Automated Analysis of Intracellular Phenotypes of <em>Salmonella</em> Using ImageJ

Abstract

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free in the cytosol (cytosolic hyper-replication). These different phenotypes of intracellular replication drive to different pathways of pathogenesis, i.e., cytosolic hyper-replication induces inflammatory cell death and extrusion into the gut lumen, while vacuolar replication leads to trans-epithelium penetration and systemic spread. Significant effort was made to create microscopy tools to study the behavior of Salmonella inside invaded cells, such as the pCHAR-Duo fluorescence reporter plasmid that allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP. However, intracellular phenotypes are often manually scored, a time-consuming procedure that limits analysis to a small number of samples and cells. To overcome these limitations, two complementary and automated image analyses were developed using ImageJ, a freely available image analysis software. In the high-throughput protocol, epithelial cells were infected with Salmonella carrying pCHAR-Duo using 96well plates. Imaging was performed using an automated fluorescence microscope.Then, two image analysis methods were applied to measure the intracellular behavior of Salmonella at different detail levels. The first method measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication. It is fast and allows the scoring of a high number of cells and samples, making it suitable for highthroughput assays such as screening experiments. The second method performs single-cell analysis to determine the percentage of infected cells, the mean vacuolar load of Salmonella, and the cytosolic hyper-replication rate giving greater details about Salmonella behavior inside epithelial cells. The protocols can be performed by specifically designed ImageJ scripts to automatically run batch analyses of the major steps of Salmonella-enterocyte interaction.

Salmonella Derby adaptation to swine and simultaneous attenuation for humans go through decay of Salmonella Pathogenicity Island I

“…We thus performed cell culture infection assays on human-derived INT-407 intestinal epithelial cells. Automated fluorescence microscopy was used to quantify at the single cell level the extent of invasion, vacuolar load, and cytosolic hyper-replication ( 25 ) ( Fig. 2A ).…”

“…The bacterial infection of INT-407 and IPEC-J2 cells for the quantification of Salmonella intracellular phenotypes was performed according to Berni et al ( 25 ). Briefly, INT-407 and IPECJ-2 cells were seeded at a density of 3 × 10 4 and 1 × 10 4 cells/well, respectively, in antibiotic-free media 20–24 hours prior to infection in 96-well imaging plates (CellVis) coated with collagen I from rat tail (Invitrogen).…”

“…The automated fluorescence image analysis was performed with FIJI (NIH) ( 50 ). The image analysis workflow and scripts for the automation were described in detail by Berni et al ( 25 ). Briefly, epithelial cells were first segmented and defined as a region of interest (ROI), uniquely labelled with their y - x coordinates.…”