Bioimage analysis ofShigellainfection reveals targeting of colonic crypts

Abstract

Few studies within the pathogenic field have used advanced imaging and analytical tools to quantitatively measure pathogenicity in vivo. In this work, we present a novel approach for the investigation of host–pathogen processes based on medium-throughput 3D fluorescence imaging. The guinea pig model forShigella flexneriinvasion of the colonic mucosa was used to monitor the infectious process over time with GFP-expressingS. flexneri. A precise quantitative imaging protocol was devised to follow individualS. flexneriin a large tissue volume. An extensive dataset of confocal images was obtained and processed to extract specific quantitative information regarding the progression ofS. flexneriinfection in an unbiased and exhaustive manner. Specific parameters included the analysis ofS. flexneripositions relative to the epithelial surface,S. flexneridensity within the tissue, and volume of tissue destruction. In particular, at early time points, there was a clear association ofS. flexneriwith crypts, key morphological features of the colonic mucosa. Numerical simulations based on random bacterial entry confirmed the bias of experimentally measuredS. flexnerifor early crypt targeting. The application of a correlative light and electron microscopy technique adapted for thick tissue samples further confirmed the location ofS. flexneriwithin colonocytes at the mouth of crypts. This quantitative imaging approach is a novel means to examine host–pathogen systems in a tailored and robust manner, inclusive of the infectious agent.