A fluorescence microscopy based genetic screen to identify mutants altered for interactions with host cells.
The study of microbial intracellular pathogenesis has benefited from the application of immunofluorescence microscopy to characterize interactions of the pathogen with host cells. Unfortunately, immunofluorescence microscopy is impractical for screening the large number of bacterial mutants necessary to represent the entire genome of the pathogen. Screening has been limited due to the lack of materials suitable for high-throughput processing (e.g. 96-well plates) that also possess the optical features needed for high resolution fluorescence microscopy. Recently marketed 96-well Special Optics (SO) plates provide both the 96-well template ideal for high-throughput analysis and optical features suitable for fluorescence microscopy. Until this work, mutants needed for the study of a fluorescence-based virulence phenotype could not be obtained by direct screening approaches. In this study, SO plates were used to examine 11520 individual Salmonella typhimurium MudJ mutants for the loss of the ability to disrupt host cell endocytic compartments. The direct application of the fluorescence phenotype for screening allowed us to obtain a set of mutants to characterize the formation of lysosomal membrane glycoprotein (lgp) containing tubules upon Salmonella infection of HeLa epithelial cells. This approach will facilitate the characterization of a wide range of microbial phenotypes detectable by fluorescence microscopy.
Guy
R L
RL
Department of Microbiology, University of Vermont, Burlington, VT 05405-0068, USA.
Gonias
L A
LA
Stein
M A
MA
D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D001431:Bacteriological Techniques; D004705:Endocytosis; D006367:HeLa Cells; D006801:Humans; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009154:Mutation; D055095:Optics and Photonics; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity*; D014774:Virulence
D015703:Antigens, CD; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins