expression under anaerobiosis) were characterized at different post-infection times. High intracellular expression levels were detected for the iroA and mgtB genes, suggesting that the concentrations of free Fe2+ and Mg2+ in the vacuole may be low. cadA activity was detected only at early post-infection times (4 h), suggesting that the vacuole may have a mild-acidic pH, and oxygen and lysine present at this time. Globally, the results reported indicate that the use of a highly sensitive beta-galactosidase substrate can provide information about the micro-environment within which an intracellular pathogen, such as S. typhimurium, resides.
Garcia-del Portillo
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Biotechnology Laboratory, University of British Columbia, Vancouver, Canada.
Foster
J W
JW
Maguire
M E
ME
Finlay
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BB
D000332:Aerobiosis; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D004285:Dogs; D004848:Epithelium / Q000382:microbiology* / Q000648:ultrastructure; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000502:physiology; D007668:Kidney; D008239:Lysine / Q000378:metabolism; D008274:Magnesium / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012486:Salmonella typhimurium / Q000235:genetics / Q000502:physiology*; D014617:Vacuoles / Q000382:microbiology*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D007501:Iron; D008274:Magnesium; D008239:Lysine