Bioimage analysis of Shigella infection 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 for Shigella flexneri invasion of the colonic mucosa was used to monitor the infectious process over time with GFP-expressing S. flexneri. A precise quantitative imaging protocol was devised to follow individual S. flexneri in a large tissue volume. An extensive dataset of confocal images was obtained and processed to extract specific quantitative information regarding the progression of S. flexneri infection in an unbiased and exhaustive manner. Specific parameters included the analysis of S. flexneri positions relative to the epithelial surface, S. flexneri density within the tissue, and volume of tissue destruction. In particular, at early time points, there was a clear association of S. flexneri with crypts, key morphological features of the colonic mucosa. Numerical simulations based on random bacterial entry confirmed the bias of experimentally measured S. flexneri for early crypt targeting. The application of a correlative light and electron microscopy technique adapted for thick tissue samples further confirmed the location of S. flexneri within 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.Shigella flexneri | host-pathogen interactions | intestinal crypts | bioimage analysis | tissue microbiology

Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells

“…6), consistent with the data of Arena et al (29). The Alexa-488 labeled polysaccharide shows clear overlap with a monoclonal antibody to glycoprotein 2 (GP2), an M-cell specific marker (Fig.…”

Stress‐induced host membrane remodeling protects from infection by non‐motile bacterial pathogens

“…Shigella infection of guinea pigs (120–250 g; Charles River Laboratories) and sample preparation were performed as described previously (Arena et al , 2015). Infections were performed in accordance with Institut Pasteur animal protocols (no.…”

The Gut Microbiome: Connecting Spatial Organization to Function

“…Preservation of endogenous fluorescence is not compatible with denaturing fixatives required for maintenance of mucus. Cross-linking fixatives such as 10% formalin or 4% paraformaldehyde are compatible with fluorescent reporters, and coupled with sucrose immersion and OCT embedding and cryosectioning, high-resolution images of bacteria in morphologically intact tissue with luminal contents have been achieved (Arena et al, 2015; Muller et al, 2012). In cases where mucus preservation is desired, methacarn fixation coupled with immunofluorescence against cloned or native bacterial proteins enables the simultaneous visualization of mucus and gut bacteria.…”

“…Fluorescent protein expression has been used successfully to study several pathogens (Arena et al, 2015; Muller et al, 2012) and commensals (Amar et al, 2011; Jemielita et al, 2014), although such in vivo bacterial localization studies require some persistence of the strain within the community. Moreover, to engineer protein expression within such a strain requires that the species be culturable and genetically tractable.…”