Computational protocol: Cardiolipin Synthesis and Outer Membrane Localization Are Required for Shigella flexneri Virulence

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Protocol publication

[…] The size of infection foci formed in plasma membrane-YFP-expressing HT-29 cells grown in McCoy’s medium (Gibco, Life Technologies) and infected with the listed CFP-expressing S. flexneri strains was determined in a 96-well plate format (catalog no. 3904; Corning). After fixation, the plates were imaged using the ImageXpress Micro imaging system (Molecular Devices), and image analysis for focus size determination was performed with the ImageXpress imaging software (Molecular Devices) as previously described ().Bacterial dissemination was monitored using time-lapse confocal microscopy. Plasma membrane-YFP-expressing HT-29 cells were grown in McCoy’s medium in eight-well chambers (Lab-Tek II [catalog no. 155409; Thermo Fisher Scientific]) at 37°C in 5% CO2. Cells were infected with the listed CFP-expressing S. flexneri strains and imaged with a Leica DMI 8 spinning-disc confocal microscope driven by the iQ software (Andor). Z-stacks were captured 2 h postinfection every 2 min for 6 h. The corresponding movies were generated with the Imaris software (Bitplane).IcsA localization was monitored as previously described (). Briefly, bacteria were grown to mid-logarithmic phase and fixed in 4% (vol/vol) paraformaldehyde in PBS. The cells were then labeled by indirect immunofluorescence, using rabbit polyclonal antibody against IcsA (rabbit 35) diluted 1:100, provided by Edwin Oaks (Walter Reed Army Institute of Research), and a fluorescent isothiocyanate-conjugated goat anti-rabbit secondary antibody diluted 1:100 (). Images were acquired using a DP73 digital microscope camera (Olympus) and processed using cellSens software (Olympus). Images were postprocessed with Lightroom (Adobe) to increase contrast. All images were processed using the same settings. […]

Pipeline specifications

Software tools Imaris, cellSens
Application Conventional fluorescence microscopy
Organisms Shigella flexneri, Bacteria, Homo sapiens
Diseases Bacterial Infections, Infection
Chemicals Cardiolipins, Phosphatidylglycerols